Pharmacology of Muscle-Type Nicotinic Receptors

C H A P T E R

34 Pharmacology of Muscle-Type Nicotinic Receptors Armando Alberola-Die, Rau´l Cobo, Isabel Ivorra, Andres Morales Division of Physiology, Department of Physiology, Genetics and Microbiology, Universidad de Alicante (Spain), Alicante, Spain

Abbreviations A ACh AM CCh CI ECD epp FCS IACh ICD LGIC nAChRm NAM NCI NMJ PAM SCS TMD

agonist acetylcholine allosteric modulator carbamylcholine competitive inhibitor extracellular domain end-plate potential fast-channel syndrome membrane current elicited by ACh intracellular domain ligand-gated ion channel muscle-type nicotinic acetylcholine receptor negative allosteric modulator noncompetitive inhibitor neuromuscular junction positive allosteric modulator slow-channel syndrome transmembrane domain

34.1 INTRODUCTION For over a century, some of the most brilliant neuroscientists (many of them are Nobel Prize laureates) have been involved in deciphering the mechanisms underlying synaptic transmission. For this purpose, the synapsis between a motor nerve and a striated muscle fiber, the neuromuscular junction (NMJ), became one of the most useful models. At the NMJ, muscle-type nicotinic acetylcholine (ACh) receptors (nAChRms) are key elements localized in the own muscle fiber. Likely, the first pharmacological study involving nAChRms was carried out in the 19th century by C. Bernard, who discovered the lack of skeletal muscle contraction after nerve stimulation in curarized frogs. Later on, Langley (1905) found that nicotine, an alkaloid present in Nicotiana tabacum leaves, induces potent contractions of fowl muscle, which are

Neuroscience of Nicotine https://doi.org/10.1016/B978-0-12-813035-3.00034-4

suppressed by curare; he concluded that “muscles have some accessory substance that combines with nicotine and curare.” Then, Dale, Feldberg, and Vogt (1936) showed that stimulation of motoneuron fibers in perfused voluntary muscles induces the appearance of ACh in the venous fluid, even when preventing the muscle contraction by curare. Soon after, Eccles, Katz, and Kuffler (1941) explored the neuromuscular transmission by extracellularly recording end-plate potentials (epps) and tested the effects of eserine, an anticholinesterase, on them. Afterward, Katz’s group studied in detail the characteristics of epps by recording muscle fibers with intracellular microelectrodes, establishing the basis for synaptic transmission (Fig. 34.1). Besides, they accurately released ACh by iontophoresis at the NMJ using ACh-filled micropipettes. By this localized ACh application, they could mimic the effect of nerve stimulation on muscle (Del Castillo & Katz, 1955) and mapped the distribution of nAChRms on the muscle fiber, which lead them to detect junctional and extrajunctional receptors (Miledi, 1960). A deeper knowledge of structural and functional properties of nAChRms, at a molecular level, has been possible thanks to several landmarks: (i) Introduction of the patchclamp technique by Neher and Sakmann (1976), which enables recording the activity of a single nAChRm. (ii) Biochemical purification of the nAChRm from Torpedo marmorata electroplaques by Miledi, Molinoff, and Potter (1971), using radioactive labeled α-bungarotoxin, a component of Bungarus multicinctus snake venom that specifically and irreversibly blocks the depolarizing effect of ACh on the NMJ (Lee & Tseng, 1966). Afterward, purified nAChRms were functionally reconstituted in artificial lipid matrices, allowing detailed studies of its activity either in cell-free models, as lipid bilayers (Nelson, Anholt, Lindstrom, & Montal, 1980) or in host cells, as

267

Copyright © 2019 Elsevier Inc. All rights reserved.

268

34. PHARMACOLOGY OF MUSCLE-TYPE NICOTINIC RECEPTORS

FIG. 34.1 Scheme of synaptic transmission at the neuromuscular junction (NMJ). (A) A motoneuron axon branches off and ends on the surface of each muscle fiber that it controls, at the NMJ. (B) Expanded view of the NMJ region indicated in (A), showing the presynaptic nerve terminal containing ACh vesicles and voltage-dependent Ca2+ channels involved in transmitter release. ACh is released to the synaptic cleft, rich in acetylcholinesterase, and eventually reaches junctional folds containing numerous (up to 20.000/μm2) junctional nAChRms. Scarce extrajunctional (fetal subtype) nAChRms are beyond this synaptic region.

