Binding sites for exogenous and endogenous non-competitive inhibitors of the nicotinic acetylcholine receptor

Binding sites for exogenous and endogenous non-competitive inhibitors of the nicotinic acetylcholine receptor

Biochimica et Biophysica Acta 1376 (1998) 173^220 Binding sites for exogenous and endogenous non-competitive inhibitors of the nicotinic acetylcholin...
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Biochimica et Biophysica Acta 1376 (1998) 173^220

Binding sites for exogenous and endogenous non-competitive inhibitors of the nicotinic acetylcholine receptor Hugo Rube¨n Arias * Instituto de Investigaciones Bioqu|¨micas de Bah|¨a Blanca, Consejo Nacional de Investigaciones Cient|¨¢cas y Te¨cnicas, and Universidad Nacional del Sur, Camino La Carrindanga Km. 7, C.C. 857, 8000 Bah|¨a Blanca, Argentina Received 21 October 1997; revised 13 March 1998; accepted 18 March 1998

Abstract The nicotinic acetylcholine receptor (AChR) is the paradigm of the neurotransmitter-gated ion channel superfamily. The pharmacological behavior of the AChR can be described as three basic processes that progress sequentially. First, the neurotransmitter acetylcholine (ACh) binds the receptor. Next, the intrinsically coupled ion channel opens upon ACh binding with subsequent ion flux activity. Finally, the AChR becomes desensitized, a process where the ion channel becomes closed in the prolonged presence of ACh. The existing equilibrium among these physiologically relevant processes can be perturbed by the pharmacological action of different drugs. In particular, non-competitive inhibitors (NCIs) inhibit the ion flux and enhance the desensitization rate of the AChR. The action of NCIs was studied using several drugs of exogenous

Abbreviations: UV, ultraviolet; AChR, nicotinic acetylcholine receptor; GABA, Q-aminobutyric acid; 5-HT, 5-hydroxytryptamine ; ACh, acetylcholine; CCh, carbamylcholine ; SubCh, suberyldicholine; d-TC, d-tubocurarine; NMDA, N-methyl-D-aspartate; AChSL, spin-labelled acetylcholine; K-BTx, K-bungarotoxin; NCI, non-competitive inhibitor; CPZ, chlorpromazine; TPMP‡ , triphenylmethylphosphonium; PCP, phencyclidine; HTX, histrionicotoxin; QX-222, NP-(trimethylaminomethyl)-2P,6P-xylidide; QX-314, NP-(triethylaminomethyl)-2P,6P-xylidide; [125 I]TID, 3-(tri£uoromethyl)3-m-([125 I]iodophenyl)diazirine ; Alphaxalone, 3K-hydroxy-5K-pregnane-11,20 dione ; Betaxalone, 21-acetoxy-3L-hydroxy-5K-pregnane-11,20 dione; EPR, paramagnetic electron spin resonance; fa , apparent fraction of available £uorophores; KQ , apparent Stern-Volmer constant; K/KPCSL , association constant of hydrophobic spin-labels relative to spin-labelled phosphatidylcholine; 5-, 7-, 12-, 14- and 16-SAL, n-doxyl stearate positional isomers (n-SAL) ; LASL, spin-labelled local anesthetic; PCSL, spin-labelled phosphatidylcholine; 4-, 5-, 12-, 14-, and 16-PCSL, 4-, 5-, 12-, 14-, and 16-doxyl phosphatidylcholine; ASL, spin-labelled androstane; CSL, spin-labelled cholestane; diBrCHS, dibromocholesteryl-hemisuccinate ; DOPC, dioleoylphosphatidylcholine; FRET, £uorescence resonance energy transfer; dansyl-C6 -choline, 6-(5-dimethylaminonaphthalene-1-sulfonamido) hexanoic acid-L-(N-trimethylammonium) ethyl ester; C12 -eosin, 5-(N-dodecanoylamino)eosin; C18 -rhodamine, octadecylrhodamine B; C12 -£uorescein, 5-(dodecanoylamino)£uorescein; Di10ASP-PS, N-(3-sulfopropyl)-4-(p-didecylaminostyryl)pyridinium ; C12 -Texas Red, N-(Texas Red0 sulfonyl)-5 (and 6)-dodecanoylamine; 7-AS, 7-(9-anthroyloxy)stearate; AC7 , [NP-methyl,NP-4-diazoniumphenyl][N-8-octanoic acid, 2-(trimethylammonium bromide)ethyl ester]urea ; d, electrical distance; PIP, 1,1-dimethyl-4-acetyl-piperazinium iodide; MTSEA, methanethiosulfonate ethylammonium; 16:0, palmitic acid; 18:0, stearic acid; 19:0, nonadecanoic acid; 20:0, arachidic acid; 18:1[n-9], oleic acid; trans-18:1[n-9], elaidic acid; 18:1[n-7], vaccenic acid; trans-18[n-7], trans-vaccenic acid; 18:1[n-12], petroselenic acid; 18:2[n-6], linoleic acid; 18:3[n-3], linolenic acid; 20:4[n-6], arachidonic acid; 22:6[n-3], docosahexanoic acid; Testosterone-3, testosterone-3-3-(Ocarboxymethyl)oxime; P-3-BSA, progesterone-3-(O-carboxymethyl)oxime-bovine serum albumin; P-11-BSA, 11 K-hydroxyprogesterone n-hemisuccinate-bovine serum albumin; Kp , apparent partition coe¤cient; Kd , apparent dissociation constant; IC50 , concentration to inhibit 50% activity; Ki , apparent inhibition constant; EC50 , concentration to enhance 50% activity; KB , agonist concentration which causes 50% self-inhibition; vG³, free energy change; vH³, enthalpy change; don , apparent mean open time; kon , association rate constant; koff , dissociation rate constant * Corresponding author. Fax: +54 (91) 861 527; E-mail: [email protected] 0304-4157 / 98 / $ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 5 7 ( 9 8 ) 0 0 0 0 4 - 5

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origin. These include compounds such as chlorpromazine (CPZ), triphenylmethylphosphonium (TPMP‡ ), the local anesthetics QX-222 and meproadifen, trifluoromethyl-iodophenyldiazirine (TID), phencyclidine (PCP), histrionicotoxin (HTX), quinacrine, and ethidium. In order to understand the mechanism by which NCIs exert their pharmacological properties several laboratories have studied the structural characteristics of their binding sites, including their respective locations on the receptor. One of the main objectives of this review is to discuss all available experimental evidence regarding the specific localization of the binding sites for exogenous NCIs. For example, it is known that the so-called luminal NCIs bind to a series of ring-forming amino acids in the ion channel. Particularly CPZ, TPMP‡ , QX-222, cembranoids, and PCP bind to the serine, the threonine, and the leucine ring, whereas TID and meproadifen bind to the valine and extracellular rings, respectively. On the other hand, quinacrine and ethidium, termed non-luminal NCIs, bind to sites outside the channel î from the lipid^water interface and lumen. Specifically, quinacrine binds to a non-annular lipid domain located V7 A î ethidium binds to the vestibule of the AChR in a site located V46 A away from the membrane surface and equidistant from both ACh binding sites. The non-annular lipid domain has been suggested to be located at the intermolecular interfaces of the five AChR subunits and/or at the interstices of the four (M1^M4) transmembrane domains. One of the most important concepts in neurochemistry is that receptor proteins can be modulated by endogenous substances other than their specific agonists. Among membrane-embedded receptors, the AChR is one of the best examples of this behavior. In this regard, the AChR is non-competitively modulated by diverse molecules such as lipids (fatty acids and steroids), the neuropeptide substance P, and the neurotransmitter 5-hydroxytryptamine (5-HT). It is important to take into account that the above mentioned modulation is produced through a direct binding of these endogenous molecules to the AChR. Since this is a physiologically relevant issue, it is useful to elucidate the structural components of the binding site for each endogenous NCI. In this regard, another important aim of this work is to review all available information related to the specific localization of the binding sites for endogenous NCIs. For example, it is known that both neurotransmitters substance P and 5-HT bind to the lumen of the ion channel. Particularly, the locus for substance P is found in the NM2 domain, whereas the binding site for 5-HT and related compounds is putatively located on both the serine and the threonine ring. Instead, fatty acid and steroid molecules bind to non-luminal sites. More specifically, fatty acids may bind to the belt surrounding the intramembranous perimeter of the AChR, namely the annular lipid domain, and/or to the high-affinity quinacrine site which is located at a non-annular lipid domain. Additionally, steroids may bind to a site located on the extracellular hydrophilic domain of the AChR and/or at the lipid^protein interface. Specifically, at the annular lipid domain or close to the non-annular quinacrine binding site. The self-inhibitory action of ACh at millimolar concentrations can be also considered an endogenous mechanism for the functional modulation of the AChR. Studies on the localization of the agonist self-inhibitory locus suggest that agonists at very high concentrations may bind to the ion channel (a luminal site) and/or to the quinacrine site (a non-luminal site). Focusing on the premise that certain structural domains of the AChR involved in NCI binding account for the functional effect of the ligand under study, the existence of luminal and non-luminal binding sites supports the idea of at least two distinct mechanisms of action for NCIs: a steric mechanism where the drug obstructs the ion permeation and an allosteric process where the AChR, upon ligand binding, suffers such a conformational change that the ion channel becomes closed and thus the ion flux is impeded. ß 1998 Elsevier Science B.V. All rights reserved. Keywords: Nicotinic acetylcholine receptor; Luminal non-competitive inhibitor binding site; Non-luminal non-competitive inhibitor binding site; Exogenous non-competitive inhibitor; Endogenous non-competitive inhibitor; Agonist self-inhibitory binding site

Contents 1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

175

2.

Structural components of the nicotinic acetylcholine receptor . . . . . . . . . . . . . . . . . . . . . .

177

3.

The nicotinic acetylcholine receptor-associated ion channel . . . . . . . . . . . . . . . . . . . . . . . .

180

4.

Functional properties of nicotinic acetylcholine receptors . . . . . . . . . . . . . . . . . . . . . . . . .

182

5.

Exogenous and endogenous non-competitive inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Pharmacological properties of non-competitive inhibitors . . . . . . . . . . . . . . . . . . . . . . 5.2. Low- and high-a¤nity non-competitive inhibitor binding sites . . . . . . . . . . . . . . . . . .

183 185 185

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5.3. Voltage-sensitive and voltage-insensitive non-competitive inhibitors . . . . . . . . . . . . . . .

187

6.

Luminal high-a¤nity non-competitive inhibitor binding sites . . . . . 6.1. The serine ring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. The leucine ring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. The threonine ring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. The valine ring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5. The intermediate and the cytoplasmic or inner ring . . . . . . . . 6.6. The outer or extracellular ring . . . . . . . . . . . . . . . . . . . . . . . .

... ... ... ... ... ... ...

187 188 189 190 190 191 191

7.

Luminal localization of binding sites for endogenous non-competitive inhibitors . . . . . . . . 7.1. The 5-hydroxytryptamine binding site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2. The substance P binding site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

192 192 194

8.

Non-luminal high-a¤nity non-competitive inhibitor binding sites . . . . . . . . . . . . . . . . . . . 8.1. Localization of the ethidium binding site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2. Localization of the quinacrine binding site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

196 197 197

9.

Non-luminal localization of binding sites for endogenous non-competitive inhibitors . . . . . 9.1. Localization of fatty acid binding sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2. Localization of steroid binding sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

201 201 206

..... ..... ..... ..... ..... ..... .....

..... ..... ..... ..... ..... ..... .....

..... ..... ..... ..... ..... ..... .....

10. Agonist self-inhibition of the nicotinic acetylcholine receptor . . . . . . . . . . . . . . . . . . . . . . 10.1. Similarity between non-competitive inhibition and agonist-self inhibition of the nicotinic acetylcholine receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2. Luminal localization for the agonist self-inhibitory binding site . . . . . . . . . . . . . . . . . 10.3. Non-luminal localization for the agonist self-inhibitory binding site . . . . . . . . . . . . .

211 212 213

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

214

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Nicotinic acetylcholine receptors (AChRs) are a family of transmembrane glycoproteins including both the muscle-type and the neuronal-type AChR. Both AChR subtypes share several structural and functional properties with other ligand-gated ion channel receptors, namely the Q-aminobutyric acid (GABAA and GABAC type), the glycine, and the 5-hydroxytryptamine (5-HT3 type) receptor (reviewed in [1^4]). The neurotransmitter-gated ion channel superfamily has physiological importance since all their members play pivotal roles in fast synaptic transmission throughout both the central and the peripheral nervous system (e.g., see review [5]). The observed functional properties of this receptor superfamily can be described in terms of four main attributes: (1) the receptors have the ability to recognize and to bind their speci¢c neurotransmitters; (2) upon ligand binding, the intrinsic ion channel

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associated with each particular receptor protein opens; (3) after channel opening, speci¢c ions (e.g., Cl3 for glycine, GABAA and GABAC receptors, and Na‡ , K‡ and Ca2‡ for both nicotinic and 5-HT3 receptors) are selectively conducted through the lipid membrane (K‡ is conducted to the outside of the cell); and ¢nally, (4) in the prolonged presence of agonists, the receptors desensitize and in this conformational state the receptor-associated ion channel is closed [6]. Most of the structural information about the ligand-gated ion channel receptor superfamily was obtained from studies with the muscle-type AChR (for more details see Sections 2 and 3). Nevertheless, it is believed that the basic constituents of the AChR are common to the other members as well. For the sake of illustration, the structural motifs of the ligandgated ion channel superfamily are depicted in Fig. 1. The fundamental architecture of these receptors is as follows. (a) Each receptor is an oligomer of

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Fig. 1. (A) Schematic representation of the primary sequence of each member of the ligand-gated ion channel receptor superfamily: the nicotinic acetylcholine receptor (AChR), the 5-hydroxytryptamine receptor (type 5-HT3 ), the Q-aminobutyric acid receptor (type GABAA ), and the glycine receptor. SP, signal peptide; M1^M4, transmembrane domains; C-C, Cys-Cys bridge found in the receptor superfamily; Y, oligosaccharide groups. (B) Diagram of the tertiary organization of the AChR subunits. Each AChR subunit includes: (1) a long NH2 -terminal hydrophilic extracellular region which bears oligosaccharide groups (Y); (2) four highly hydrophobic domains named M1, M2, M3, and M4. It is postulated that the intrinsic AChR ion channel is composed by ¢ve M2 segments (one from each subunit). Moreover, M1 and M2 as well as M2 and M3 are connected by minor hydrophilic stretches; and (3) a major hydrophilic segment facing the cytoplasm which has several phosphorylation sites (P). Additionally, the M4 domain orientates the COOH-terminus to the synaptic side of the membrane.

¢ve subunits (see [7^10]). (b) Based on hydrophobicity analysis it is suggested that the receptors present three well di¡erentiated domains: a hydrophilic extracellular region, a hydrophobic region proposed to reside in the membrane, and a hydrophilic cytoplasmic portion. (c) The extracellular domain contains both the N- and the C-termini. In the N-terminus, the agonist binding sites (reviewed in [1,11,12]), and two disul¢de-linked cysteines separated by 13 other amino acids forming a 15-residue loop [3] have been found. These receptors are called Cys-loop receptors due to the existence of this loop. The N-terminus domain also bears, depending on the receptor subunit, one, or more than one putative glycosylation site (reviewed in [13]). The exception is the subunit of the 5-HT3 receptor which has no glycosylation

site. (d) The primary sequence of each receptor subunit crosses the lipid membrane four times forming the transmembrane (hydrophobic) domain M1^M4. (e) The M2 sequences from each subunit combine to form the wall of the receptor-associated ion channel. In cation-selective channels the M2 sequences contain negatively charged residues, whereas in anionselective channels they contain positively charged residues. (f) The intracellular domain is formed by a hydrophilic stretch bracketed by the M3 and M4 transmembrane sequences. In turn, (g) the M1^M2 and M2^M3 domains are linked by short hydrophilic stretches either located in the cytoplasm or extracellularly, respectively. To address all available information on each member of the ligand-gated ion channel superfamily lies

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outside the scope of this review. For this purpose, we refer the reader to other thematically wider reviews (i.e., see [1,2,4,14]). The present review attempts to de¢ne the fundamental architecture and the basic function of the paradigm of this ion channel receptor superfamily: the AChR, with the ¢nal intention of clarify the mechanism of action for non-competitive inhibitors (NCIs). Much e¡ort has been expended elucidating the key domains of the AChR responsible for agonist binding, ion conduction, and gating. An emerging concept has been to consider the receptor protein as a signal transducer that undergoes modulation control by allosterically acting ligands. Of particular interest are molecules that inhibit the agonist-evoked ion £ux activity in a non-competitive manner: the so-called NCIs. Some relevant points regarding the pharmacological process of non-competitive inhibition, but not the general pharmacology of NCIs, is considered in this review. For example, we outline the progress evidenced on the localization of NCI binding sites for drugs from exogenous origin and, what is much more important, for those endogenous molecules that have been found to directly interact with the AChR in a non-competitive fashion including lipids such as neurosteroids and fatty acids, the neurotransmitter 5-hydroxytryptamine (5-HT) and related compounds, as well as the neuropeptide substance P. In this regard, we review the available experimental evidence indicating the existence of both luminal (located into the ion channel) and non-luminal (located outside the ion channel) binding sites for NCIs. In turn, the existence of both NCI binding site classes support the idea of at least two di¡erent mechanisms of action for NCIs: a steric mechanism where the drug obstructs the ion pore and an allosteric process where the AChR, upon e¡ector binding, undergoes a conformational change in which the probability of the ion channel being open declines and thus, the ion £ux is impeded. Since the inhibitory process of the AChR elicited by agonists at high concentrations (in the millimolar concentration range) is pharmacologically very similar to that evidenced for classical NCIs, and considering that the neurotransmitter acetylcholine (ACh) is also an endogenous molecule, the localization of the agonist self-inhibitory locus is also addressed.