Xenopus oocytes, after their microtransplantation (Morales et al., 1995). (iii) With the progress of molecular biology techniques, it was possible to solve the α-subunit sequence (Noda et al., 1982) and later to clone the cDNA coding for other nAChRm subunits (reviewed by Changeux & Edelstein, 2005), which paved the way for mutagenesis experiments addressed to unravel the functional role of key structural nAChRm residues. (iv) Highresolution electron microscopy of Torpedo electroplax membranes, in which nAChRms are densely packed, enabling to develop atomic-scale models of the nAChRm structure (Unwin, 2005), which have become crucial tools for in silico studies of nAChR function and modulation.

34.2 OVERALL STRUCTURE AND FUNCTION OF nAChRm The nAChRm is a ligand-gated ion channel (LGIC) belonging to the Cys-loop subfamily of receptors, which are involved in fast synaptic transmission. All of them share a pentameric structure, four transmembrane (M1–M4) hydrophobic regions (TMD) in each subunit, and a disulfide bond between two cysteine residues, forming a distinctive loop of 13 amino acids at the extracellular side. nAChRms from adult vertebrates are constituted by 2α1, 1β1, 1δ, and 1ε subunits, being the ε subunit (junctional type) equivalent to the γ subunit from Torpedo nAChRs. In embryonic and extrajunctional (Fig. 34.1) nAChRms, the ε

subunit is substituted by a different γ subunit. This heterogeneity of nAChRm subunit combinations is relevant, because it determines, at least in part, both their functional and pharmacological properties (see below). Structural models from high-resolution electron microscopy images of Torpedo nAChRm show its five subunits arranged around a central axis, constituting the channel pore, and both extracellular and intracellular domains (ECD and ICD, respectively) protruding from the membrane bilayer (Unwin, 2005; Fig. 34.2A1 and A2). The channel pore is lined by the M2 segments of each subunit (channel inner ring), whereas M4 segments constitute the outermost ring, interacting directly with bilayer lipids (see Fig. 34.2A2). In the ECD, the nAChRm has two ligand-binding (orthosteric) sites located at α-γ (or α-ε) and α-δ interfaces (Fig. 34.2A1). nAChRms are allosteric proteins that may adopt, at least, three different interconvertible conformational states (reviewed by Albuquerque, Pereira, Alkondon, & Rogers, 2009; Bouzat & Sine, 2017; Changeux, 2012; Fig. 34.2B). In the absence of agonist, the hydrophobic channel gate, located deep in the membrane, is closed, and the nAChRm is in the resting state. When ACh or other agonists, as carbamylcholine (CCh), bind to the orthosteric sites, the nAChRm quickly activates (in the microsecond range), and the channel opens, allowing the flux of, mainly, Na+ and K+ through the membrane, generating ionic currents (see Fig. 34.2C1 and C2). A prolonged exposure to ACh elicits a conformational

34.3 MECHANISMS OF nAChRm MODULATION

269

FIG. 34.2

Structure and function of nAChRms. (A) Lateral (A1), at the membrane plane, and top (A2), from the synaptic cleft, views of the nAChRm, showing its pentameric structure. Red square in A1 shows α-γ orthosteric binding site. Each subunit contains four transmembrane segments (M1–M4, shown as spirals in γ subunit of panel A2) arranged around the channel pore. (B) nAChRm can exchange between three functional states: (i) resting, without bound agonist and channel closed; (ii) active, after binding of two agonist molecules the channel opens; and (iii) desensitized, channel closes though agonist remains bound. (C) Functional activity of nAChRms monitored by recording single-channel (C1) or whole-cell membrane (C2) currents. Carbamylcholine (CCh) binding activates the nAChRm, eliciting channel openings (C1, discrete downward deflections at
80 mV). nAChRm desensitization is better shown in slower, whole-cell recordings; ACh-elicited current (IACh) decreases during steady presence of agonist because of desensitization, which increases with agonist concentration (C2). Bars above recordings indicate the time of agonist application, and downward deflections denote inward currents, here and thereafter. Panels A1 and A2 derived from Torpedo nAChRm structure (code 2BG9; Unwin, 2005). Recordings in this and subsequent figures are authors’ unpublished results from Torpedo nAChRms.

shift in the nAChRm called desensitized state (Thesleff, 1955), which is characterized by a nonconducting conformation with an enhanced affinity for the agonist. Most likely, there are several intermediate desensitized states, each one with its own kinetics, but the overall desensitization rate is markedly dependent on agonist concentration, increasing when ACh concentration rises (Fig. 34.2C2).