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2. Structural components of the nicotinic acetylcholine receptor The muscle-type AChR is a heteromeric membrane-embedded protein formed by four subunits: K1, L1, Q (or O), and N. Depending on the developmental stage of mammalians, the AChR subunit stoichiometry changes from K12 L1QN (embryonic) to K12 L1ON (adult). The receptor from Torpedo electric organs is always present in the embryonic form. Additionally, a shorter Qs subunit encoded by exon 5 was found in the C2C12 cell line [15]. The missing 52 residue portion seems to be important for the modulation of the AChR channel gating kinetics [16]. Neuronal-type AChRs are formed by only two subunit classes, the K and the non-K (or L). Exploiting the possibilities provided by several molecular biological approaches, seven K (K2^K8) and three L (L2^L4) subunits have been found so far (reviewed in [17]). The pharmacology of the K9 subunit, speci¢cally found in rat cochlear hair cells, is di¡erent from both muscle- and neuronal-type receptors [18]. The immense subunit diversity found for neuronaltype AChRs opens the possibility for the existence of several heteromeric combinations (reviewed in [1]). For example, one of the most abundant species found in the brain is the K4L2 receptor. The putative stoichiometry of this receptor is (K4)2 (L2)3 [19], a subunit ratio similar to that found for the muscletype AChR: (K)2 (non-K)3 . In addition, homomeric nicotinic receptors such as K7 and K8 [20] as well as K9 [21] have been found in several cell types. Nevertheless, K7-, K8-, or K9-containing heteromeric receptors might also exist (reviewed in [1]). Concomitantly, the observed variety in subunit composition for neuronal-type AChRs contributes to the functional diversity existent in nature (reviewed in [5,17]). Such receptor diversity gives to each neuronal- and muscle-type AChR di¡erent ligand sensitivity. Moreover, receptor distribution is tissue-, regional-, cellular-, and subcellular-dependent. Thus, each AChR class has the potential for physiologically distinct functions. Electron microscopy has been used to elucidate the overall structure of the muscle-type AChR. Viewed from outside the cell, the AChR appears as a rosette î in diameter with a central depression of of 70^80 A î V25 A wide. The ¢ve subunits are arranged pseudo-

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symmetrically around an axis that passes through the ion pore, perpendicular to the plane of the lipid membrane [22,23]. The observed rosette is formed by the extracellular hydrophilic domain of the receptor containing both the N- and the C-termini (Fig. 1B). The N-terminus of each subunit is formed by approximately 210 amino acids and protrudes 60^70 î from the membrane. In the N-terminus of the K A subunits exists the main immunogenic region (comprising at least residues 67^76) which is the binding target for more than 60% of the antibodies raised against the AChR protein in the autoimmune disease myasthenia gravis (reviewed in [24]). This particular epitope was recently observed in three dimensions by high resolution electron microscopy [25]. The observed central depression is the extracellular portion of the ion channel, also called the vestibule. This domain has the binding site for the NCI ethidium (see Section 8). Deeper within the receptor vestibule the lumen of the ion channel is found. The wall of the ion pore is formed by the M2 transmembrane region from each subunit. The other transmembrane domains of the muscle-type AChR, M1, M3, and M4, have been shown to be in close contact with the lipid phase [26^29] (reviewed in [30]). The most hydrophobic domain, M4, is the least conserved among the existent transmembrane domains. The M1 and M3 domains are believed to be important for the process of AChR assembly [31] and channel gating [32], respectively. The transmembrane domain from each muscle-type AChR subunit is formed by 19^27 residues, giving a calculated length î if the sequence is assumed to form an of about 40 A K-helix (Fig. 1B). The transmembrane sequences have traditionally been considered as K-helical. However, this is not known with certainty. Analyzing î resolution, Unwin electron microscopy images at 9 A [8] proposed the existence of L-strands in M1, M3, and M4. Based on this observation, he hypothesized that the L-strands from all subunits form a L barrel surrounding the ¢re M2 K-helices. The existence of transmembrane L-strands has received support from other studies as well. In this regard, enzymatically digested AChRs with the extracellular and the cytoplasmic portions `shaved' o¡ contained 50% K-helices and 40% L-strands and turns [33]. However, completeness of digestion problems and the possibility of conformational changes induced by removal of

the extramembrane portions has to be taken into account when considering such results. Moreover, the use of either hydrophilic or hydrophobic probes to label the transmembrane area from the ion pore or from the lipid membrane, respectively, allows one to infer the secondary structure of this particular portion of the AChR. Particularly, the labelling pattern of methanethiosulfonate ethylammonium (MTSEA), a positively charged sulfhydryl-speci¢c reagent, on residues previously substituted by Cys, indicated that the M2 transmembrane domain is essentially K-helical, except for three residues in the middle of the ion channel [34]. This coincides with the observation of a kink in the middle of the ion pore [8]. On the other hand, the 3-(tri£uoromethyl)3-m-([125 I]iodophenyl)diazirine ([125 I]TID)-labelled residues on M1, M3, and M4 suggest a secondary structural motif in which part of both M3 and M4 are K-helical, whereas the middle of M1 is non-helical [28]. The latter observation is not totally consistent with the MTSEA labelling on Cys-substituted residues indicating that the extracellular portion of M1 is non-helical [35]. The mixed helix-L model of the AChR transmembrane domain has received support from modelling studies where a membrane-embedded structure formed only by K-helices is less probable [36]. The interest in the segment KM1, from the point of view of NCI binding site location, is that it has been shown to be labelled by the derivative [3 H]quinacrine azide [37] (see Section 8 for more details). Regarding the surface of the AChR in intimate contact with the lipid membrane, two well-di¡erentiated lipid binding regions have been identi¢ed: the annular lipid domain and the non-annular lipid domain [38]. The annular lipid domain is surrounded by a belt of V45 lipid molecules abutting the intramembranous perimeter of the AChR [39^41]. Although the exact place for non-annular lipid binding sites is unknown, these are proposed to be located at the intramolecular interfaces of the ¢ve subunits and/or at the interstices of the four transmembrane domains [36,38,42], close to the hydrophilic portions of the AChR [43]. The intracellular hydrophilic domain of the AChR, the major cytoplasmic loop, measures 15^20 î wide and is composed of, depending on the subA unit, 109^142 amino acids (Fig. 1B). In the case of

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the muscle-type AChR, this loop contains the region that interacts non-covalently with the peripheral membrane protein rapsyn, originally called 43 kDa protein. This protein functions in receptor clustering and receptor-cytoskeleton communication [44]. Additionally, in the Torpedo N subunit, a Cys residue forming a disul¢de bridge between two AChR protein monomers, each with a molecular mass Mr of V290 kDa, was found [45]. However, the exact physiological importance of the existence of AChR protein dimers (Mr V580 kDa) in electrocytes (typical cells of electric organs from electric ¢shes of the order Torpediniformes) has not been elucidated as yet. The dimeric form does not exist in muscle and the pharmacological characteristics of ligand binding and ion channel activity from both monomers and dimers were shown to be practically identical [46]. Nevertheless, patch-clamp data from monomers and dimers puri¢ed from Torpedo californica and reconstituted in large lipid vesicles indicated that ion channels from dimers present larger (double) conductances (84 þ 6 pS) than that from monomers (42 þ 3 pS) [47]. The cytoplasmic domain also contains one tyrosine phosphorylation site on the L, the Q, and the N subunits [48] (Fig. 1B). In addition, the existence of both protein kinases A and C in AChR-rich membranes suggests that Ser residues from di¡erent receptor subunits may be phosphorylated (reviewed in [49]). For example, a conserved phosphorylation site was recently identi¢ed as Ser342 in the K7 receptor subtype [50]. Since AChR tyrosine phosphorylation is involved in the intrinsic process of desensitization [51] and some NCI molecules are found to enhance the rate of AChR desensitization with the concomitant occurrence of ion channel inhibition [52], it is conceivable that NCIs may modulate the desensitization process through phosphorylation of the AChR. This is a possible mode of action for some NCIs, but most NCIs show a direct allosteric mechanism for inhibition. Indirect allosteric modulation, in which the NCI binding enhances phosphorylation is certainly possible, but has not been observed. Thus, allosteric enhancement of phosphorylation will not be discussed further in this review. Interestingly, studies using chimeric forms of the AChR have indicated that the three main domains of the AChR (i.e., the extracellular, the hydrophobic,

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and the cytoplasmic), each one with distinctive structural characteristics, possess functional speci¢city. For instance, the chimeric AChR constructed from the N-terminus of the neuronal-subtype K7 and the rest of the protein from the 5-HT3 receptor preserves the functional property of recognizing and binding ACh with subsequent cation £ux activity [53]. In addition, data from studies using K3/K4 chimeras where the N-terminus of one subunit has been exchanged for the other chain support the view that the apparent sensitivity di¡erence found between both K3and K4-containing receptors can be ascribed to the N-terminal segment of the K subunit [54]. Finally, chimeric K7/glycine or K7/GABAA receptors in which three amino acids from the M2 transmembrane domain of either the glycine or the GABAA receptor were introduced into the M2 domain of the K7 were gated by ACh but the £ux activity was found to be speci¢c for anions, indicating that the M2 domain is involved in the ion speci¢city elicited by each receptor [55]. The arrangement of the AChR subunits around the ion channel has been studied by using di¡erent approaches. One model where the L chain is inserted between both K subunits was proposed on the basis of cross-linking of subunit image reconstruction technique [22], of image analysis of tubular crystals of AChR-rich membranes from T. marmorata electric organ [8,56^58], and of cholinergic agonist-dependent toxin cross-linking of AChR subunits [59,60]. This subunit arrangement has also received support by using electron microscopy and the monoclonal antibody 247G which is considered a marker of the high-a¤nity d-tubocurarine (d-TC) binding site [61,62]. On the contrary, by using d-TC as a probe to label the competitive antagonist binding sites on the AChR, either the N or the Q subunit, but not the L chain, was inferred to reside between both K subunits [63]. This latter possibility was corroborated by means of several lines of experimental arguments [64^69]. In this regard, the actual consensus is that the Q subunit resides between both K subunits (reviewed in [2,70,71]). However, the KQK subunit order allows two possible subunit arrangements: KQKNL or its minor image KQKLN. Recent evidence by using di¡erent photoactivatable analogues of the K-neurotoxin II from Naja naja oxiana has indicated that the clockwise subunit arrangement KQKNL is the most

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plausible and its mirror image KQKLN can be discarded [72]. Successful studies using several methodologies such as photoa¤nity labelling and mutagenesis in combination with electrophysiological measurements have allowed us to obtain information on the localization of the agonist binding sites on the muscletype AChR (reviewed in [1,2,13,73]. For example, it has been demonstrated that the neurotransmitter ACh binds with unequal a¤nity to two di¡erent sites. One of these sites, the high-a¤nity locus, is located at the interface between the K and N subunits, and the low-a¤nity binding site is positioned at the KQ interface. For the case of the competitive antagonist d-TC, the position of both high- and low-a¤nity binding sites is opposite to that for ACh. Moreover, the agonist/competitive antagonist binding sites have been shown to be formed by a principal component located in the K subunit and a complementary component found in the N (or in the Q) chain. Regarding homomeric AChRs (e.g., K7, K8, and K9 receptors), both components should be located at adjacent K subunits. The principal component is formed by three loops: the loop containing the residue Tyr93 (sequence number from the Torpedo receptor), the loop containing Trp149 , and another loop formed by amino acids Tyr190 , Cys192 , Cys193 , and Tyr198 . Additionally, the complementary component of the high-a¤nity ACh binding site (or the low-af¢nity d-TC binding site) located in the N subunit is probably formed by three loops: a negatively charged loop containing Asp180 and Glu189 , and two more loops, each one contains the respective residue Thr119 or Trp57 . Likewise, the complementary component of the low-a¤nity ACh binding site (or the high-a¤nity d-TC binding site) located in the Q chain is formed by homologous loops: the negatively charged loop containing Asp174 and Glu183 , in addition to both Tyr117 - and Trp55 -containing loops. Concurrently, the use of biophysical approaches such as electron microscopy and £uorescence spectroscopy has helped to elucidate the transversal localization of both agonist binding sites with respect to the plane of the lipid membrane [8,74]. In particular, î the ACh binding sites are positioned at 30^40 A above the membrane surface. Regarding other members of the ligand-gated ion channel receptor superfamily, it is likely that they too

contain a series of homologous loops (matching AChR loops) forming the neurotransmitter binding area at the N-terminal receptor domain [75,76] (reviewed in [2,11,12]). 3. The nicotinic acetylcholine receptor-associated ion channel Since the ion channel of the AChR is one of the target sites for the pharmacological action of several NCIs and this is one of the issues to be covered in this review, a more detailed description of the structural components of the ion channel is addressed. During the determination of NCI binding site locations, much information about the structure of the ion channel has emerged. For example, determining the photocrosslinking of either radiolabelled NCIs or photoactivatable NCI derivatives with speci¢c photoreactive groups such as azide, mustard, or diazirine on the AChR, an important conclusion emerged: the ion channel wall is principally comprised of the transmembrane M2 domain of each AChR subunit. The ion channel is apparently a cylindrical tube with î that protrudes above the lipid a diameter of 20^25 A î height, including the phospholipid bilayer (V40 A headgroup region) (see Fig. 1B). Although the structural characteristics of the transmembrane portion of the channel have not been resolved in detail, the narrowest portion of the cylinder, the so-called ion channel gate, has been determined by permeation î [77,78]. This studies to have a diameter of 7^8 A diameter is large enough to allow the passage of a partially hydrated Na‡ cation. In addition, the length of this particular region has been estimated î by potential streaming measurements to be 3^6 A long [79], approximately the extension of one K-helical turn. The neuronal-type ion channel has been also suggested to be funnel-shaped by means of structure-blocking activity relationship studies [80,81]. Basically, the ion channel was proposed to î [80], be formed by two rectangles separated by 7.5 A î î or alternatively by another rectangle of 6.1 AU8.3 A [81]. The rectangle located close the extracellular porî U8.3 tion has the dimension of approximately 6.1 A î (or alternatively 7.0 A î U8.4^9.0 A î [81]) and the A other rectangle close to the cytoplasmic domain î U6.3 A î (or alternatively 5.8 measures about 5.5 A

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î U8.0 A î [81]). Regarding other members of the liA gand-gated receptor family, the narrowest part of the GABAA , the glycine, and the 5-HT3 ion channel was î found to measure approximately 6, 5 [82], and 7.6 A [83], respectively. Thus, the dimensions of the narrowest part of ion channels follow the sequence: glycine receptor 6 GABAA receptor 6 5-HT3 receptorWAChR.

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There appear to be two distinct environments within the AChR channel (reviewed in [73,84,85]). Fig. 2 (bottom and right panel) depicts three loci providing two very di¡erent environments. An uncharged region formed by a series of di¡erent rings of amino acids (one from each subunit) is vectorially disposed from the extracellular to the intracellular channel portion in the order: valine ring, leucine

Fig. 2. Transverse schematic representation of the muscle-type AChR showing the domains involved in the speci¢c binding of nonî competitive inhibitors from exogenous origin. Top and right panel: Schematic representation of a section of the AChR at V46 A from the membrane surface viewed from the synaptic cleft. The ethidium locus can be putatively located in the AChR vestibule between both acetylcholine (ACh) binding sites. The ethidium site includes either a region near the KQ interface, being this K subunit the one that bears the high-a¤nity ACh binding site, or a portion of the L subunit. Bottom and left panel: Schematic representation of a section of the AChR at the lipid^aqueous interface viewed from the synaptic cleft. The amino acid chain from each subunit (K, L, Q, and N) crosses the lipid membrane four times (M1-M4). The perimeter of the AChR is surrounded by V45 lipid molecules, i.e., the annular lipid domain. The 23 empty circles around the AChR represent the phospholipid headgroups on the extracellular lea£et of the lipid bilayer. The small black circles between subunits and between domains M1/M4 and M3/M4 represent the possible locations for non-annular lipid domains. The high-a¤nity quinacrine binding site is located at the KM1 transmembrane domain, near the L subunit, based on previous experimental evidence [70,224,228]. Bottom and right panel: Schematic representation of two M2 transmembrane domains of the AChR subunit. The amino acid side chains from both KM2 domains represent the transmembrane K-helix. A consensus exists that the interaction of certain residues from the K subunit (shown here as ¢lled spheres), in addition to the respective homologous amino acids from the other subunits (not shown for simplicity), form a series of strati¢ed rings disposed from the extracellular to the intracellular side in the following manner: outer or extracellular, valine, leucine, serine, threonine, intermediate, and cytoplasmic or inner ring. In addition, some of them are involved in the binding of the so-called luminal non-competitive inhibitors. In particular, the binding site for the local anesthetic meproadifen is located close to the mouth of the ion channel, probably at the negatively-charged outer or extracellular ring. The binding site for cembranoids and tri£uoromethyl-iodophenyldiazirine (TID) is related to both the valine and the leucine ring. Finally, the binding site for chlorpromazine (CPZ), triphenylmethylphosphonium (TPMP‡ ), the local anesthetic QX-222, and phencyclidine (PCP) includes the leucine, the serine, and the threonine ring.

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ring, serine ring, and threonine ring. In turn, this uncharged portion is framed by two negatively charged regions: an anionic ring located at the extracellular portion of the channel (the outer ring) and two more anionic rings located near the cytoplasmic portion of the channel (the inner ring and the intermediate ring).

minimum of four interconvertible states (reviewed in [1,49,86]). The tetrahedral diagram indicating the existence of at least four receptor states is shown in Fig. 3. In the absence of agonists, most Torpedo receptors (V80%) are in the resting state (R) [87,88]. In addition, V20% of receptors are in the desensi-

4. Functional properties of nicotinic acetylcholine receptors Nicotinic acetylcholine receptors, as well as other members of the ligand-gated ion channel superfamily, present a very simple repertoire of functional properties. The AChR recognizes the neurotransmitter ACh, and upon binding, the intrinsically coupled ion channel is opened, augmenting in turn the possibility of cations to cross the lipid membrane. Thus, after channel opening, a new ionic concentration is found at the aqueous solutions bathing the opposed faces of the lipid bilayer of the cell. In particular, the extracellular liquid presents now a higher concentration of K‡ (e¥ux) and the cytoplasmic compartment has a higher content of Na‡ (in£ux). The concentration change to reach equilibrium produces membrane depolarization. Depolarization of the membrane provokes speci¢c physiological responses by each involved cell. For instance, if the muscle membrane depolarization is large enough, an action potential is elicited. This action potential propagates from the neuromuscular junction all over the muscle ¢ber. During the propagating action potential the release of Ca2‡ ions from intracellular stores in the muscle cell is stimulated. The ¢nal response in the muscle is the contraction of its myo¢brils. On the other hand, in neurons, the excitatory signal provided by the activation of the cation channel is summed with other signals (including inhibitory ones provided by the activation of anionic channels from either GABAA or glycine receptors) and subsequently re-directed to another neuron or to an endocrine gland cell as a nerve action potential. All these biologically relevant AChR properties are triggered by the binding of the neurotransmitter ACh. Upon binding, the receptor protein undergoes a conformational change. Several lines of experimental evidence suggest that the AChR may exist in a

Fig. 3. Diagram showing the dynamic of the multiple conformational states of the nicotinic acetylcholine receptor (AChR) (modi¢ed from [223]). In the absence of the neurotransmitter acetylcholine (ACh, shown here as empty circles) the AChR is in the resting (R) state, a conformational state where the ion channel is closed. The closed ion channel can be opened upon binding of two ACh molecules to the AChR. This active (A) state presents low a¤nity for agonists (Kd s from 10 WM to 1 mM) (shown here as a loosed interaction between ACh and the AChR). The transition from the R to the A state is a fast process which proceeds in the microsecond to millisecond time regime. In the prolonged presence of agonists, the AChR becomes refractive to the agonist pharmacological action and thus, to the activation of its ion channel. In the Torpedo AChR there exists two refractive closed ion channel states, the intermediate (I) and the desensitized (D) state. Both states show high a¤nity for agonists and some antagonists (Kd s from 10 nM to 1 WM) (shown here as a tight interaction between ACh and AChR). The transition from the A to the I state is a slow process which is produced in the 1^100 ms time range. Additionally, the transition from the A to the D sate has a much slower time course (in the second to minute timeframe).

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tized state (D). The resting state is de¢ned by the existence of an activatable closed ion channel. In the presence of agonists, the receptor is activated (A) in the microsecond to millisecond time frame. This state presents an open ion channel and a low a¤nity for agonists [apparent dissociation constant (Kd ) between 10 WM and 1 mM]. Concomitantly, in the prolonged presence of agonists the activated receptor is commuted to an intermediate state (I) in the 1^100 ms timescale and further to a D state in the second to minutes timeframe. No energy source nor ionic gradient is needed to induce the conformational shift from the R to the D state. Both I and D states are refractory to the pharmacological action of agonists and the ion channel remains closed. However, mutating the Leu247 residue to Thr located in the M2 transmembrane segment of the K7 neuronal AChR, a desensitized but activatable ion channel was found [89]. In addition, both I and D states have a high a¤nity for agonists and some antagonists (apparent Kd ranging from about 10 nM to 10 WM). The recovery process from the D to the R state is slower than the forward rate. A recent picture of the change in conformation from the closed to the open-channel AChR by means of ACh binding has been determined by electron î resolution [57]. More premicroscopy images at 9 A cisely, the hollow forming the ACh binding pocket on the K subunit located next to the N chain (the high-a¤nity locus) disappeared by the e¡ect of ACh released onto tubular vesicles containing AChRs. More recently, the projection structure of î resolution indithe non-activated AChR at 7.5 A cated that the two K subunits, more precisely the K-helices encircling the putative agonist cavities, are unequally inclined [58]. Although both muscle- and neuronal-type AChRs present the same basic functional attributes, two properties have been assigned as representative for neuronal AChRs. Neuronal-type AChRs have higher Ca2‡ permeability (P) [90]. For example, the PCa /PNa ratio ranges from V1.1 (for the K3L4 subtype) to s 10 (for the K7 subtype) (reviewed in [5]). Additionally, the positive modulation of the opening probability of the neuronal-type ion channel is mediated by external Ca2‡ [91,92]. Interestingly, di¡erent categories of regulatory Ca2‡ binding sites are located on the K7 subtype [93]. One Ca2‡ binding

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site is found in the residue sequence 161^172 from the K7 subunit, and two other at the level of the Glu18 and Glu44 . 5. Exogenous and endogenous non-competitive inhibitors Non-competitive inhibitors of the AChR comprise a wide range of structurally di¡erent compounds with speci¢c physical and chemical properties such as £exibility, distance between relevant groups (e.g., between ammonium and carbonyl in local anesthetics), charge density, size, hydrophobicity, and shape. There is not a unique structural feature of drugs with NCI properties (reviewed in [73]). The molecular structures of some classical NCIs are shown in Fig. 4. Among them are compounds such as the neuroleptic chlorpromazine (CPZ), the lipophilic cation triphenylmethylphosphonium (TPMP‡ ), the hallucinogen phencyclidine (PCP), the antimalarial drug quinacrine, the DNA probe ethidium, the toxin histrionicotoxin (HTX) obtained from the skin of the Colombian frog Dendrobates histrionicus, the permanently positively charged derivative of the local anesthetic lidocaine NP-(trimethylaminomethyl)-2P,6P-xylidide (QX-222), the hydrophobic probe tri£uoromethyl-iodophenyldiazirine (TID), general anesthetics, and barbiturates. The speci¢city of these NCIs for the AChR has been also established. For example, classical NCIs of the AChR such as QX222 and CPZ [94], and procaine [95] blocked the activity of 5-HT3 receptors from di¡erent sources. However, the evidence indicates that the mechanism of inhibition involves binding to the competitive antagonist 5-HT3 receptor site. Moreover, contrary to the inhibitory action of both volatile general anesthetics and n-alcohols on the AChR, it was shown that, depending on the concentration used, general anesthetics may potentiate or inhibit the 5-HT3 receptor [96]. More precisely, the general anesthetics iso£urane, halothane, en£urane, and methoxy£urane, as well as low concentrations of lower n-alcohols such as butanol and hexanol, potentiated the pharmacological response elicited by 5-HT. On the other hand, high concentrations of the same lower n-alcohols and a broad range of concentrations of higher nalcohols (higher than octanol), as well as the barbi-

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turate thiopentone, inhibited the 5-HT3 receptor in a non-competitive fashion. Concurrently, cyanotriphenylborate, the negatively charged structural analogue of TPMP‡ , has been shown to be speci¢c for the K1containing glycine receptor but not for the AChR ion channel [97]. Finally, classical NCIs such as quinacrine [98] and NP-(triethylaminomethyl)-2P,6P-xylidide (QX-314) [99] have also been shown to potently block Ca2‡ channels, from outside or inside of the lipid membrane, respectively. The `non-classical' NCIs include compounds such as L-eudesmol [100], antibiotics [101], cembranoids [102], the intracellular Ca2‡ antagonist 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester [103], insecticides [104], acetylcholinesterase inhibitors [105], the sympathomimetic agent ephedrine [106], ( þ )-pseudophrynaminol [107], and drugs used in AChR phosphorylation studies [108]. However, this inventory of very di¡erent substances with activity on the AChR increases daily. This extended register of compounds from exogenous origin complements a variety of endogenous molecules that posses the pharmacological property of inhibiting the AChR ion £ux. The molecular structure of several endogenous NCIs is displayed in Fig. 4. These include lipids such as free fatty acids [109] and steroids [110], the neuropeptide substance P [111], as well as the neurotransmitter 5-HT and related compounds [112]. 5.1. Pharmacological properties of non-competitive inhibitors Pharmacologically, NCIs exert their blocking action on the ion channel without changing maximal agonist binding, while reducing the mean duration of channel open time (don ). However, the mechanism of channel inhibition is still a matter of controversy. Experimental evidence supports the idea of channel blocking by a steric mechanism in which the drug enters into the channel lumen and plugs it. For NCIs to gain access to the lumen, the channel must

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be in the open state. In this scenario, agonists could modulate the accessibility of NCI molecules to the channel lumen. There also exists evidence for an allosteric mechanism of non-competitive drug action. In this kind of mechanism, it is proposed that the ligand binds to speci¢c sites other than in the channel lumen, i.e. non-luminal binding sites (see Section 8). The interaction between the receptor and the ligand induces a conformational change on the AChR. In the proposed conformational state, the channel is closed, inhibiting the ion £ux previously elicited by the action of cholinergic agonists. The e¡ect of NCIs on AChR conformational transitions and desensitization rate has been studied using a variety of indirect methods [52,113,114]. There are two interesting properties of NCI molecules that should be appreciated. (1) In general, NCIs bind preferably to the desensitized AChR (reviewed in [84]). However, it has also been shown that the hydrophobic probe TID, which is considered a higha¤nity NCI, binds the AChR mainly in the resting state [115]. (2) The mutually exclusive action between two di¡erent NCIs: the binding of one molecule prohibits the interaction of a second ligand from a different class with the AChR (e.g., the binding of the local anesthetic procaine to the AChR impedes the interaction of PCP, and vice versa). However, preliminary evidence indicate the possibility that two di¡erent NCIs bind to their respective high-a¤nity binding sites on the AChR at the same time [116]. 5.2. Low- and high-a¤nity non-competitive inhibitor binding sites Non-competitive inhibitor binding sites are located on regions of the AChR that are topologically di¡erent from agonist and competitive antagonist binding sites. Based on the number of binding sites, on the sensitivity to the pharmacological action of the higha¤nity NCI HTX, and on the Kd ranges, two main binding site classes for NCIs have been proposed

6

Fig. 4. Molecular structure of some non-competitive inhibitors of the nicotinic acetylcholine receptor. (A) Non-competitive inhibitors from exogenous origin: PCP, phencyclidine; TPMP‡ , triphenylmethylphosphonium ; HTX, histrionicotoxin; CPZ, chlorpromazine; QX-222, NP-(trimethylaminomethyl)-2P,6P-xylidide; TID, tri£uoromethyl-iodophenyldiazirine. (B) Non-competitive inhibitors from endogenous origin: 20:4[n-6], arachidonic acid; 5-SASL, 5-doxyl stearic acid; 5-HT, 5-hydroxytryptamine.

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[113] (reviewed in [73,84]). (a) Low-a¤nity NCIs (Kd s up to hundred micromolar) comprise a population of 10^30 binding sites which are not sensitive to HTX, located probably at the lipid^protein interface. (b) High-a¤nity NCIs (Kd s from submicromolar to several micromolar concentration range) representing several binding sites (one per each speci¢c NCI) displaceable by HTX. Experimental evidence for both high- and low-af¢nity binding site classes was obtained by using a wide range of di¡erent techniques. In particular, the evidence that high-a¤nity NCIs from exogenous origin present a stoichiometry of one binding site per functional AChR was determined by using equilibrium binding of radioactive NCIs or by titration of £uorescent NCIs. Averaging almost all experimental values obtained to date a stoichiometric ratio of V1 NCI binding site/AChR was calculated (reviewed in [84]). Low-a¤nity NCI binding sites were studied, among other methods, by using electron paramagnetic resonance (EPR) spectroscopy (for methodological details see Section 8). This spectroscopic technique has revealed a highly anisotropic component in the spectra of local anesthetics and PCP spin-labels which corresponds to a restriction of the molecular motion of the spin-labelled analogue by the AChR protein (reviewed in [73,84]). The quanti¢cation of the fraction of spin-labelled molecules undergoing restricted motion at the lipid^protein interface allows the calculation of the association constant of spinlabelled local anesthetics (LASL) relative to the nonselective phospholipid spin-labelled phosphatidylcholine (PCSL) [39], (KLASL /KPCSL ) [see Eq. 2]. In this regard, it was possible to discriminate among local anesthetics interacting with high (KLASL /KPCSL s 2), intermediate (KLASL /KPCSL =1.6^1.9), and low (KLASL /KPCSL 91.3) speci¢city with the AChR [117] (reviewed in [73,84]). The ¢rst group includes local anesthetic analogues such as procaine thioester and benzocaine spin labels. The second group includes spin-labelled procainamide and the third, spin-labelled procaine (for molecular structures see [117]). Another spectroscopic method to study the interaction of local anesthetics with the transmembrane domain of the AChR has been successfully used. The use of £uorescence-quenching has allows measurement of the e¤ciency of di¡erent spin-labelled local

anesthetics for quenching the intrinsic £uorescence of the AChR protein [117]. The protein intrinsic £uorescence originates from Trp and Tyr residues. Interestingly, only one Trp residue is found in the transmembrane domain of the AChR, speci¢cally Trp453 in the M4 domain of the Q subunit. However, due to the hydrophobic environment of Trp453 the quantum yield of this residue is higher than that for the rest of the Trp groups [118]. Thus, Trp453 produces V30% of the observed AChR intrinsic £uorescence intensity. The transversal distance of this Trp from the inner lipid membrane surface (from the phospholipid headgroup region) was measured by the parallax î [118]. Thus, hydromethod and found to be V10 A phobic quenchers such as spin-labelled local anesthetics (and spin-labelled and brominated lipids; see Section 9 for more details) may interact with this residue from the lipid phase. In fact, both spin-labelled benzocaine and thioprocaine were found to e¤ciently quench the AChR intrinsic £uorescence. Since £uorescence-quenching is a short range process [119,120], a higher quenching e¤ciency indicates more accessibility of the quencher to the area where the £uorophore is attached. For this purpose, the quenching parameters were graphically calculated by plotting [Io /(Io 3I)] vs. 1/[Q] [117] according to the modi¢ed Stern-Volmer equation [121]: I o =…I o 3I† ˆ 1=…f a K Q ‰QŠ† ‡ 1=f a

…1†

where Io and I are the intensities of the intrinsic AChR £uorescence in the absence or in the presence of di¡erent concentrations of the quencher ([Q]), respectively. KQ is the apparent Stern-Volmer quenching constant and fa is the apparent fraction of available £uorophores. The calculated quenching parameters are summarized in Table 1. By comparing the quenching parameters it is possible to deduce that the benzocaine derivative is closer to Trp453 than procaine thioester spin-label. This is in accord with the putative position of the benzocaine binding site(s) at the triple aqueous^lipid^protein interface deduced spectroscopically by means of competition experiments with the hydrophobic £uorescent probe merocyanine 540 [122]. Nevertheless, another explanation may be inferred: considering that Trp453 is located in the non-annular lipid domain [38,123], then, the benzocaine binding site(s) would be closer to this particular lipid domain. Further studies to

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Table 1 Quenching e¤ciency of spin-labelled local anesthetics on the AChR intrinsic £uorescence Spin-labelled local anesthetic

AChR conformational statea

fa b

Procaine thioester

Resting Initially activated Desensitized Resting Initially activated Desensitized

0.40 0.51 0.44 0.67 0.99 0.65

Benzocaine

KQ c (mM31 ) 180 50 110 33 18 45

1/KQ d (WM) 6 20 9 30 56 22

a

Increasing concentrations of spin-labelled local anesthetics were incubated with AChR-rich membranes. Considering that each di¡erent AChR suspension was in the absence of CCh, or alternatively was co-incubated or pre-incubated with 100 WM CCh, the AChR should be in the resting, initially activated, or desensitized state, respectively. b The apparent fraction of available £uorophores in the protein was determined from the y-intercept of the modi¢ed Stern-Volmer plot, fa = 1/y-intercept, according to Eq. 1 [117]. c The apparent Stern-Volmer constant was calculated from both the y-intercept and slope of the modi¢ed Stern-Volmer plot, KQ = y-intercept/slope, according to Eq. 1 [117]. d Relative concentration of spin-labelled local anesthetic molecules at which 50% of the £uorescence intensity is quenched assuming that AChR-£uorophores are totally accessible to local anesthetic quencher (fa = 1).

demonstrate the interaction of benzocaine molecules with the quinacrine locus, which is considered to be located at a non-annular lipid domain (see Section 8), might help to elucidate this question. The elicited agonist e¡ect on AChR-local anesthetic interactions is also shown in Table 1. For example, the di¡erence in the concentration at which 50% of the initial intensity is quenched considering that all £uorophores are fully available to quencher (1/KQ ) indicates that a higher concentration of both spin-labelled local anesthetics is necessary to quench a greater fraction of AChR-attached Trp residues when the receptor is initially activated by 100 WM carbamylcholine (CCh). This may be explained by proposing that the receptor protein changes its conformational state upon agonist activation and subsequently, this structural change is sensed by the local anesthetic interacting with the lipid^protein interface. 5.3. Voltage-sensitive and voltage-insensitive non-competitive inhibitors On the basis of electrophysiological studies, NCIs can be discriminated based on voltage dependence or voltage independence (reviewed in [73,124]). The sensitivity of NCI molecules to membrane potential changes has been considered as evidence for the existence of an open channel-blocking mechanism (see also Section 10). Among voltage-sensitive NCIs are drugs such as local anesthetics, the antiviral drug

amantadine, HTX, quinacrine, ephedrine, hexamethonium, decamethonium, and ethidium. However, PCP, CPZ, benzocaine, cembranoids, and cyclothiazide are all examples of voltage-insensitive drugs. There also exists contrasting evidence on PCP [125] and CPZ [126], suggesting that their action may be voltage-sensitive. Regarding endogenous NCI molecules, the inhibitory e¡ect elicited by the native neurotransmitter 5-HT and derivatives is considered relatively voltage-sensitive [112,127] (see Section 7 for more details). Progesterone is evidently a voltage-insensitive molecule [110], while hydrocortisone was apparently slightly voltage-sensitive [128] (see Section 9 for more details). 6. Luminal high-a¤nity non-competitive inhibitor binding sites The evidence for a unitary stoichiometry and the mutually exclusive competition behavior of NCIs described in the above section suggested the possibility of a unique binding site, perhaps located in the channel lumen which, when occupied, could sterically block the ion £ux activity. However, detailed analysis of the results is more consistent with the notion of several high-a¤nity binding sites at di¡erent rings disposed along the lumen of the ion channel. By photocrosslinking studies it was possible to demonstrate that one NCI molecule is in contact

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with the ¢ve subunits of the AChR. The speci¢city of the photoa¤nity labelling experiments was established by these criteria: (1) displacement of the photolabel (prior to activation) by other known higha¤nity NCIs such as PCP or HTX; (2) enhancement of a¤nity and labelling of the NCI under study in the presence of agonists; and (3) inhibition of the e¡ect elicited by agonists [see (2)] in the presence of competitive antagonists. Analysis by peptide mapping of the AChR subunits photolabelled with radioactive NCI analogues indicated that the homologous residues of Ser248 from the transmembranous domain M2 of each subunit are the preferred targets of several NCIs (reviewed in [1,2,73,84,85]). The position of Ser residues labelled with [3 H]CPZ are at K248 , N262 , L254 , and Q257 . In addition, CPZ also labelled the Leu residues at position L257 and Q260 , and Thr at Q253 [129^132]. Interestingly, taking into account these photolabelled residues and using computational modelling, it was possible to ¢t the plane formed by the triple ring structure of the CPZ molecule parallel to the axis of the channel within the crevice formed by two adjacent AChR subunits [133]. The CPZ photolabelling results were basically the same as the observed by using the lipophilic cation TPMP‡ . In this case, the Ser residues were labelled at positions K248 , L254 , and N262 [134,135]. The uncharged domain of the ion channel (see Section 2) is the principal labelling site for exogenous substances such as CPZ, TPMP‡ , and TID (Fig. 2, bottom and right panel), as well as for the endogenous polypeptide substance P (see Section 7). In addition, this domain has been proposed as the target for the pharmacological action of the local anesthetic QX-222, cembranoids, and PCP (Fig. 2, bottom and right panel), as well as for the native neurotransmitter 5-HT and related compounds (see Section 7). This speci¢c ion channel domain is framed by two negatively charged loci (see Fig. 2, bottom and right panel). Evidence supporting these ideas will be presented below, strati¢ed by the various rings comprising the uncharged and charged portions of the channel lumen. 6.1. The serine ring The NCI labelling observations expounded in the previous section have been complemented using site-

directed mutagenesis in combination with patchclamp technique. The localization of the QX-222 binding site was proposed on the basis of several studies using this approach [55,136,137] (reviewed in [73,84,124,138,139]). For instance, it was demonstrated that the pharmacological activity of the openchannel blocker QX-222 is a¡ected when a Ser-rich domain is mutated. The drug was AChR-bound for shorter periods and its Kd was increased when the polar amino acid KSer248 was mutated to the nonpolar amino acid Ala. In comparison, the NSer to Ala mutation also a¡ected these properties but with a two-fold lower extent than detected in the K subunit. This e¡ect can be interpreted in the light of the difference in the number of residues mutated (there are two K subunits per each N). A double mutation (actually a triple mutation, one on each K subunit and another one on the N subunit) produced an additive e¡ect on the Kd of QX-222 (in non-mutated AChRs the Kd was 27 WM, whereas the triple mutated AChR presented a Kd of 134 WM [138]). However, the bound lifetime of the local anesthetic did not change appreciably with respect to the N mutation. In contrast with the experimental evidence on serine ring mutations, when KSer252 , which is positioned near the leucine ring, was mutated to Ala, the a¤nity of QX-222 and the lifetime of the AChR^QX-222 complex were enhanced [136]. The increased a¤nity was principally due to a decrease in the dissociation rate constant (koff ) value and was not a¡ected by membrane voltage changes. Mutations of LPhe259 to Ser and of KSer252 to Ala, in KLN-containing receptors, showed similar relative e¡ects on QX-222 blockade as compared to KLQN-containing receptors [140]. Assuming that the M2 transmembrane domain of the K subunit is an K-helix (so Ser252 would be positioned one turn away from Ser248 ) and, that the arî apart omatic moiety of QX-222 molecule is 5.7 A from the quaternary amine, it has been suggested that QX-222 is located into the lumen channel (reviewed in [73,84,124,138,139]) (Fig. 2, bottom and right panel). More speci¢cally, the ammonium group of the local anesthetic is positioned close to the serine î away, ring and its hydrophobic portion is some 5.7 A near the leucine ring. Interestingly, taking into account the susceptible residues a¡ecting binding a¤nity and using molecular modelling, the QX-222 mol-

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ecule was ¢tted into the channel lumen of the AChR [133]. Nonetheless, it is necessary to take into account that, in addition to the existence of K-helix conformation for the domain M2 [141], other observations suggested that a portion of the KM2 domain from the mouse muscle AChR (residues Ser248 to Thr254 ) is formed by a L-strand [34,142]. The same amino acids, KSer252 and homologous residues from the other subunits, seem to be involved in the binding site for the general anesthetic hexanol [143]. More speci¢cally, the mutational e¡ect elicited by substitution of each of the residues was additive indicating that the hydrophobic molecule makes contact with all ¢ve M2 residues. However, mutation on either KSer252 or its homologous residue LThr to Ile, produced a larger increase in apparent hexanol binding (4.3- and 2.2-fold, respectively) than the mutation on each Ala from the respective Q and N subunit (1.2fold each), suggesting that the extent of contact is not identical for each side chain. Starting from the observation that [3 H]PCP binds with 60 times higher a¤nity to muscle-type AChRs from electric organ than from mouse, Eterovic's laboratory [102] has hypothesized that the binding site for PCP is located at a luminal locus. The di¡erence in PCP a¤nity is based on three amino acids found in the mouse AChR (Phe254 and Thr258 in the L subunit, and Asn257 in the Q subunit) which are changed in the Torpedo AChR (Ser254 and Ala258 in the L subunit, and Ser257 in the Q subunit). They studied six hybrid formed by di¡erent combinations of Torpedo and mouse AChR subunits and con¢rmed that PCP binds with higher a¤nity to the receptor containing L and Q subunits from Torpedo than to that from mouse. Interestingly, a mutant mouse Q subunit where Asn257 of the M2 domain was replaced by Ser showed a PCP a¤nity equivalent to the Torpedo Q subunit. Considering the molecular structure of PCP, its binding site was putatively located at residues homologues to QAsn257 (the serine ring) and QAla261 (one residue away from the leucine ring). The importance of the serine ring in regards to NCI binding is also based on the fact that mutations distant from this locus (e.g., Cys418 to Trp at KM4) have shown no alteration in PCP and tetracaine noncompetitive inhibition properties [144]. The functional importance of this uncharged ring with respect to channel activity has also been ad-

189

dressed by means of site-directed mutagenesis and patch clamp. For instance, it was evidenced that when KSer248 and NSer262 are mutated to Ala in both subunits and to Phe in the L chain, there is a selective decrease in outward single-channel currents and an increased recti¢cation. This suggests that the hydroxyl groups of the serine ring present an energy barrier to ion permeation [137]. The amino acid speci¢city is correlated with several experiments where it was demonstrated that mutations KSer252 and LThr258 to Ala, which are not located at the serine ring, produced no change in channel conductance [140,145] (reviewed in [71,138]). From the clinical point of view, it is interesting to note that mutation of Ser248 to the Phe on the K4 subunit of the neuronal-type K4L2 receptor has been implicated in the human idiopathic epileptic disease: the autosomal dominant nocturnal frontal lobe epilepsy [146]. In comparison with the wild type K4L2 receptor, the Ser248 to Phe mutation provoked an enhancement of the desensitization rate and a slower recovering from the desensitized state. 6.2. The leucine ring This ring of highly conserved leucines has been proposed to play two possible roles. One model suggests that the leucines from each M2 sequence point into the channel lumen occluding the conduction pathway, but in the open state this kink rotates in such a way that the channel is no longer closed [57]. The fact that the replacement of Leu by Ser in the K7 receptor still gates is in disagreement with this model [89]. The second model suggests that the leucines move into the channel during the desensitization process to form the non-conducting con¢guration state. This model has received support from mutational studies. Mutation of Leu247 to Thr in the K7 neuronal-type AChR, produced a desensitized AChR with a new conducting state that can be activated at lower ACh concentrations than the wild type AChR, and in addition, by competitive antagonists such as d-TC, hexamethonium, and dihydro-L-erythroidine [89,147]. Thus, the existence of a desensitized AChR where the channel is in the open state has been suggested. Further work with this mutant also suggests that the non-competitive antagonist 5-HT activates the AChR through binding to the non-com-

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petitive agonist site [148] (i.e., the physostigmine binding site; for a review on this site see [73]). More recently, oocyte expression of mouse AChR subunits containing one to ¢ve (one per each subunit) Leu to Ser mutations indicated that mutations are independent, equivalent, and multiplicative [149]. The same results have been found when Leu was exchanged for Thr [150]. The evidence suggests that the leucine ring plays an important role in the gating process by setting the mean open time of the channel by means of interactions with other domains of the AChR. Further information about the role of the leucine ring is provided by the use of the nonsense suppression method to make mutants at this position [151]. Incorporating a total of four natural and six unnatural residues at the leucine ring of the muscletype AChR, structural features such as side chain length, branching, and substitution of oxygen for methylene groups were studied. The main conclusion is that by increasing polarity at the level of the leucine ring, the ACh sensitivity is augmented. In addition, the data suggest an especially strong interaction between L and N subunits in the ion channel region. Concerning the localization of NCI binding sites, the pharmacological e¡ect of QX-222 was abolished in chick brain K7 receptors by mutation of the leucine ring, in particular Leu247 to polar residues such as Thr or Ser [89]. However, it is necessary to take into account that these experiments were performed in the presence of Ca2‡ . Thus, part of the detected ACh-evoked conductances on K7-expressing oocytes might be carried by Cl3 currents through Ca2‡ -activated Cl3 channels, which are known to be present in oocytes [152]. Taking into account the proposed location for the PCP binding site (see Section 6), new experimental evidence regarding the location of cembranoid binding site has emerged [102]. Cembranoids are cyclic diterpenoids isolated from Gorgonian corals which inhibit both muscle- and neuronal-type AChRs in the subnanomolar to submicromolar concentration range. The ion channel inhibition elicited by cembranoids was studied by voltage clamp on AChRs from Torpedo or mouse expressed in Xenopus oocytes. These studies indicated that inhibition by cembranoids is reversible, non-competitive, incomplete, and membrane voltage-independent (from 380 to +20 mV). Cembranoids increase the desensitization

rate of oocyte-expressed K4L2, K3L2, and K2L2 neuronal subtypes by an increase in the rate of fast desensitization at the expense of slow desensitization. Additionally, cembranoids completely displace PCP from its high-a¤nity binding site with apparent inhibition constants (Ki s) in the order of submicromolar to several micromolar concentration range. This suggests that the binding site for cembranoids partially overlaps with the PCP locus [102]. By using several cembranoid analogues it was found that the minimum moiety of cembranoids that e¤ciently displaces PCP is the cembrane ring and a single hydroxyl group located on carbon 14. This suggests that these molecules interact primarily with a hydrophobic domain on the AChR, perhaps with residues located in both the leucine and the valine ring such as Ala, Ile, and Val (see Fig. 2, bottom and right panel). 6.3. The threonine ring The physiological importance of this ring has been addressed by mutational studies. Mutations of the threonine ring in rat or Torpedo AChRs do a¡ect channel conductance [145,153^155]. In particular, when KThr244 is replaced by Val, the selectivity sequence Rb‡ s Cs‡ s K‡ s Na‡ is changed to Rb‡ s K‡ s Cs‡ s Na‡ , and when it is replaced by Gly the permeability ratios are greater for larger cations, indicating that ions are selected according to their dehydrated size [145,155]. This was not the case when the proper serine ring (speci¢cally Ser248 ) and other neighboring residues (Ser246 and Ser252 ) were mutated [136,137,145]. The threonine ring, in addition to the serine ring, has been implicated as part of the binding site for pentamethylenebisammonium derivatives by means of a combination of electrophysiological and molecular mechanical methods [156]. Concerning neuronal AChRs, the mutation Thr244 to Asp on the K7 homomeric receptor exhibited larger currents and higher permeability to Ba2‡ than wild-type receptors [157]. Additionally, Mg2‡ was found to be permeable only in the mutant. 6.4. The valine ring This ring, in addition to the leucine ring, is labelled

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by the hydrophobic probe TID. The labelling elicited by TID was slightly di¡erent from the labelling by CPZ or TPMP‡ [141]. In the absence of agonist, TID labelled Leu257 and Val261 on the L subunit, and Leu265 and Val269 on the N subunit. In the presence of agonist, this labelling was reduced 90%, but other amino acids in the L subunit such as Ser250 , Ser254 , Leu257 , and Val261 were now labelled, concomitantly with Ser258 and Ser262 in the N subunit, and other residues in the NM1 domain. This change in labelling pattern has been proposed to result from a rearrangement of the M2 helices elicited by agonists. The speci¢c labelling of TID was competitively inhibited by tetracaine and dibucaine in the presence of K-bungarotoxin (K-BTx) [158]. Instead, procaine, CPZ, PCP, and lidocaine blocked TID incorporation at lower concentrations in the absence of K-BTx, suggesting that the NCI-induced inhibition of TID binding is elicited by a NCI-dependent desensitization process. Interestingly, tetracaine photolabelled NAla268 (J.B. Cohen, personal communication in [159]). This location is almost coincident (one amino acid apart) with the TID-labelled residue on the N subunit. Mutations on the adjacent Leu254 and Leu255 of the K7 subtype AChR, which form part of the outer leucine ring (see Fig. 2 from review [85]), increased the apparent ACh a¤nity, reduced the extent of desensitization, and increased the Hill coe¤cient of ACh dose-response relationships [160]. These mutations might a¡ect agonist-gating mechanisms (reviewed in [85]). Regarding the muscle-type K1 subunit, the amino acids involved in this ring are Leu254 and Val255 and they are located between the valine and the outer ring (see Fig. 2, bottom and right panel). From the clinical point of view, it is interesting to note that mutation of Thr264 to Pro on the human O subunit has been implicated as responsible of one of the congenital myasthenic syndromes [161]. This mutation is located close to the valine ring and causes markedly prolonged openings of the ion channel. This mutation does not change the inhibitory properties of the local anesthetic QX-222, indicating that Thr264 is not involved in the QX-222 binding site [128]. Speci¢cally, QX-222 binds to normal adult and mutated AChRs with Kd s of 23 and 35 WM, respectively.

191

6.5. The intermediate and the cytoplasmic or inner ring The distance between the intermediate and the î and probably correthreonine ring is about 5 A sponds to the narrowest part of the ion channel (see [79]). Mutations of the intermediate ring (located in the border of the M2 transmembrane sequence, very close to the hydrophilic portion formed between M1 and M2) decreased the conductance, relative to Na‡ or K‡ , of monovalent cations such as NH‡ 4, Cs‡ , and Rb‡ [145,162^164], and of organic cations such as methylamine, ethylamine, diethanolamine, and Tris‡ [78,164]. The selectivity of these ions is altered while maintaining the same channel conductance for the physiological ions Na‡ and K‡ . Removing charges in this ring from the K7 receptor, by exchanging Glu237 for Ala, abolishes channel permeability for divalent cations [55]. In conclusion, the obtained permeability ratios are dependent of the net charge of the ring. The residues forming the segment between M1 and M2 as well as its length seem to be important in cation/anion selectivity [55]. Speci¢cally, a total conversion of a cation channel into an anion channel was elicited by three structural changes in the K7 homooligomer AChR: (a) substitution of Val251 by Thr, (b) exchange of Glu237 for Ala, and (c) addition of a Pro (or Ala) residue at the end (the one connected to the M1 domain) of the M2 segment. Considering this and other evidence, Karlin and Akabas [2] suggested that the relative movement of M1 and M2 and the putative change in its secondary structure are responsible for the modulation of the gate. 6.6. The outer or extracellular ring The outer ring is the labelling site for the potent local anesthetic derivative meproadifen mustard. Meproadifen mustard was initially found in a fragment beginning at KSer173 . More precisely, the meproadifen derivative labelled the position KGlu262 [165,166]. Based on the condensed four transmembrane AChR structural model (see Fig. 1), this ring of negative charges would be located between the synaptic membrane and the extracellular domain of the AChR (between M2 and M3 domains but closer to M2), probably at or near the internal mouth of the chan-

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nel (Fig. 2, bottom and right panel). Interestingly, the lipophilic probe TID labelled amino acids Ile274 and Tyr277 in a non-speci¢c manner [28]. Both residues are included in the hydrophilic region between the M2 and the M3 domain of the K subunit. This ¢nding suggests an exposed hydrophobic nature for the fragment comprising residues Ile274 to Met278 , which is included in the M2-M3 loop. Alternatively, modelling studies have predicted that part of this loop might be in the transmembrane section [36]. The side chains of NCI-photolabelled amino acids such as Ser248 for CPZ and Glu262 for meproadifen î apart from mustard should be approximately 15 A each other. This fact provides support for the extension of the early hypothesis of only one binding site for structurally unrelated high-a¤nity NCIs to acknowledge the existence of several binding sites for di¡erent NCIs, all located in the channel lumen (reviewed in [70,71,73,84,124]). Functional experiments with chimeric AChRs have shown that the segment between M2 and M3 of the N subunit is involved in the conductance di¡erence observed for Torpedo and calf channels [167]. Mutations on this ring, in addition to the inner ring, in£uenced the single-channel K‡ conductance [162]. For instance, mutations that added negative charge raise the conductance and mutations adding positive charge lower it. More particularly, point mutations of the charged residues Glu or Lys, doubled the conductance of the K4L2 neuronal-subtype AChR. The e¡ects of extracellular ring mutations are independent on ionic strength suggesting that the mechanism is not based on simple electrostatic components [168]. Finally, comparisons between native receptors (e.g., KLNO and KLNQ), the KLNQ/O chimera, and the mutated receptor containing the QLys272 to OMet point mutation, indicated that this single site on the O subunit is responsible for the increased channel conductance during maturation of the amphibian neuromuscular junction [169]. It has been suggested that the contribution of the cytoplasmic and extracellular charged rings to charge selectivity is insigni¢cant (reviewed in [2]). This assumption is based on the fact that: (1) the cytoplasmic ring from both cation (e.g., 5-HT3 and nicotinic receptors) and anion (e.g., GABAA and glycine receptors) channels is negatively charged; (2) although the extracellular ring is negative for cationic recep-

tors whereas the net charge for anionic receptors is between 31 and +1, mutations on this ring do not produce a great e¡ect on conductance; (3) di¡erentially charged reagents gain access to the external portion of either cation or anion channels; (4) although the intermediate ring is negative in cationconducting receptors and uncharged in anion-conducting receptors, and mutations on this ring change the conductance properties of the AChR (see Section 6), other evidence [55] indicate that this is not the only domain involved in ion conductance. 7. Luminal localization of binding sites for endogenous non-competitive inhibitors 7.1. The 5-hydroxytryptamine binding site The basic pharmacological action of neurotransmitters has been considered to be mediated by the activation of their own receptors and in general they fail to trigger the opening of channels gated by other endogenous agonists. The cross-reaction elicited by 5-HT on the AChR is a notable exception to this rule. One of the earliest works showed that 5-HT antagonized the action of ACh at the neuromuscular junction of the adult frog [170]. This e¡ect was later con¢rmed in peripheral autonomic ganglia and the mechanism was ascribed to a competitive action of 5-HT for the ACh binding site [171]. Moreover, on the basis of the existence of at least 14 metabotropic 5-HT receptors in both peripheral and central nervous systems (reviewed in [172]), several mechanisms have been considered to explain the reported functional regulation of AChR by 5-HT. One proposed mechanism is that 5-HT might bind to any of its G-protein-coupled receptors activating intracellular second messenger systems, which in turn, would alter the AChR function. Nevertheless, the existence of coupling between the 5-HT-mediated modulation of AChR through a 5-HT receptor-phosphoinositide pathway has been discarded [173]. Alternatively, it has been postulated that 5-HT may modulate the AChR by direct binding. In fact, the action of 5-HT on both the muscle-type [127,173] and the neuronal-type AChR [112,171,173^177] appears to be mediated by a non-competitive inhibitory process. Some of the observed pharmacological properties

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Table 2 Pharmacological properties of 5-hydroxytryptamine and related compounds on both muscle-type and neuronal-type AChRs Serotonergic agent

AChR type

Receptor-containing or -expressing cell

IC50 a (WM) (membrane potential)

nH b

Voltage dependence

Agonist-induced desensitization

Reference

5-HT

K7 K2 LQN K2 LQN KLN KLQ K2L4 K2 LQN

oocyte oocyte oocyte oocyte oocyte oocyte C2 myotube human TE671/RD cell oocyte rat diaphragm muscle ¢ber bovine adrenal chroma¤n cell oocyte oocyte oocyte oocyte oocyte oocyte oocyte

56 (350 mV) 40.3 (350 mV) 1191 þ 143 (0 mV) 123 þ 15 (0 mV) 440 þ 113 (0 mV) 269 þ 27 (360 mV) ^ ^ ^ ^ V100 (370 mV)

1.2 1.7 ^ ^ ^ 0.48 ^ ^ ^ ^ ^

No ^ Yes Yes No Yes Yes Yes Yes ^ ^

^ ^ ^ ^ ^ Accelerated Accelerated Accelerated Accelerated Accelerated Protected

[148]

21 þ 2 (0 mV) 18 þ 1 (0 mV) 14 þ 1 (0 mV) 9 þ 2 (0 mV) 4 þ 1 (0 mV) 3 þ 1 (0 mV) 30 þ 2 (360 mV)

^ ^ ^ ^ ^ ^ 0.85

Weak Weak Weak Weak No No Weak

^ ^ ^ ^ ^ ^ ^

Methysergide

Spiperone

K2 LON K7 or K3, K5, L4 subunits K2 LQN KLN KLQ K2 LQN KLN KLQ K2L4

[127]

[112] [173]

[175] [127]

[112]

a

These values indicate the inhibitory e¡ect of 5-hydroxytryptamine (5-HT) and related compounds on ion £ux. b These values correspond to the Hill coe¤cient.

of 5-HT and related compounds on di¡erent AChR subtypes have been summarized in Table 2. An interesting point is that a broad range of concentrations (from micromolar to millimolar) is necessary for the non-competitive e¡ect of 5-HT and related compounds on AChRs from di¡erent sources. For example, the potency for distinct serotonergic drugs to inhibit the neuronal-subtype K2L4 follows the order: ( þ )-8-hydroxy-2-(di-n-propylamine)tetralin (agonist of the 5-HT1A receptor) s methysergide (antagonist of 5-HT2 and 5-HT1C receptors) s spiperone (antagonist of 5-HT1A and 5-HT2 receptors) s ketanserin (antagonist of 5-HT2 and 5-HT1C receptors) s 5-HT [112]. Interestingly, £uoxetine, most known as prozac, a widely used antidepressant whose pharmacological action is believed to be mediated by blockage of the 5-HT uptake system, inhibits non-competitively both muscle- and neuronal-type AChRs [178]. In addition, the voltage dependence of the observed AChR inhibition by serotonergic agents is a property not clearly de¢ned (see Table 2). It seems that the drug sensitivity to membrane potential is dependent on both the serotonergic molecule tested and the type of AChR, and that the

magnitude of the voltage e¡ect is not correlated to the elicited drug potency. Finally, it is also evident that 5-HT, in addition to £uoxetine, exerts its e¡ect by accelerating the rate of AChR desensitization (see Table 2). On the contrary, 5-HT protected AChR desensitization in bovine adrenal chroma¤n cells [175]. This incongruent fact might be the consequence, the same as for substance P (see next section), of a mechanism secondary to AChR activation. From the physiological point of view, the neurotransmitter-mediated modulation of the AChR is a relevant issue. In order to understand how 5-HT exerts its inhibitory action on the AChR, the localization of its binding site must be accomplished. However, there is not much information on the localization of the 5-HT binding site on the AChR. For example, there is no experimental evidence indicating the existence of one high-a¤nity binding site or several low-a¤nity sites. However, taking into account the electrical distance (d) of di¡erent serotonergic agents to block the muscle-type AChR, it has been suggested that the 5-HT binding site (dW0.72) is putatively located at the serine ring [127]. These

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experiments also suggested that the binding sites for both methysergide (dW0.23) and spiperone (dW0.17) are located more externally than the site for 5-HT, probably close to both the leucine and the valine ring. Additionally, by using di¡erent subunit compositions of the muscle-type AChR it was evidenced that the N subunit is essential for the 5-HT non-competitive inhibition of the AChR and for the voltage dependence of the inhibitory e¡ect. Concurrently, an extracellular domain has been implicated in the binding of 5-HT to both neuronal-subtype K4L2 and K7 receptors [171,176]. This evidence suggest more than one mechanism of action. A putative mechanism mediating neuronal-type AChR modulation by activation of serotoninergic terminals is represented in Fig. 5. The basic tenets of this model are the following: (1) the number of 5-HT molecules at the synaptic cleft is raised after an evoked potential on the presynaptic membrane is elicited and in turn, (2) this concentration might be enough to block the ion £ux evoked by the activation of the AChR-containing postsynaptic membrane by two molecules of ACh originating from an adjacent cholinergic neuron terminal. Additionally, the concentration of 5-HT in the serum of mammalians has been estimated to be in the micromolar concen-

tration range [179]. Thus, 5-HT may be interacting with the muscle-type AChR at the neuromuscular junction as well, which might have implications in neuromuscular in£ammatory processes [173]. 7.2. The substance P binding site In addition to the modulatory e¡ect elicited by 5-HT on AChRs, substance P, another neurotransmitter, has been involved in the endogenous modi¢cation of the AChR properties. Substance P is present in more than 30 neuronal centers as well as in many peripheral tissues (reviewed in [180]). In addition, this undecapeptide has been involved in painprocessing mechanisms. Regarding the AChR, one of the earliest works demonstrating the modulatory effect of substance P was performed at the Mauther ¢ber-giant ¢ber synapse in the hatchet ¢sh [181]. The mechanism of action of this neuropeptide on the AChR was shown not to be mediated by binding to the agonist/competitive antagonist sites. For instance, substance P, at concentrations of V100 WM, had no e¡ect on either the initial rate of [125 I]K-BTx binding [182] or [3 H]ACh binding [183]. On the contrary, it appears that substance P a¡ects the cooperative interactions between both agonist

Fig. 5. Putative model for the endogenous modulation of the nicotinic acetylcholine receptor (AChR) by di¡erent neurotransmitters (taken from [343]). First, acetylcholine (ACh) molecules are released to the synaptic cleft from cholinergic terminals. When the ACh concentration is in the micromolar range, the AChR is activated and thus, cations £ow across the lipid membrane. Instead, when the ACh concentration reaches the millimolar range, the cation £ux is inhibited (blocked state). Two possible agonist inhibitory mechanisms have been suggested: one where the ACh molecules enter into the ion channel and occlude it, and another where ACh interacts with the high-a¤nity quinacrine binding site located at a non-annular lipid domain (see Fig. 2, bottom and left panel) inhibiting the AChR through an allosteric mechanism. Second, both the neurotransmitter 5-hydroxytryptamine (5-HT) and the neuropeptide substance P may co-exist at serotoninergic terminals. After releasing to the synaptic cleft, both neurotransmitters can interact with the AChR ion channel in a non-competitive manner inhibiting ion £ux (blocked state).

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binding sites [182]. For instance, the Hill coe¤cient for ACh binding at equilibrium changed from 1.25 to 1.70 in the presence of 10 WM substance P. In addition, substance P has been considered to act through a second messenger system enhancing AChR desensitization [184]. However, the structural speci¢city of the elicited pharmacological e¡ect is not consistent with the activation of any of the G-protein-coupled neurokinin receptor subtypes [185]. Most studies have concluded that substance P increases ACh-dependent desensitization by direct binding to the AChR [111,182,186^188]. Conversely, substance P protected AChR desensitization in bovine adrenal chroma¤n cells [185,186]. However, this divergence has been ascribed to an e¡ect secondary to AChR

195

activation ([3 H]norepinephrine secretion and 45 Ca2‡ £ux). Moreover, substance P produces a slow blockade of the channel from both muscle-type [189^192] and neuronal-type AChRs [187,188,193^199]. This latter evidence strongly agrees with a non-competitive inhibitory mechanism. Nevertheless, the presence of agonists to cause these pharmacological properties is necessary. In this regard, the obtained concentration values of substance P to inhibit (IC50 ) and to enhance (EC50 ) 50% of the ion channel activity on di¡erent AChR-containing cells are shown in Table 3. From these data it is evident that substance P elicits their e¡ects in the 1^20 WM concentration range. Interestingly, other tachykinins such as neurokinin A and eledoisin, but not neurokinin B, also

Table 3 Substance P-elicited e¡ect on both muscle-type and neuronal-type AChRs AChR source

IC50 a (WM)

EC50 b (WM)

Methodology

K1L1QN-expressing TE671/RD cells K3L4-expressing SH-SY5Y cells K3L4-expressing PC12 cells PC12 cells

V21 V2.3 V2.1 1.8

^ ^ ^ ^

86

Rb‡ e¥ux

22

Na‡ uptake

1.3 (1.0)c

^

V7 1 8 ^

^ ^ ^ 0.7

22

Na‡ uptake Na‡ uptake

V0.5

^

15.5 þ 5.9 8.3 þ 2.6 3.5 þ 1.5 7 (4 mM)d

^ ^ ^ ^

^ ^

5 6 10

V300

V4

V3

^

^

V5

BC3 H-1 cells Avian sympathetic ganglia Bovine adrenal chroma¤n cells (K3, K5, and L4 subunits) Torpedo native membranes

22

Experimental conditions

Reference [194]

pre-incubation with substance P, washing, and uptake determination in the absence of substance P uptake determination in the presence of substance P 0.5 mM CCh as agonist 1 mM CCh as agonist

rate of desensitization [3 H]noradrenaline 50 WM ACh as agonist release 22 Na‡ e¥ux no K-BTx 50% agonist sites blocked by K-BTx 75% agonist sites blocked by K-BTx [3 H]PCP in the presence of 200 WM CCh binding in the absence of agonist rate of desensitization in the presence of 150 mM NaCl [3 H]ACh binding in the presence of agonist [3 H]PCP binding in the absence of agonist

a

These values indicate the inhibitory e¡ect of substance P on both ion £ux and NCI binding. These values indicate the positive e¡ect of substance P on both desensitization rate and NCI binding. c The value in parentheses corresponds to the Hill coe¤cient. d The value between parentheses is a second IC50 . b

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[188]

[201] [193] [184] [196] [191,192]

[182]

[183]

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inhibit both neuronal- and muscle-type AChRs but with lower a¤nities (IC50 W60^160 WM) than substance P [194]. Contrary to the scarce evidence on the localization of the 5-HT binding site on the AChR, there exists more complete information on the localization of the binding site for substance P. Blanton et al. [200] used the p-benzoyl-L-phenylalanine derivative of substance P and were able to speci¢cally label fragment Ser253 to Glu280 contained in the NM2 domain of the AChR. This indicates that the binding site for substance P is within the channel lumen. However, although substance P allosterically modulates [3 H]PCP binding, positively in the absence of agonist and negatively in the presence of agonist (see Table 3), it seems that the neuropeptide does not directly interact with the serine ring (see Section 6) [182,183,201]. In addition, the inhibitory e¡ect of substance P on neuronal-type AChRs is determined by the presence of the L subunit [199]. The pharmacological property of substance P is ubiquitous since this undecapeptide also a¡ects both Ca2‡ and K‡ channels [202]. Furthermore, the speci¢c binding site for this peptide was found to be Met181 at the second extracellular loop of the metabotropic neurokinin-1 receptor by using the same p-benzoyl-L-phenylalanine substance P derivative [203,204]. Interestingly, other peptides have been shown to a¡ect AChR function by direct binding. For example, both the thymic hormone thymopoietin [205] and its pentapeptide derivative thymopentin (ArgLys-Asp-Val-Tyr) [206,207] have been implicated in the enhancement of the rate of ACh-induced AChR desensitization, probably by binding to an allosteric NCI site (reviewed in [49]). Moreover, the neuropeptide calcitonin gene-related peptide has also been shown to accelerate AChR desensitization. However, this e¡ect was ascribed to the activation of secondmessenger-operated metabolic cascades and the phosphorylation of speci¢c AChR subunits [208]. Finally, the neuropeptide Y blocked the AChR ion channel in a non-competitive fashion [209]. Other examples where one neurotransmitter class modulates the action of a di¡erent agonist have been documented. The allosteric potentiation of the Nmethyl-D-aspartate (NMDA) receptor by the action of glycine [210], the non-competitive inhibition of

nicotine-induced catecholamine release from bovine chroma¤n cells by K2 -adrenoceptor agents [211], the potent activation of 5-HT receptors by dopamine [212], or the blocking e¡ect of 5-HT on AChRs (see previous section) are all examples of this e¡ect. The future development of this new research ¢eld might help to understand how the agonist-mediated activation of one receptor category is modulated by a foreign neurotransmitter and in turn, how the crosstalk between di¡erent neuron classes is performed. To date, more than 20 neurotransmitters and approximately 30 neuropeptides have been identi¢ed as responsible for chemical signaling. In general, it is known that a kind of neuron can biosynthesize, store, and release more than one chemical involved in synaptic transmission. For example, both substance P and 5-HT may co-exist in the nerve ending of some neuron classes (i.e., neurons from the ventral medulla oblongata; reviewed in [180]). Interestingly, neuropeptide Y, another endogenous NCI of the AChR [209], is also known to exist in gabaergic neurons from both cortex and hippocampus. Thus, a model where both neurotransmitters co-exist in a serotoninergic terminal is shown in Fig. 5. It is plausible that, upon presynaptic membrane depolarization, both biomolecules are released to the synaptic cleft in a concentration range high enough to produce the non-competitive inhibition of the AChR by occluding the cation channel. 8. Non-luminal high-a¤nity non-competitive inhibitor binding sites In addition to luminal binding sites there exist non-luminal loci for NCIs. The localization of nonluminal NCI binding sites has been addressed for exogenous as well as for endogenous molecules. For instance, the naturally occurring venoms of wasps contain toxins that block the activity of the AChR in a non-competitive manner. In an attempt to localize the toxin binding site, the toxin from the wasp Philanthus, philanthotoxin, was used [213]. The data indicated that the K AChR subunit was preferentially photocrosslinked by employing the radiolabelled philanthotoxin-433 derivative containing a photolabile group on one of its aromatic rings. Proteolysis of the AChR^philanthotoxin complex indi-

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cated that the hydrophobic portion of the toxin interacts with the cytoplasmic loop (the large hydrophilic stretch between M3 and M4) of the K chain. Interestingly, after removing the 43 kDa non-receptor protein all subunits were labelled. Further studies are necessary to understand how this toxin reaches its intracellular binding site and how the molecule is structurally orientated on the AChR. A recent report has suggested that the ethanol binding site is also located at a non-luminal site [214]. For this purpose, the e¡ect of ethanol on the K7 AChR, on the 5-HT3 receptor, and on a chimeric receptor containing the amino terminal domain from the K7 and the transmembrane and the carboxyl domain from the 5-HT3 receptor was studied. These experiments showed that ethanol inhibits the K7 AChR (IC50 = 33 mM) and potentiates the 5-HT3 receptor (EC50 = 57 mM). The fact that ethanol also inhibits the chimeric receptor suggests that the ethanol binding site is located on the extracellular amino terminal domain of the K7 AChR. Aside from the documentation on the above-mentioned exogenous molecules, there exists a larger body of experimental evidence on the localization of the binding sites for two £uorescent high-a¤nity NCIs, ethidium and quinacrine, which is addressed below. 8.1. Localization of the ethidium binding site Spectroscopic studies have demonstrated that the ethidium binding site is non-luminal [215]. The distance between the ethidium (donor) binding site and the long wavelength £uorescent hydrophobic probe N-(Texas Red0 sulfonyl)-5(and 6)-dodecanoylamine (C12 -Texas Red) as acceptor at the surface membrane was determined by using £uorescence resonance energy transfer (FRET). A transverse distance of V46 î (see Fig. 2) was calculated by assuming a model A where the donor is attached at a certain distance from the major axis of symmetry [215,216] (reviewed in [73]). The calculated transverse distance is compatî ible with the ethidium binding site located 15^40 A away from the major axis of symmetry on the AChR vestibule wall, well above the transmembrane region and slightly above the level of the two agonist binding sites. By de¢nition, the AChR vestibule is a domain di¡erent from the channel lumen and thus,

197

ethidium would bind to a non-luminal site. Interestingly, there exists a predominant negative charge in the vestibule [62] which might contribute to ethidium binding. Unfortunately, attempts at photolabelling have failed to discriminate which amino acids are involved in the high-a¤nity ethidium binding site [217]. Three azido ethidium derivatives labelled the high-a¤nity locus for d-TC, with some preference for the Q subunit. Since the distance between the ethidium and the î [218], agonist binding sites is thought to be 21^40 A it was proposed that the domain for ethidium binding on the AChR may be located either at the KQ interface or at the L subunit [217] (see Fig. 2, top and right panel). The evidence that ethidium binds to the KQ interface suggests an allosteric interaction between ethidium and agonist binding sites. However, the putative location of the ethidium binding site by means of FRET measurements where the KQ interface is formed by the K subunit which shapes the low-a¤nity binding site for d-TC [215] is incongruent with the labelling studies where the labelled K subunit is instead related to the d-TC high-a¤nity binding site [217] (see Section 2 for more details). 8.2. Localization of the quinacrine binding site By means of FRET measurements between the quinacrine (donor) binding site and the lipophilic acceptors 5-(N-dodecanoylamino)eosin (C12 -eosin) and N-(3-sulfopropyl)-4-(p-didecylaminostyryl)pyridinium (Di10ASP-PS) [219] the quinacrine binding î from the site is located at a distance less than 10 A lipid membrane. If it is taken into account that the distance between the center of the channel and the membrane surface in the transmembrane region of î [8], the speci¢c site for the AChR is about 20 A quinacrine could be located at the lipid^protein interface (see Fig. 2, bottom and left panel). In agreement with this location, AChR-bound quinacrine £uorescence was e¡ectively quenched by lipid derivatives with a nitroxide group attached to its molecular structure. The quenching mechanism is dependent on the distance between £uorophores in the excited state and nitroxide radicals [119,120]. A relatively high quenching can be observed over distances as î from the center of the £uorophore far as 8^20 A molecule to the center of the quencher [119]. Thus,

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an elicited high quenching e¤ciency means that the used spin-labelled lipid is particularly accessible to the ligand binding site. Within this perspective, the evidence indicating a higher e¤ciency of the steroid analogue spin-labelled androstane (ASL) [219^221] and of the fatty acid analogue 5-doxyl stearate (5-SASL) [220^222], and in a lesser extent of the phospholipid derivative 5-doxyl phosphatidylcholine (5-PCSL) [220], to quench AChR-bound quinacrine £uorescence than that the e¤ciency to quench AChR-bound ethidium £uorescence, strongly supports di¡erent locations for both NCI binding sites (Fig. 2, compare top and right panel vs. bottom and left panel). In order to determine how deep the quinacrine binding site is located in the hydrophobic domain of the AChR, a series of di¡erent positional isomers of the fatty acid analogue spin-labelled stearate (nSASL [222]) were used. The stearate analogues with the paramagnetic group positioned at carbon 5, 7, 12, and 16, are respectively named 5-, 7-, 12-, and 16-SASL. Each isomer showed a di¡erent pattern in the extent of AChR-bound quinacrine £uorescence quenching. The elicited 5- and 7-SASL quenching e¤ciency was double to that of the 12- and 16SASL, indicating that the quinacrine binding site is located near to carbons 5 and 7 of the fatty acyl chains of the membrane-forming phospholipids. Therefore, the quinacrine binding site would be loî below the aqueous^lipid interface (V12 cated V7 A î A considering the phospholipid headgroup) (Fig. 2). The fact that AChR-bound quinacrine £uorescence is almost completely insensitive to quenching by iodide from the aqueous phase supports a lipid-buried location for the quinacrine binding site. Our data are at odds with reports that use the photolabelling approach. Photolabelling experiments using quinacrine azide as a probe for the localization of the high-a¤nity quinacrine binding site have proved that quinacrine azide speci¢cally labelled amino acids Arg209 and Pro211 of the KM1 transmembrane domain [37,70,224]. Based on the proposed structure of the M1 transmembrane extension (Fig. 1), the labelled residues should be located at the lipid^aqueous interface. A possible explanation for the di¡erence between techniques is that the £uorescence experiments were performed with receptors in the desensitized state and with native quinacrine

whereas the photolabelling experiments were accomplished with AChRs in the open state and with a quinacrine derivative. However, another possibility might be evoked: it may be possible for the quinacrine binding site to move from the channel lumen to the lipid^protein interface when the receptor changes from the open to the desensitized state. Nevertheless, recent stopped-£ow experiments showed that AChR-bound quinacrine £uorescence was quenched by 5-SASL when the channel was in the open con¢guration, indicating that the quinacrine locus is outside the ion channel domain [225]. The photolabelling pattern observed for quinacrine azide di¡ers from that found for other NCIs such as CPZ, TPMP‡ , and TID (Fig. 2, compare bottom and left panel vs. bottom and right panel), which label a luminal location at the M2 transmembrane sequence. One important di¡erence between quinacrine azide and CPZ labelling is that the former labelled AChR in the open state whereas the latter reacted with the AChR in the closed sate. The combination of site-directed mutagenesis and voltage-clamp approaches have helped to elucidate, at least partially, whether there exist other residues of the KM1 domain from Torpedo AChR involved in the quinacrine binding site [226]. The exchange of the Arg209 for Lys or His increased the sensitivity of the AChR to the elicited non-competitive inhibitory effect of quinacrine. Other mutations on amino acids positioned very close to the proposed quinacrine azide-labelled residues such as Ile210 or Leu212 to Ala did not show any e¡ect on quinacrine binding. However, substitutions at positions Pro211 and Tyr213 a¡ect quinacrine binding suggesting that these residues in fact contribute to the quinacrine site. Additionally, residue mutations a¡ecting quinacrine activity were not shown to a¡ect the pharmacological action of CPZ. On the other hand, newer experimental data suggest that Pro211 is oriented against the channel lumen [35]. This conclusion is based on the fact that MTSEA reacted with this site when Pro211 had been substituted by Cys. A deeper insight into the location of the quinacrine binding site was obtained by using several spin-labelled lipids, each one sensing di¡erent lipid domains of the AChR to quench the AChR-bound quinacrine £uorescence [220]. In this regard, we used both the fatty acid derivative 5-SASL and the cholesterol-like

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analogue ASL, which preferentially interact with non-annular binding sites, and the phosphatidylcholine derivative 5-PCSL which only binds to annular binding sites. From this set of experiments, the quenching e¤ciency sequence: 5-SASL s ASLE 5-PCSL was obtained. Since the relative position of the nitroxyl group on the lipid molecule can produce di¡erent quenching e¤ciencies on AChR-bound quinacrine [222] and distinct pharmacological e¡ects on ion £ux measurements [109], we used 5-SASL and 5-PCSL both with the covalently attached nitroxide group at carbon 5. This structural feature makes evident the existence of causes other than the relative position of the paramagnetic group as responsible for the observed quenching e¤ciencies between both spin-labelled lipids. The 4^6 times higher accessibility to 5-SASL and ASL than to 5-PCSL is in agreement with the proposal that the quinacrine binding site is structurally near to a non-annular lipid domain of the AChR. Two more data support such a non-annular location [220] (reviewed in [73]): (1) the evidence that 5-PCSL does not displace quinacrine from its binding site in the concentration range of 200^450 WM, and (2) the observed lower accessibility of 5-PCSL to the quinacrine binding site than to the membrane-partitioned £uorescent probe 7-(9-anthroyloxy)stearate (7-AS). Concomitantly, other conclusions may be drawn. (1) The observed higher accessibility of 5-PCSL to the quinacrine binding site than to the ethidium locus agrees in large part with previous experimental evidence indicating a lipid^protein interface location for the quinacrine locus which is far apart from the ethidium binding site [219^222]. Contrarily, it was suggested by means of competition studies that both NCIs bind to the same locus [227]. However, in a recent work using stopped-£ow instrumentation it was suggested that both NCIs bind to their respective high-a¤nity sites at the same time [116]. The evident confusion on this point indicates that further e¡ort is necessary to elucidate this issue. (2) Taking into consideration that the accessibility of a hydrophobic molecule such as 5-PCSL to a hydrophilic domain such as the channel lumen is not thermodynamically allowed, the demonstrated (although low) accessibility of 5-PCSL to the quinacrine locus argues against the possibility that the quinacrine binding site is located in the channel lumen [220].

199

In Fig. 2 (bottom and left panel) we illustrate the most probable location of the quinacrine binding site at the M1 sequence of the K subunit. In this scheme, the quinacrine binding site is closer to the non-annular than to the annular lipid domain. The annular lipid domain is represented by 23 open circles corresponding to the phospholipid molecules from the extracellular lea£et of the lipid bilayer surrounding the AChR. The quinacrine binding site is accessible from intersubunit and/or interhelical non-annular lipid sites. The relatively closer location of the quinacrine binding site to the L subunit is based on the fact that, in addition to the labelling of the K subunit by both tritiated quinacrine azide [70,224] and quinacrine mustard [228], the L chain was also labelled, albeit on di¡erent AChR functional states and time regimes. More speci¢cally, the labelling on the L chain was potentiated by agonists by approximately 20%. Another example in which a NCI binding site is postulated to be located at a non-annular lipid domain is the site for the inhalational anesthetic halothane [229]. The photoa¤nity labelling data by using AChR-rich membranes and [14 C]halothane indicate that this anesthetic binds to the AChR with a Kd of 150 þ 40 WM in a stoichiometric ratio of 2.5 þ 0.4 sites per AChR. Although the principal target site for halothane binding was found to be at the transmembrane M1^M4 domain the exact localization of this site remains unknown. However, comparing these results with the photolabelling pattern of a photoactivatable phosphatidylserine analogue [26] it is postulated that the halothane binding site is located at a non-annular lipid domain. Finally, the importance of the KM1 region regarding the ion channel function has been supported by mutation of Cys230 to Thr, Ser, Ala or Gly, respectively, which altered the duration of channel opening and closing [230]. Furthermore, all residues of the KM1 sequence were individually mutated to Cys [35]. Cysteine substitution of residues Pro211 , Leu212 , Tyr213 , and Ile220 all caused V10-fold increases in the apparent Kd for ACh. In contrast, other mutations (e.g., Phe to Cys) decreased the apparent Kd of ACh in the same extent. The exchange of residues at the extracellular border of the M1 domain such as Arg209 for His or Lys only produced a slight modi¢cation on the ACh-induced activation [226].

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8.2.1. Proposed mechanism of quinacrine binding to its high-a¤nity locus on the nicotinic acetylcholine receptor To elucidate the binding mechanism of the antimalarial drug quinacrine to its high-a¤nity site located at the lipid^protein interface of the AChR, the binding of quinacrine was determined at di¡erent temperatures [231]. The relative constancy in quinacrine's Kd values at 4^15³C is in agreement with the fact that very little change in the association of [3 H]PCP to its high-a¤nity NCI site on the AChR was observed between 0 and 37³C [232]. By using the data from Oswald et al. [232] approximately 1.4-fold increase in the PCP Kd by increasing the temperature from 15 to 23³C was calculated [231]. This e¡ect was lower than that seen for quinacrine's Kd which increased V4.8 times in the same temperature range. Opposite to these data, the binding of the DNA probe ethidium to the AChR is not sensitive to temperature in the range of 15^23³C. The di¡erent temperature dependence of quinacrine and ethidium binding is another piece of evidence that supports the conjecture that the quinacrine and the ethidium binding site are located on di¡erent parts of the AChR. The sign and the magnitude of the thermodynamic parameters allow some qualitative conclusions about quinacrine binding to the receptor [231]: (1) the small but negative free energy change (vG³) values observed between 4 and 15³C indicates that quinacrine binds with greater a¤nity at these temperatures than at 20^23³C where the vG³ values were positive; and (2) the enthalpy change (vH³) value suggests that a signi¢cant enthalpic barrier to binding exists and that the binding of quinacrine to the AChR is an exothermic reaction. This is contrasted with a minor enthalpic component for the PCP equilibrium a¤nity [232]. These di¡erences might be accounted for by subtle structural features in the respective binding sites for quinacrine and PCP (see Fig. 2, compare bottom and left panel vs. bottom and right panel). The existence of an enthalpic barrier might be explained by the following dynamic model [231]: when quinacrine is already in the lipid membrane, the molecule needs ¢rst to be sterically well oriented to fur-

ther enters into its binding site located at the lipid^ protein interface. This molecular ¢t is accomplished at temperatures where quinacrine does not move very fast in the lipid phase (between 4 and 15³C). However, when the temperature is raised (to 20^23³C), the mean orientation of quinacrine in the lipid membrane is diminished as consequence of a slight increasing in its anisotropic movement. Under this circumstance, the molecule is spatially aligned with its binding site for shorts periods of time, thus, the diffusion of properly oriented quinacrine into the locus is restricted. The environmental characteristics of the quinacrine locus might perfectly account for a partially restricted di¡usion-controlled mechanism of quinacrine binding. For instance, the existence of a crevice for the quinacrine binding site at the lipid^ protein interface was based on the lack of aqueous solute accessibility to AChR-bound quinacrine [222]. Accordingly, we have recently postulated that the quinacrine binding site is located at a non-annular lipid domain of the AChR [220]. By taking into consideration the preliminary observation that quinacrine binding is sensitive to ionic strength [222], the quinacrine Kd was further determined at di¡erent NaCl concentrations at a ¢xed temperature of 4³C [231]. From these experiments, we can assess that quinacrine binding was not affected by salt concentrations as high as 100 mM. Further increases in salt concentration (250 mM NaCl) caused a screening of the electrostatic interactions between quinacrine and the AChR giving rise to an increase in the apparent quinacrine Kd [231]. This is consistent with the existence of an electrostatic component in the quinacrine locus. An electrostatic component has been also demonstrated in the interaction of a series of spin-labelled local anesthetic derivatives with the hydrophobic domain of the AChR [117]. In addition, based on the V2.3-fold increase in the emission of quinacrine upon binding to the AChR, a hydrophobic microenvironment around the acridine moiety of the quinacrine molecule was suggested [219]. On the other hand, ethidium binding was sensitive to NaCl at these concentrations [231,233]. In addition, ethidium was displaced by a variety of mono and divalent cations such as Ca2‡ , Mg2‡ , Tl‡ , Rb‡ , K‡ , Cs‡ , and Li‡ [233].

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9. Non-luminal localization of binding sites for endogenous non-competitive inhibitors 9.1. Localization of fatty acid binding sites An increasing volume of data indicates that free fatty acid molecules may a¡ect excitable tissues including di¡erent neurons and muscle cells. In general, fatty acids in£uence the activity of ion channels comprising NMDA and GABAA receptors [234], as well as L-type Ca2‡ [235], Cl3 [236,237], Na‡ [238^ 241], and distinct K‡ channels [242^245]. There is not a unique pharmacological e¡ect but a broad range of e¡ects. In fact, fatty acids may activate (e.g., see [244]) or inactivate (e.g., see [243]) ion channels. Additionally, fatty acids can exert their e¡ects through either direct or indirect mechanisms (for a review see [246]). An indirect mechanism includes the formation of metabolic subproducts from the enzymatic conversion of arachidonic acid (20:4[n-6]) by either the lipoxygenase or the cyclooxygenase pathway which in turn, may a¡ect ion channels by activation of di¡erent protein kinases (e.g., see [247]). The pharmacological e¡ects of di¡erent fatty acids on both muscle- and neuronal-type AChRs [109,248^ 251] is shown in Table 4. Early work demonstrated that fatty acids do not a¡ect agonist binding to the AChR [109,251,252]. In addition, fatty acids do not greatly change the physical properties of the bulk lipid membrane [109]. For instance, the order parameter, S, of three spin-labelled fatty acid isomers, 5-SASL, 12-SASL, and 16-SASL, did not change in the presence of, among other fatty acids, 1 mM petroselenic acid (18:1[n-12]), suggesting that membrane £uidity is not perturbed by fatty acids. Thus, it is possible that the observed e¡ect may be mediated by either a direct blockade of the ion channel or a perturbation of the lipid^protein interface. In this regard, the latter possibility has received much experimental support. For example, various phospholipases of the A2 type from exogenous origin that hydrolyze the sn-2 acyl chain of membrane phospholipids inactivate AChR ion channel [248, 252^254] with a concomitant structural perturbation of the protein [255]. Moreover, the observed phospholipase A2 -induced ion channel inhibition is believed to be achieved by the hydrolyzed free fatty acids and not by lysophospholipids [252].

201

The agonist-evoked ion channel activity was inhibited by a large variety of structurally di¡erent fatty acids, including those with distinct chain lengths and number of double bonds (Table 4). Both the degree of unsaturation and the acyl chain length seem not to be an important requisite for the ion channel inhibition of muscle-type AChRs [248]. For instance, the mean open time (don ) of the AChR ion channel measured by the patch-clamp technique was decreased by all fatty acids tested including palmitic (16:0), nonadecanoic (19:0), 20:4[n-6], and docosahexanoic (22:6[n-3]) acids. This is in agreement with the fact that di¡erent fatty acids inhibit the 22 Na‡ e¥ux activity elicited by ACh when experiments are performed above the melting temperature of each fatty acid [109]. In contrast, in the neuronal-subtype K7 receptor, polyunsaturated fatty acids such as linoleic (18:2[n-6]), linolenic (18:3[n-3]), and 20:4[n-6], demonstrated a higher relative inhibitory response than both saturated and monounsaturated fatty acids [249]. In this regard, the apparent IC50 for 20:4[n6] was calculated to be 1 WM. Within this scenario it is reasonable to think that the lipid^protein interface of the AChR is the target site for the physiological action of fatty acids. The hydrophobic surface of the AChR is a large area of the protein in intimate contact with the lipid phase. Hydrophobic probes as well as lipid derivatives were found to label this particular domain of the AChR (reviewed in [30]). Among transmembrane segments, M1, M3, and M4 were the most labelled [26^29]. In addition, lipid molecules bind with distinct speci¢cities to the hydrophobic surface of the AChR. Such di¡erences were addressed by using EPR spectroscopy, a useful technique to study lipid interactions with integral membrane proteins (reviewed in [256^ 258]). Taking into account that lipid motion becomes slower when the molecule interacts with the protein, the signal provided by spin-labelled lipids at the lipid^protein interface is distinguishable from the signal in the bulk lipid membrane. In practice, the signals can be separated by spectral subtraction. This consists of subtracting the signal provided by spin-labelled lipid molecules in a membrane suspension of previously extracted protein free lipids (the membrane mobile component) from the tissue or cell under study from the total signal (both protein perturbed and membrane mobile components) of the

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Table 4 Pharmacological e¡ects elicited by several fatty acids on both muscle-type and neuronal-type AChRs Fatty acid (nomenclature)a

Saturated: Palmitic (16:0) Stearic (18:0) Spin-labelled stearic acids (5-SASL and 16-SASL) Nonadecanoic (19:0) Arachidic (20:0) Unsaturated: Oleic (18:1[n-9]) Elaidic (trans-18:1[n-9]) Petroselenic (18:1[n-12]) Vaccenic (18:1[n-7]) trans-Vaccenic (trans-18:1[n-7]) Linoleic (18:2[n-6]) Linolenic (18:3[n-3]) Arachidonic (20:4[n-6]) Docosahexanoic (22:6[n-3])

Pharmacological e¡ect Ion £ux inhibitionb

Mean open time reductionc (control/treated)

Whole-cell currentd (% initial response)

^ No (0^33³C) Yes (33^37³C) ^ ^

3.0 ^ ^ 4.4 ^

^ V108 ^ ^ V97

Yes ^ Yes Yes Yes ^ ^ Yes ^

^ ^ ^ ^ ^ ^ ^ 2.9 4.1

V82 V98 ^ ^ ^ V47 V28 V13 ^

(25^33³C) (33³C) (25^33³C) (33³C)

(0^33³C)

a

In the nomenclature of fatty acids, n indicates the position of the ¢rst double bond from the methyl end (IUPAC-IUB Commission on Nomenclature). b Carbamylcholine-induced 22 Na‡ e¥ux was determined in AChR-rich membranes from Torpedo electric organ [109]. The fatty acid ¢nal concentration was 2 mM (for spin-labelled analogues the ¢nal concentration was 1 mM). The temperature shown in parentheses indicates the incubation temperature range at which the ion £ux was completely blocked. c Single-channel activity of AChR-containing BC3 H-1 cells was recorded at 370 mV in the inside-out patch con¢guration before and 10 min after the addition of 60 WM of each fatty acid in a bu¡er containing 1.5% fatty acid-free bovine serum albumin [248]. Mean open times were obtained from the dwell-time histograms, and correspond to the major component. The mean open time after 10 min incubation with no addition of fatty acid is 4.30 þ 0.92 ms. d Whole-cell currents induced by 100 WM ACh were measured in K7-containing oocytes [249]. Fatty acids (20 WM) were applied 30 s prior to measuring the ACh response.

same spin-label in protein-containing native or reconstituted membranes. In this regard, di¡erent laboratories have used either AChR-rich membranes [41,259^262] or AChR-dioleoylphosphatidylcholine (DOPC) reconstituted systems [39,263,264], and liposomes furnished with either electric organ-extracted total lipids or pure DOPC. Since fatty acid molecules inhibit the AChR ion channel in a non-competitive fashion and since we are interested in elucidating the pharmacological mechanism of inhibition, among all lipid classes found in native membranes, we show in Table 5 the EPR data for several spin-labelled stearate positional isomers. The data indicate that there is an appreciable fraction (f) of spin-labelled fatty acid molecules interacting with the lipid^protein interface of the AChR. However, depending on the AChR

membrane preparation used, the temperature at which the EPR measurement was performed, or the lipid/protein molar ratio of the reconstituted system, a broad range of f values is obtained. Thus, we compared the f value of each spin-labelled stearate isomer with the value obtained for PCSL (fPCSL ) containing the same fatty acid isomer and measured in the same experimental conditions. In this regard, the relative association constant (K/KPCSL ) for each fatty acid was calculated according to the equation: K=K PCSL ˆ ‰…13f PCSL †=f PCSL Š=‰…13f †=f Š

…2†

The calculated values were summarized in Table 5. In reconstituted membranes, the association constant of spin-labelled lipids relative to DOPC was obtained from the y-intercept (3KDOPC /K) of the plot mobile/ perturbed lipid fraction vs. lipid/protein molar ratio

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203

Table 5 Fatty acid and steroid speci¢cities for the hydrophobic surface of the AChR Spin-labelled lipid Fatty acid 4-SASL 12-SASL 14-SASL

16-SASL

16-SESL Steroid ASL

CSL Phosphatidylcholine 4-PCSL 14-PCSL

16-PCSL

K/KPCSL g

vG-vGPCSL i (kJ/mol)

Reference

0.35 þ 0.10 0.48 þ 0.04 0.30 0.44 0.54 0.49 þ 0.02 0.44 þ 0.03 0.35 0.25 0.38 0.60 0.35

1.14 2.43 1.60 1.17 (3.0)h 2.14 4.85 2.91 (4.10)h 4.85

30.3 32.2 31.1 30.4 (32.5)j 31.7 33.5 32.4 (33.2)j 33.5

[43] [341] [41] [261] [263] [264] [341] [259] [260] [39]

22 15 23 0 0 22

0.43 þ 0.03 0.38 þ 0.01 0.25 þ 0.01 0.59 0.58 V0.16

4.27 1.30 2.79 (4.3)h 1.38 (3.5)h V1.08

33.6 30.6 32.3 (33.3)j 30.7 (32.8)j V30.2

[341] [43] [39] [263] [262]

4 22 0 0 0 34 0

0.32 þ 0.01 0.15 0.33 0.31 þ 0.02 0.50 6 0.10 0.34

1.0 1.0 1.0 1.0 (1.1)h 1.0 (1.0)h

0 0 0 0 0 0 0

[43] [41] [261] [264] [264] [259] [39]

AChR membranea

Temperature (³C)

f

native native native native reconstitutedb reconstitutedb native native native native reconstitutedc native

10 29 22 0 0 0 0 34 0-20 0 0 0-17

native native native reconstitutedd reconstitutedb native native native native reconstitutedb reconstitutede native reconstitutedc

f

[259]

a

EPR measurements were performed in both native and reconstituted membranes. Reconstituted membranes containing AChR were prepared in dioleoylphosphatidylcholine at the following lipid/protein molar ratios: 198:1b , 142:1c , 155:1d , and 115:1e . f Fraction of protein-associated component. This component was quantitated by spectral subtraction. g Association lipid constant relative to spin-labelled phosphatidylcholine. The relative constants were calculated according to Eq. 2. For this purpose, we used the corresponding f and fPCSL (the protein-associated component for spin-labelled phosphatidylcholine) values for each particular experimental condition (e.g., temperature and AChR membrane preparation). h Alternatively, the association constants were obtained from the y-intercept (3KDOPC /K) of the plot mobile-perturbed lipid fraction ratio vs. lipid/protein molar ratio (see [39,263,264]). i Di¡erential free energy of association. The values were calculated according to Eq. 3. j These values were obtained taking into account the association constants relative to DOPC (values in parentheses).

(see [39,263,264]). Interestingly, the values calculated by this method were higher than that calculated directly by Eq. 2. Another interesting detail is the fact that both 16SASL and its ester analogue 16-SESL present the same a¤nity for the hydrophobic surface of the AChR [260] (Table 5). This suggests that the hydroxyl group of the carboxyl moiety of the fatty acid molecule is not physicochemically involved in the interaction with the AChR. This evidence is in agreement with the fact that increasing monovalent salt

concentration (up to 2 M NaCl) on the 14-SASL^ protein complex did not cause a screening of the electrostatic interactions between free fatty acid and AChR positively charged amino acids at pH 5.5 [264]. However, a partial electrostatic component was manifested by pH titration [264]. Concomitantly, the energetics of the selectivity of the lipid-protein interaction can be obtained relative to PCSL. For this purpose, the di¡erential free energy of association of each spin-labelled fatty acid respect to PCSL (vG-vGPCSL ) was calculated by us-

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ing the expression: vG3vG PCSL ˆ 3RT ln…K=K PCSL †

…3†

where R is the universal gas constant and T is the absolute temperature used in the EPR experiment. The calculated values were also included in Table 5. In addition to the EPR technique, another spectroscopic approach has been used to study the interaction of fatty acids with the lipid^protein interface of the AChR. Taking advantage of the quenching properties elicited by nitroxide fatty acid analogues, it was possible to demonstrate that fatty acids gain access to the hydrophobic domain of the AChR. For this purpose, both native and reconstituted membranes containing intrinsic or extrinsic £uorophores were used. The intrinsic £uorescence of the transmembrane AChR domain is assumed to arise from the Trp453 residue located in the QM4 portion (see Section 5). In general, the extrinsic £uorescence is provided by a £uorophore covalently attached to

the protein. For this end, McNamee's laboratory used a pyrene group attached to the QCys451 residue î apart from Trp453 which is thought to be V18 A [123]. Interestingly, from the fa values summarized in Table 6 it is possible to suggest that fatty acid molecules gain access to both Trp453 and to Cys451 bound pyrene residues. The fact that spin labels quench Cys451 -labelled pyrene £uorescence more e¤ciently than AChR-Trp453 £uorescence may be ascribed to a higher quenching intrinsic sensitivity for pyrene than for Trp. Nevertheless, the fatty acid accessibility to both £uorophores follows the sequence: 5-SASL s 12-SASL s 16-SASL, indicating that both £uorophores are located close to the phospholipid headgroup^water interface. The elicited agonist e¡ect on AChR-fatty acid interactions is also shown in Table 6. For example, the di¡erences in the 1/KQ values indicate that the required concentration of 16-SASL to quench the intrinsic AChR £uorescence when the receptor is de-

Table 6 Quenching e¤ciency of lipid quencher analogues on both AChR intrinsic £uorescence and pyrene-labelled AChR £uorescence Lipid quencher

Modulator

AChR intrinsic £uorescence Fatty acid: 5-SASL ^ SubCh 12-SASL ^ 16-SASL ^ SubCh Steroid: ASL ^ CSL ^ CCh diBrCHS ^ CCh K-BTx Pyrene-labelled AChR £uorescence Fatty acid: 5-SASL ^ 12-SASL ^ Steroid: diBrCHS ^ CCh K-BTx

Concentration

fa a

KQ b (mM31 )

1/KQ c (WM)

Reference

^ 20 nM ^ ^ 20 nM

0.39 0.33 0.20 0.20 0.20

7 6 7 9 23

143 167 143 111 43

[342]

^ ^ 100 WM ^ 1 mM 0.4 WM

0.10 0.59 0.28 0.27 þ 0.01 0.23 þ 0.01 0.31 þ 0.05

15 71 333 43 þ 3 32 þ 3 37 þ 3

67 14 3 23 31 27

[342] [262]

^ ^

1.00 þ 0.09 0.65 þ 0.04

187 þ 18 72 þ 5

5 14

[123]

^ 1 mM 0.4 WM

0.65 þ 0.05 0.90 þ 0.08 0.70 þ 0.07

123 þ 10 167 þ 15 134 þ 12

8 6 7

[42]

[42]

diBrCHS, dibromocholesteryl-hemisuccinate. Apparent fraction of £uorophores accessible to lipid quencher. For more details see Table 1. b Apparent Stern-Volmer quenching constant for the accessible fraction of £uorophores. c Relative concentration of lipid quencher molecules at which 50% of the £uorescence intensity is quenched assuming that AChR-£uorophores are totally accessible to lipid quencher (fa = 1). a

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sensitized is lower than that in the resting state. However, in the 5-SASL^AChR complex this e¡ect is not apparent. This suggests that the conformational changes of the receptor protein are more evident at a deeper domain of the lipid^protein interface. Interestingly, the structural change at the lipid^protein interface elicited by the agonist-triggered desensitization process was not reproduced by phosphorylation of the AChR tyrosine groups [43]. Although spectroscopic experiments demonstrated an e¡ective interaction of fatty acid molecules with the lipid^protein interface, there is no evidence indicating whether a speci¢c binding site(s) for fatty acids exists or not. In this regard, both (a) the e¡ect elicited by di¡erent NCIs on the binding of 14-SASL to the lipid^protein interface [41,261,263,264], and (b) the e¡ect elicited by high concentrations of both 5-SASL and its unlabelled analogue stearic acid (18:0) on the binding of two high-a¤nity NCIs, quinacrine and ethidium [220^222], were measured. From the former experiments (a), it was determined that local anesthetics such as procaine and benzocaine [41], general anesthetics such as hexanol [261] and ethanol [263], as well as the openchannel blocker PCP [264], did not perturb the protein-immobilized component for 14-SASL. In addition, the local anesthetic quotane did not change the protein-associated fraction of 16-SASL [260]. All this EPR evidence is consistent with the existence of fatty acid binding site(s) not overlapping with that for anesthetic-like molecules. However, since tetracaine seems to partially inhibit 14-SASL binding to the lipid^protein AChR interface [41], more experiments will be necessary to ¢nally determine the localization of the fatty acid binding site(s). From the second set of experiments (b), it was demonstrated that 5-SASL competitively inhibited quinacrine, but not ethidium, binding with an apparent inhibition constant (Ki ) of 160^170 WM [221,222]. Since the binding sites of both NCIs have been already localized on the AChR (see Fig. 2), then, it is plausible that fatty acids may interact with a non-luminal binding site located at the non-annular lipid domain (the quinacrine binding site) but may not interact with that non-luminal site located at the vestibule of the ion channel (the ethidium binding site). Interestingly, the fatty acidinduced displacement of quinacrine from its binding site was shown to be steric in nature since di¡erent

205

spin-labelled stearate positional isomers gain access to the quinacrine locus [220^222]. In addition, 18:0 provoked a lower displacement of quinacrine from its binding site than 5-SASL at 15³C. At a ¢rst glance, this result suggests that 5-SASL would have greater a¤nity for the quinacrine binding site than its parent 18:0. Functional experiments measuring CCh-induced 22 Na‡ e¥ux in the absence or in the presence of either 18:0 or 5-SASL are in agreement with these results [109,252]. These authors observed that 5-SASL inhibited ion £ux at extents of 30 and 100% when measurements were carried out at temperatures of 25 and 33³C, respectively, whereas 18:0 did not inhibit CCh-evoked response at any temperature (Table 4). The possibility that 18:0 had not been e¤ciently incorporated into AChR-containing membranes was excluded [109]. The activation of K7 receptors trigger the production of 20:4[n-6] probably by a Ca2‡ -mediated process [249]. The total amount of 20:4[n-6] released in the neuron by nicotinic stimulation has been considered to be V400 WM in a space equivalent to the cell volume. Thus, the 20:4[n-6] local concentration would be in the micromolar range, a concentration high enough to cause the inhibitory e¡ect on ion £ux. The combination of these ¢ndings argues in favor of a model where the concentration of free fatty acid in the lipid membrane is raised by the activation of a Ca2‡ -dependent phospholipase A2 after the agonist-triggered access of Ca2‡ to the cytoplasm. A diagram of this model is shown in Fig. 6. This model indicates that the Ca2‡ -activated phospholipase A2 hydrolyzes phospholipid molecules to produce lysophospholipids and free fatty acids. The fatty acid molecule produced in the cytoplasmic membrane lea£et may di¡use laterally to ¢nally interact with the lipid^protein interface at the annular lipid domain. Alternatively, lipid molecules may cross the AChR native membrane from the inner to the extracellular lea£et (or vice versa) by transversal di¡usion (£ip-£op) [259,265]. The existence of a very active fatty acid deacylation-reacylation enzyme system in the electrocyte [266,267] supports the concept that this model can be extrapolated to the muscle-type AChR. In this regard, it has been shown that fatty acid molecules interact with the high-a¤nity quinacrine binding site located at a non-annular lipid domain (see Section 8). Thus, the

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Fig. 6. Diagram depicting the putative binding site locations for free fatty acid molecules on the nicotinic acetylcholine receptor (AChR) (taken from [343]). The opening of the AChR ion channel allows the movement of Ca2‡ ions with the concomitant increasing in the intracellular concentration (u[Ca2‡ ]i ). This raised concentration allows the activation of a phospholipase A2 located in the lipid membrane. The activation of the enzyme provokes the hydrolysis of phospholipids with the subsequent production of lysophospholipids and free fatty acids. The produced fatty acid molecules may di¡use laterally through the lipid membrane to further interact with its binding site located at the annular lipid domain. Nevertheless, free fatty acid molecules may cross the lipid membrane from the inner to the outer lea£et (and vice versa) by transversal di¡usion (£ip-£op). Additionally, fatty acids might be delivered on the extracellular surface of the lipid membrane by circulating albumin (shown with four bound fatty acid molecules) or lipoproteins (shown with only one bound fatty acid molecule), or on the intracellular surface of the membrane by fatty acid binding proteins (shown with only one bound fatty acid molecule). Finally, fatty acid molecules may interact with the high-a¤nity quinacrine binding site located at a non-annular lipid domain (see Fig. 2, bottom and left panel).

increased free fatty acid concentration in the bilayer would allow the interaction of these endogenous molecules, probably sterically, with the quinacrine locus generating the observed pharmacological noncompetitive inhibition of the AChR. Aside from the increase in the membrane fatty acid concentration by a Ca2‡ -activated phospholipase A2 system, it is necessary to take into consideration the possibility that fatty acid molecules can be delivered from both the extracellular and the intracellular surface by proteins such as albumin and lipoprotein, as well as by fatty acid binding proteins (see Fig. 6). 9.2. Localization of steroid binding sites Classically, the principal site for steroid binding is found in intracellular receptors and the physiological mechanism elicited by them are mediated by a slow process which involves the modulation of genetic transcription. In fact, steroids modulate the expression of the AChR protein probably by a genomic mechanism (reviewed in [73]). However, certain ste-

roids exert some of their biological e¡ects on excitable membranes too fast (in the seconds to minutes time regime) to involve only a genomic process. Thus, an emerging concept in neuroendocrinology is that steroids rapidly a¡ect neurons by binding to membrane receptors and channels [268^270]. For example, synthetic steroids such as 3K-hydroxy-5Kpregnane-11,20-dione (alphaxalone) and its structural analogue 21-acetoxy-3L-hydroxy-5K-pregnane11,20-dione (betaxalone) blocked both K‡ and Na‡ channels from frog myelinated axons [271,272]. More interestingly from the physiological point of view is that endogenous steroids blocked Ca2‡ currents [273,274]. Regarding ligand-gated ion channel receptors, alphaxalone binds the GABAA receptor at low concentrations (30 nM^1 WM) and modulates their properties [275]. Recently, it has been shown that alphaxalone does not interact with the GABA, the barbiturate, the bicuculline, or the gabazine binding site [276]. Interestingly, betaxalone at low micromolar concentrations did not produce any e¡ect on the

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Table 7 Inhibitory potency of structurally di¡erent steroids on muscle-, ganglionic-, and neuronal-type AChRs Steroid

IC50 (WM) Muscle-type AChRa

Progesterone Progesterone-3 Progesterone-3-BSA Dexamethasone Testosterone Testosterone-3 Estradiol Corticosterone Pregnenolone 5K-Pregnan-3K-hydroxy-20-one Cholesterol Alphaxalone Betaxalone

d

6.1 (0.8) 12 (5.3)d 1.4 (0.5)d 39 ^ 44 56 92 ^ ^ ^ 6e ^

Ganglionic-type AChRb d

11 (3.0) s 100 (60)d 5.6 (3.5)d 45 ^ s 100 43 94 ^ ^ ^ ^ ^

Neuronal-type AChRc 9 2.9 2.9 ^ 46 ^ ^ ^ No e¡ect No e¡ect No e¡ect 20f 30f

Testosterone-3, testosterone 3-(O-carboxymethyl)oxime; Progesterone-3, progesterone 3-(O-carboxymethyl)oxime; Progesterone-3-BSA, progesterone 3-(O-carboxymethyl)oxime-bovine serum albumin conjugate. a Each embryonic muscle-type and K3L4b ganglionic-type AChR was expressed in the human clonal cell lines TE671/RD or SH-SY5Y, respectively [299]. The 50% inhibition (IC50 ) for the di¡erent steroids was determined by 86 Rb‡ e¥ux experiments where receptor-expressing cells were treated with 1 mM CCh for 5 min in the absence or in the presence of steroid at the concentration range of 10 nM to 100 WM. c The neuronal-type K4L2 AChR was expressed in oocytes and the IC50 values were obtained by steroid dose-response inhibition experiments by voltage-clamp at a holding potential of 3100 mV [110,297]. d The values in parentheses were obtained by pre-incubating the cells with each steroid for 5 min [299]. e This value was obtained from cultured rat myobals [277]. f These values were obtained from bovine chroma¤n cells by voltage-clamp at a holding potential of 360 mV [275].

GABAA receptor but at higher concentrations (10^ 100 WM) it blocks both the GABAA and the AChR ion channel [275]. The same blocking e¡ect on AChinduced currents was observed by alphaxalone at high concentrations [277]. Both steroid derivatives inhibit 50% of the AChR ion channel activity (IC50 ) from bovine chroma¤n cells in the concentration range of 20^30 WM (Table 7). More important is the fact that the pharmacological e¡ect of the neurotransmitter GABA is potentiated by endogenous steroid molecules such as 3K-hydroxy ring A-reduced pregnane derivatives (e.g., the major metabolites of progesterone or deoxycorticosterone) as well as progesterone or corticosterone in the physiological concentration range (nanomolar) reviewed in [73,278^ 281]). An interesting point is that this positive modulatory e¡ect is considered to be mediated by the binding of neurosteroids to enantioselective sites [282]. Moreover, at higher concentrations (micromolar), allopregnanolone increases Cl3 conductance and 36 Cl3 in£ux into synaptoneurosomes in the ab-

sence of GABA [283], suggesting that neurosteroids may behave as agonists. Another interesting issue is the experimental evidence indicating that the gabaergic transmission inhibition enhances the neurosteroid content in the rat brain [284]. On the other hand, pregnanediols, and pregnenolone and dehydroepiandrosterone sulfates inhibit GABAA function [285^ 287]. One of these sites, the pregnenolone locus, has been demonstrated to be di¡erent from that for the anesthetic propofol [288]. It is possible that the steroid binding site on the transmembrane GABAA receptor presents a similar topology with intracellular corticoid receptors. Indeed, the fact that tetrahydrodeoxycorticosterone, the neuronal endogenous metabolite of deoxycorticosterone, produces its e¡ect on the GABA-activated type A receptor with a Kd of V40 nM and the fact that deoxycorticosterone binds to both mineralocorticoid (type I corticoid receptor) and glucocorticoid (type II corticoid receptor) receptors with Ki s in the concentration order of 0.55^1.2 and 15^23 nM, respectively [289], support the con-

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Fig. 7. Schematic representation of the possible locations of steroid binding sites (taken from [343]). A single steroid binding site was proposed to exist on the extracellular hydrophilic domain of the nicotinic acetylcholine receptor (AChR). However, there is no evidence of the exact location of this locus. Instead, a series of steroid binding sites were suggested to be located at the lipid^protein interface of the AChR, probably at the annular lipid domain, on the basis of the electrophysiological similarities between steroids and both general and local anesthetics (see Section 9). Finally, we suggest a structural correlation between the steroid binding site (speci¢cally for spin-labelled androstane) and the high-a¤nity quinacrine locus (see Section 8) at the non-annular lipid domain.

jecture of a similar binding site on both receptor classes. In this regard, a residue homology comparison between both GABAA and glucocorticoid receptors will help to elucidate which amino acid sequence is involved in the steroid locus. Aside from the GABAA receptor, progesterone, and pregnenolone and dehydroepiandrosterone sulfates reduce glycineevoked currents in spinal cord neurons [286,290]. Additionally, dehydroepiandrosterone, but not progesterone [291], and pregnenolone sulfate or other pregn-5-ene derivatives potentiate, whereas other steroids inhibit [292^295] NMDA-type glutamate receptors. Regarding the AChR, endogenous steroids such as glucocorticoids [128,296], corticosteroids [297,299], and sex steroids [110,298^304] inhibit the ion channel activity. The acute application of the synthetic glucocorticoid dexamethasone to muscle- [305] or neuronal-type receptor-containing cells [299] also inhibited ion £ux. The IC50 values elicited by steroids on di¡erent AChRs are summarized in Table 7. The observed e¡ect of steroids on the AChR pharmacological properties may be explained by direct binding to a high-a¤nity non-luminal site or to several lowa¤nity binding sites. However, there is no available evidence indicating the steroid binding site stoichiometry. In addition, the large range of IC50 s (1^100 WM) does not give an indication of the involvement of either one or another binding site class. Nevertheless, the steroid IC50 concentration ranges observed

on neuronal-type AChRs are closer to the Kd s for high-a¤nity NCIs (see Section 5). An important question to be answered is how steroids mediate their pharmacological e¡ects by direct binding to membrane receptors. In this regard, the inhibitory e¡ect elicited by steroids on the AChR was found not to be mediated by a¡ecting the agonist/competitive antagonist binding sites [128,299]. In addition, it has been con¢rmed that the main mode of action of steroids is mediated neither by non-speci¢c perturbation of the membrane lipid bilayer surrounding the AChR protein nor by activation of second messenger pathways [110,297]. The possibility that steroid molecules inhibit ion channel by an open-channel-blocking mechanism has not received experimental support [110,128,298,305]. For instance, (a) the duration of open channels when K12 L1ON-containing HEK-293 cells were treated with 0.4 mM hydrocortisone is the same either in the absence or in the presence of 30 WM QX-222 [128] [it is necessary to recall that QX-222 is a high-a¤nity NCI whose binding site is located at a luminal channel domain (see Section 6)]; (b) the observed decrease in the burst length as a function of hydrocortisone concentration was not the same as that for QX-222 [128]; (c) the inhibitory e¡ect elicited by hydrocortisone is not in£uenced by the Thr264 to Phe mutation on the OM2 domain [128]; (d) the observed lack of e¡ect on the elicited inhibitory action of progesterone on the neuronal-subtype K4/non-K1 receptor

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when point mutations such as Glu266 to Lys on the K4 subunit and Lys260 to Glu on the non-K1 subunit were performed [110]. These speci¢c residues are believed to be structurally related to the outer mouth of the ion channel. In turn, this domain has been demonstrated to be involved in the conductance of both neuronal- [306] and muscle-type [162] ion channels; and ¢nally, (e) the conductance remains the same in the absence or in the presence of di¡erent steroids [128,305]. Instead, a negative heterotropic interaction between the speci¢c steroid and the agonist binding sites has been hypothesized [110]. At least two domains have been considered to be structurally related to the steroid binding site by using di¡erent steroid molecules. Nevertheless, the experimental evidence indicating two plausible locations for the steroid binding site(s) might be ascribed to the structural di¡erences in the steroid molecules (e.g., progesterone vs. hydrocortisone) and to the type of AChR under study (e.g., muscletype vs. neuronal-type AChRs). One of these plausible domains is the extracellular hydrophilic portion of the AChR (1), and the other possible locus is the lipid^protein interface (2). Both locations are shown in Fig. 7. (1) The extracellular localization was suggested since two water-soluble bovine serum albumin-attached progesterone derivatives (P-3-BSA and P-11-BSA) produced the same inhibitory e¡ect as its untagged parent [110,299] (see Table 7). (2) The steroid binding site localization at the lipid^protein interface was based on other evidence. For instance: (a) steroids are highly hydrophobic molecules which induce a dose-dependent reduction in the AChR channel don with blocking rate constants (kon ) similar to alcohols and anesthetics [128,298,305]. The kon for dexamethasone (7.3U105 M31 s31 [305]) and hydrocortisone (5.4U105 M31 s31 [298]) are practically in the same range than that for n-alcohols (2.8^5.7U106 M31 s31 [307]), iso£urane (2.1U106 M31 s31 [308]), and benzocaine (2.6U106 M31 s31 [309]); (b) an equivalent model, where blocked channels may close, has been developed for both n-alcohols [307] and iso£urane [308] as well as for di¡erent steroids [128,298,305]. Thus, taking into account this similarity and considering that alcohols and anesthetics produce their pharmacological action by binding at the lipid^protein interface of several receptor classes (reviewed in [310]), it is postulated that steroids also

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interact with this domain. This localization is also supported by the fact that, (c) application of steroids from either the extracellular or the cytoplasmic surface of the AChR produces practically the same ion channel inhibition [298]. In addition, (d) the analgesic e¡ect of both progesterone and 3-K-androstanediol at moderate concentrations has been postulated to be performed via neuronal membrane actions [311]. Finally, (e) the e¡ect elicited by progesterone was observed to be voltage-independent, it was enhanced in the presence of agonists, and it was not mediated by the desensitization process [110,229]. In contrast, the e¡ect of hydrocortisone was slightly voltage-dependent and it was not conditioned by agonist concentration [128]. For instance, the decrease in the don of hydrocortisone-treated ion channels was approximately 50% and 30% at 3100 and 340 mV, respectively, when compared with the observed voltage dependence on control cells. Thus, this is consistent with steroid molecules binding inside the lipid membrane to a site(s) that senses the electric ¢eld. Aside from the two mentioned domains on the AChR associated with steroid binding, another plausible location for steroid molecules can be considered. Taking into account the observed high quenching e¤ciency of ASL on AChR-bound quinacrine £uorescence [219,220], and considering that paramagnetic quenching is a short range process [119,120], it is possible to account for the existence of non-annular steroid binding site(s) structurally related to the high-a¤nity quinacrine locus (see Section 8 for more details). This steroid location was also included in Fig. 7. From inhibitory studies by using several steroids with subtle di¡erences in their molecular structures it was possible to demonstrate certain structure-function relationships. (1) Since both hydrocortisone and its derivative 11-deoxycortisone showed the same inhibitory potency on AChR-containing BC3 H-1 cells, it is possible to conclude that the hydroxyl group attached at carbon 11 on hydrocortisone (see Fig. 4B) is not a requisite for steroid binding to the AChR [298]. (2) The fact that the forward rate constant of the hydrocortisone inhibitory process is qualitatively similar to both adult and embryogenic AChRs indicates that neither the O or the Q subunit is responsible for hydrocortisone binding [128]. Alternatively, the steroid binding site may be

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found on homologous sequences from both chains. (3) The fact that both progesterone and testosterone present practically the same potency as their 3-labelled derivatives (see Table 7) suggests that the carbonyl moiety at carbon 3 of the A ring (see Fig. 4B) is not indispensable for steroid binding to either the muscle- or the neuronal-type AChR. However, this carbonyl group seems to be necessary for steroid binding to the ganglionic-type K3L4 receptor (Table 7). The fact that neuroactive steroids, the so-called neurosteroids, have been found to be endogenously synthesized and accumulated in the central nervous system independently from peripheral sources [270] opens the possibility of a biologically relevant role for neurosteroids on synaptic modulation. However, since the endogenous concentration of neurosteroids that can be released from oligodendrocytes is in the order of 10^100 nM [312], and the observed IC50 s are in the range of 1^100 WM (see Table 7), there is still concern whether the elicited steroid e¡ect on the AChR is a physiologically relevant process or not. However, the steroid concentration in the plasma can be raised to levels between 1 and 20 WM under stress processes and/or during the estrous cycle and pregnancy of females (e.g., see [313]). Thus, this evidence together with the fact that steroids are molecules with the capacity of crossing the blood-brain barrier might explain, at least partially, how the steroid concentration changes in£uence cognitive skills and emotions (reviewed in [269]). 9.2.1. Di¡erences between cholesterol and steroid action The lack of inhibitory e¡ect of cholesterol in comparison with other steroids in the neuronal-type AChR (see Table 7) deserves a special consideration. There is a great body of experimental evidence indicating that (a) cholesterol augments the ion channel activity and preserves the a¤nity state transitions on the AChR [260,314^319]; and that (b) the abovementioned e¡ects are mediated by cholesterol interaction with the lipid^protein interface [320,321] which stabilizes the secondary structure of the AChR [317,322]. Moreover, cholesterol and several cholesterol analogues as well as the steroid androstanol showed a similar e¡ect on ion £ux response from di¡erent reconstituted systems containing the

muscle-type AChR [314]. Thus, we may suppose that the action of steroid molecules on the AChR is also mediated through the lipid^protein interface. In this regard, con£icting results between the steroid derivative ASL and the cholesterol analogue spin-labelled cholestane (CSL) have been found. For example, a V4-fold higher relative a¤nity for ASL than that for CSL was obtained by EPR measurements (Table 5). On the contrary, a 4.8 times higher quenching e¤ciency for CSL than that for ASL was observed on Torpedo native membrane intrinsic £uorescence (see Table 6). This latter di¡erence might be explained considering a distinct transversal position of both nitroxide-containing lipids relative to the plane of the membrane. In this regard, the nitroxide group of CSL has been considered to be close to the lipid^aqueous interface [255] which might allow CSL to interact with a large population of Trp residues at the extracellular portion of the AChR augmenting, in turn, its £uorescence e¤ciency. This is in agreement with the fact that CSL is accessible to a higher population of £uorophores than ASL (see fa values in Table 6). Although the EPR and the £uorescence spectroscopic behavior of both CSL and ASL are incongruent, the observed di¡erence between both derivatives might indicate the existence of subtle structural distinctions in the environment of each particular binding site. Experiments comparing the displacement e¡ect of local anesthetics on the AChR-ASL interactions, as was previously determined for CSL [262], would be of great utility to understand the above-mentioned apparent discrepancy. For example, the interaction of ASL with the AChR was not a¡ected by added ethanol [263]. The elicited ligand e¡ect on AChR-steroid interactions is also shown in Table 6. For example, the di¡erences in the 1/KQ values indicate that a lower concentration of CSL to quench an inferior fraction of AChR-bound Trp residues from the desensitized receptor is necessary. However, this e¡ect was not observed by using dibromocholesteryl-hemisuccinate (diBrCHS), a more water-soluble cholesterol analogue with two bromines as the quencher moiety. In addition, the observed fa value for the quenching of the AChR-labelled pyrene £uorescence by diBrCHS was higher in the desensitized than in the resting state (both in the absence of ligands or in the

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presence of K-BTx). However, this trend was not observed for diBrCHS quenching on AChR intrinsic £uorescence. This suggests that the conformational changes of the protein elicited at the lipid^protein interface are more pronounced in the region containing the QCys451 residue than that containing QTrp453 . This is in agreement with the measured distance î ) between both residues [123]. Interestingly, (V18 A although tyrosine phosphorylation was shown to be involved in the modulation of the desensitization rate, the structural alteration of the lipid^protein interface evidenced by di¡erent cholesterol derivatives in the desensitized AChR was not observed in the phosphorylated receptor [43]. There is no clear evidence indicating that both the cholesterol and the steroid binding site are structurally related. However, the spectroscopic (see Tables 5 and 6) and pharmacological (see Table 7) behavior of both kind of molecules allows us to suggest that both binding sites are neither functionally nor structurally connected. This seems to be also the case for the binding site of pregnanolone and cholesterol on the GABAA receptor [323]. Speci¢cally, the addition of more cholesterol in GABAA receptor-containing membranes from cerebral cortex reduced the e¡ect of pregnanolone as a potentiator of the binding of the benzodiazepine agonist [3 H]£unitrazepam. However, the results using GABAA receptor-containing membranes from cerebellum and spinal cord were not in accord with the previous experiment. 10. Agonist self-inhibition of the nicotinic acetylcholine receptor Various approaches such as electrophysiological and ion-£ux kinetic measurements have shown that agonist concentrations in the millimolar range reduce single-channel conductance, cause frequent closure of open channels and inhibit AChR-mediated cation £ux (reviewed in [73,124]). For instance, pyrantel, which at low concentration (0.03^100 WM) acts as an agonist on somatic muscle AChR from the parasite Ascaris suum, at high concentrations ( s 100 WM), this anthelminthic drug blocks the ion channel [324]. The simplest explanation for these pharmacological properties is that agonists at high concentrations may enter the ion channel and sterically block

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the cation £ux elicited by micromolar concentration of cholinergic agonists (see Fig. 5). 10.1. Similarity between non-competitive inhibition and agonist-self inhibition of the nicotinic acetylcholine receptor The hypothesis described above is supported by the similarity of agonist self-inhibition with AChR non-competitive inhibition. In particular, (1) the conductance inhibition elicited by agonists at high concentration or NCIs (see Section 5 for more details) is enhanced at hyperpolarized membrane potential; and (2) agonists at high concentration compete in a mutually exclusive manner with di¡erent NCIs. (1) The similarity in voltage dependence between non-competitive inhibition and agonist self-inhibition provides the basis for the notion that both kinds of organic cations may inhibit ion £ux via similar mechanisms (reviewed in [73,124]). However, the experimental evidence that membrane partitioning of charged drugs is also sensitive to transmembrane potential [325] can perfectly account for the observed voltage dependence of agonist self-inhibition. This is in concordance with the evidence that, although ethidium [326] and quinacrine [327,328] inhibit ion channel £ux in a voltage-dependent manner, they bind at non-luminal loci on the AChR (see Section 8). Finally, the ACh a¤nity for the blocking site was increased two-fold by mutating Tyr190 to Phe, a residue that is not located in the channel lumen but in the hydrophilic extracellular portion of the AChR [329]. (2) Agonists at high concentrations compete in a mutually exclusive manner with di¡erent classical NCIs such as the local anesthetic procaine, HTX, hexamethonium, decamethonium, TPMP‡ , TID, ethanol, ethidium, and quinacrine (reviewed in [73,124]). The fact that the value of the apparent agonist concentration which causes 50% self-inhibition (KB ) increased in the presence of procaine, and that the apparent procaine Kd increased linearly with the concentration of suberyldicholine (SubCh), indicated that both drugs might bind at the same site within the AChR ion channel [330]. However, evaluation of the kinetic constants of the ion translocation inhibition elicited by procaine and several agonists evidenced two di¡erent inhibitory binding sites: one for ACh and the other one for procaine

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[331,332]. The exact localization of the procaine binding site on the AChR is controversial (reviewed in [73,84,124]). However, the localization of other high-a¤nity NCI binding sites, both luminal and non-luminal are more known (see Sections 7 and 8). 10.2. Luminal localization for the agonist self-inhibitory binding site As was outlined in the previous section, the pharmacological similarities in inhibiting the ion channel found for NCIs and agonists at high concentrations as well as the observed agonist-induced displacement of NCI binding, supported the conjecture that both classes of ligands enter the ion pore, bind to a unique site located in the channel lumen and sterically occlude it (reviewed in [73,124]) (see Fig. 5). However, there is no direct evidence sustaining this hypothesis. For example, the only indication that suggests a stoichiometry of one single agonist self-inhibitory binding site per AChR is indirectly provided by the unitary value of the Hill coe¤cient [333], but particularly the Hill coe¤cient of nicotine self-inhibition was found to be about two [334]. A more direct evidence can be found by using di¡erent agonist derivatives (reviewed in [73,124]). For example, (a) the labelling of several Cys-substituted amino acids on the KM2 domain by an agonist sulfhydryl-reacting derivative, and (b) the agonist size dependence of the depth to which the molecules penetrate the membrane spanning region of the ion channel. For the ¢rst item (a), an attractive approach has been used to understand the structural basis of ion conduction and blocking [34,142]. Consecutive residues from Met243 to Val261 in M2 and amino acids Glu241 , Lys242 , and Glu262 £anking the domain M2 of the mouse muscle K subunit were mutated, one at a time, to Cys. The mutated K subunit was expressed in the presence of the wild-type L, Q, and N chains in Xenopus oocytes, giving robust responses to ACh (except for Lys242 CCys mutant). Furthermore, each new Cys residue was allowed to react with MTSEA. Since this agonist derivative has a diameter î and the open channel has a diameter of of V6 A î , and since these molecules are very hydroV7 A philic, they may have access to exposed residues all along the channel lumen. In comparison with the wild-type AChR, MTSEA labelling irreversibly in-

hibited the ACh-induced response on mutants Thr244 , Leu245 , Ser248 , Leu250 , Ser252 , Val255 , and Glu262 -expressing oocytes, with statistical signi¢cance. Interestingly, some of these residues were also labelled by luminal NCIs (see Section 6). Additionally, the MTSEA inhibition on Glu241 , Leu251 , Val255 , and Leu258 mutants was increased about 5 times when the channel was open at least part of the time by the presence of ACh. All these data suggest that the reactive agonist can reach its binding domain by ¢tting into the open channel. This is in agreement with the observed accumulation of ACh molecules in the ion path near the middle of the î resolution, AChR transmembrane domain at 9 A when an ACh solution was sprayed onto an aqueous ¢lm of AChR-containing tubular vesicles reaching ¢nal concentrations in the order of 1^10 mM [57]. However, residues from the KM1 region previously mutated by Cys were also labelled by MTSEA [35]. Regarding the second item (b), by using a series of derivatives of the agonist analogue 1,1-dimethyl4-acetyl-piperazinium iodide (PIP) and studying the kinetics of channel blocking, a linear increase in the depth to which the smaller derivatives penetrate the ion channel was evidenced [335]. Although the kon remained practically constant, the koff , and thus, the calculated KB values, decreased exponentially in concordance with the increased molecular size. When the d value is plotted as a function of van der Waals volume and the hydrophobic surface area for each agonist, the constancy of d for the smaller agonists PIP, ethyl-PIP and propyl-PIP, indicates that they traverse about 75% of the membrane ¢eld. The location of the selective ¢lter is in agreement with previous measurements (e.g., see [336]). A preliminary study to elucidate the localization of the agonist self-inhibitory site is described below. Photoactivatable agonist analogues were synthesized to study the agonist binding site on the AChR [337]. From these the potent muscle cholinergic agonist derivative [NP-methyl,NP-4-diazonium phenyl][N-8-octanoic acid, 2-(trimethylammonium bromide)ethyl ester]urea (AC7 ) could be used to determine which residues are involved in the agonist self-inhibitory binding site. This possibility is based on the steric blocking mechanism suggested in the following data: (1) AC7 competed for the PCP binding site with an apparent Ki of 35 WM in the presence of

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CCh; and (2) this ligand provoked a decrease in don with increasing concentrations. In addition, at concentrations higher than 10 WM, a rapid £ickering of the single-channel openings was observed, which is reminiscent of local anesthetic action. Since there is not direct evidence for the existence of a luminal binding site that accounts for the fast agonist inhibition, part of the hypothesis is only indirectly supported by the occurrence of high-a¤nity luminal NCI binding sites (see Section 6). However, the actual data based on photolabelling and quantitative £uorescence spectroscopy approaches are not congruent with the existence of only luminal binding sites but rather implicated non-luminal loci (see Section 8). In turn, the existence of non-luminal NCI binding sites do not support the simplest open-channel-blocking mechanism but points to an alternative allosteric mode of drug action. 10.3. Non-luminal localization for the agonist self-inhibitory binding site The existence of non-luminal NCI binding sites, and thus, the regulation of cation permeation by an allosteric process (see Section 8), opens the possibility for the existence of a similar mechanism for the agonist self-inhibition. We have approached this problem by studying the localization of the agonist self-inhibitory binding site for two non-luminal NCIs [231,338]. More precisely, we quanti¢ed the agonistinduced displacement of quinacrine and ethidium from their speci¢c binding sites and determined the accessibility of the spin-labelled analogue of ACh (AChSL) to these particular NCI sites. From these experiments, it became evident that: (1) quinacrine was 3- to 40-fold more sensitive to agonist-induced inhibition than ethidium binding [339]; (2) the agonist Ki values (in mM) follow the order: SubCh (9.3 þ 0.8)IACh (142 þ 48) 6 CCh (240 þ 67) [231]. Plotting these values against the respective KB s obtained from AChR vesicles in a 10-s 86 Rb‡ e¥ux assay with 80^85% of the K-BTx sites occupied at zero membrane potential (i.e., 3.0 þ 0.2I104 þ 23 6 244 þ 28 [333]), a slope of 1.03 was determined indicating a perfect correlation. Although the experiments of Forman et al. [333] differ in the AChR conformational state when compared to the desensitized form used in our experi-

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ments [231,339,340], the observed correlation suggests a structural relationship between both the quinacrine and the agonist self-inhibitory binding site. Finally, (3) assuming that the paramagnetic quenching of quinacrine is intrinsically 1.4^3.6-fold more e¤cient than ethidium £uorescence quenching [221], AChSL was approximately three times more accessible to the quinacrine than to the ethidium binding site [339]. These data suggest that agonists at high concentrations preferably bind to the quinacrine site (see Fig. 5). 10.3.1. Plausible mechanism of agonist binding to the quinacrine locus Since the quinacrine binding site is located at the lipid^protein interface (see Section 8) and since the agonists are quaternary amines which are generally thought to be membrane impermeable, it was unexpected to ¢nd that agonists preferentially bound to the quinacrine binding site. Thus, taking into account this evidence, we can expect a scenario where the agonist at high concentration is (1) adsorbed on the aqueous^lipid interface and subsequently (2) diffuses through (or onto) the lipid membrane (3) to reach its binding site located close to the quinacrine binding domain. Experimental evidence for this hypothesis has been previously addressed (reviewed in [73,124]). Along the same perspective, we examined the ability of various agonists to partition into AChR native membranes [340]. The observed agonist partition coe¤cients (Kp s) follow the order: AChSL s SubCh s s CChWACh, where the values for AChSL and SubCh are about two orders of magnitude higher than the Kp for CCh and ACh. This correlation was also observed in the agonist Ki values obtained by AChR-bound quinacrine displacement [339]. This relevant relationship indicates that agonists with higher ability to partition into lipid membrane are more successful in inhibiting quinacrine binding on the AChR. The direct relationship between Kp and Ki values strongly suggests that agonists at high concentrations may interact with the quinacrine binding site (or a site very close to the quinacrine domain) at the lipid^protein interface via a lipid membrane approach (see Fig. 5). To understand the basic mechanism of agonist binding to its inhibitory site we determined the ionic

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strength and temperature e¡ect on agonist-induced displacement of quinacrine from its high-a¤nity binding site by measuring the agonist Ki s at di¡erent experimental conditions [231]. Each particular agonist Ki value slightly increased as the temperature decreased from 15 to 4³C. Interestingly, this slight temperature e¡ect is consistent with the behavior of quinacrine binding which showed no change between 4 and 15³C (see Section 8). Additionally, the e¡ects of high concentrations of agonists on currents through AChR channel by using single-channel recording present the same temperature sensitivity [336]. We calculated the thermodynamic parameters in spite of the fact that the temperature e¡ect on agonist-induced displacement of quinacrine binding is very low (the slopes of the van't Ho¡ plots are not signi¢cantly di¡erent than zero) [231]. Qualitatively the results suggest that a low enthalpic barrier exists on the equilibrium binding of agonist at high concentration to the quinacrine locus. Moreover, the sign of the vH³ values calculated from the temperature dependence of agonist binding to the quinacrine locus is positive, indicating an endothermic reaction. A possible explanation for the observed di¡erence in the temperature e¡ect of quinacrine binding vs. agonist-induced inhibition of quinacrine binding is that, contrary to the partially restricted di¡usioncontrolled mechanism proposed for quinacrine binding at the lipid^protein interface of the AChR (see Section 8), agonist binding to the quinacrine locus is driven by a mechanism where no requirement in alignment between the agonist molecule and the self-inhibitory binding site at the lipid^protein interface is required. In other words, once agonists are in the lipid membrane the molecules can di¡use more freely into the crevice of the quinacrine binding site at high (e.g., 15³C) than at low (e.g., 4³C) temperatures. This is consistent with the fact that cholinergic agonists are smaller than the quinacrine molecule: the fully extended length of ACh is î whereas quinacrine is about 13 approximately 6 A î A. Additionally, the observed kon for ACh binding to its self-inhibitory site (V103 M31 s31 [334]) is in agreement with the existence of a non-di¡usion-limited barrier for the agonist inhibitory binding. One possibility is that the blocking site is partially hin-

dered at the lipid^protein interface of the AChR and thus, agonist molecules need ¢rst to be partitioned in (or adsorbed on) the lipid membrane to reach its binding site. Previous works suggested that agonists in the millimolar concentration range may bind to a site structurally related to the quinacrine locus located at the lipid^protein interface close to a nonannular lipid domain (see Section 8). The agonist-induced inhibition of quinacrine binding is also dependent of the ionic strength [231]. The Ki value for each particular agonist increased by increasing the NaCl concentration from 0 to 100 mM. This is consistent with the existence of an electrostatic component in the quinacrine locus. Acknowledgements I thank Dr. W. Sandberg for his help with the English language.

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