34.3 MECHANISMS OF nAChRm MODULATION nAChRms are relevant therapeutic targets, since their dysfunction is related to the genesis of several pathophysiological processes that lead to impaired motor activity, as occurs in diverse congenital myasthenic syndromes (reviewed by Kalamida et al., 2007). Hence, it is important to understand the mechanisms by which these receptors

are modulated by different molecules and to unravel their specific binding sites (reviewed by Chatzidaki & Millar, 2015), in order to develop new therapeutic agents. In the last few decades, it has been shown that a broad number of molecules, with heterogeneous chemical structures, modulate nAChRm function by their binding to different regions of this receptor and, therefore, acting through distinct mechanisms: (i) Competitive: mediated by molecules that bind into the orthosteric binding site (Fig. 34.3; site 1), interfering with the agonist-receptor interaction (see Table 34.1 for examples). Many of these molecules can also act as full or partial agonists. The pharmacological profile of all these molecules is characterized by the rightward shift of the doseresponse curve (Fig. 34.4A2 and B). (ii) Steric: elicited by molecules that interact with (a) residues at positions close to the orthosteric

270

34. PHARMACOLOGY OF MUSCLE-TYPE NICOTINIC RECEPTORS

FIG. 34.3 Main modulating sites at the nAChRm. All three nAChRm domains, extracellular (ECD), transmembrane (TMD), and intracellular (ICD), have relevant loci for its modulation. The main modulating sites are indicated on the nAChRm structure (same template as in Fig. 34.2A) and summarized on the right.

TABLE 34.1

binding sites in such a way that they hinder/restrict agonist binding to the orthosteric site (Fig. 34.3, site 2; Table 34.1), and (b) residues located into the channel pore when the receptor is in the active state, acting as open-channel blockers (Fig. 34.3, site 6). Steric blockers elicit a noncompetitive nAChRm inhibition (NCI; Fig. 34.4A2 and B); nevertheless, open-channel blockers are continuously associating and dissociating from their interacting site, causing a dynamic blockade, which is observed as “flickering” when recording single-channel currents (Neher & Steinbach, 1978). Usually, the open-channel blockade is exerted by charged molecules at physiological pH, as tacrine, edrophonium, or fluoxetine (Table 34.1), and their extent of blockade is largely dependent on the cell membrane potential (Fig. 34.4A1 and A2). (iii) Allosteric: molecules that interact at loci distinct from orthosteric sites and modify the functional activity of nAChRms. They are classified as either negative

Selected Molecules That Modulate nAChRm Function

Category

Molecules

Action(s)/binding site(s)

Potency

Reference(s)

Muscle relaxants

Succinylcholine

A; (NCI)/1, 5, 6

EC50 ¼ 10.8 μM; IC50 ¼ 126 μM

Jonsson et al. (2006)

Pancuronium

CI/1

IC50 ¼ 15 nM

Liu and Dilger (2009)

Tacrine

NAM (NCI)/3, 6

IC50 ¼ 1.6–4.6 μM

Prince, Pennington, and Sine (2002)

Edrophonium

NAM (NCI)/6

IC50 ¼ 10 μM

Olivera-Bravo, Ivorra, and Morales (2007)

Physostigmine

PAM; NAM/2, 3

IC50 ¼ 10 mM

Hamouda, Kimm, and Cohen (2013)

Galanthamine

Acetylcholinesterase inhibitors

Cations

Endogenous molecules

Antimalarials

PAM; NAM/2, 3

IC50 ¼ 2.8 mM

Hamouda et al. (2013)

2+

NAM/4

0.1–1 mM range

Ochoa et al. (1989)

2+

Zn

PAM/3

Tested concentration ¼ 200 μM

García-Colunga, VázquezGómez, and Miledi (2004)

Progesterone

NAM (NCI)/3, 7, 8, 9

IC50 ¼ 1.0–6.1 μM

Ke and Lukas (1996)

Estradiol

NAM (NCI)/3, 7, 8, 9

IC50 ¼ 20–56 μM

Ke and Lukas (1996)

Corticosterone

NAM (NCI)/3, 7, 8, 9

IC50 ¼ 30–92 μM

Ke and Lukas (1996)

Cholesterol

AM/7, 8, 9

Barrantes (2004)

Substance P

NAM (NCI)/5

Arias (1997)

Serotonin

NAM (NCI)/6

Arias (1997)

Fatty acids

NAM (NCI)/8, 9

Barrantes (2004)

Protein kinase A

NAM/10

Hoffman, Ravindran, and Huganir (1994)

Protein kinase C

NAM/10

Ochoa et al. (1989)

Quinacrine

NAM (NCI)/8, 9

10–100 μM

Arias (1997) and Kaldany and Karlin (1983)

Quinine

NAM (NCI)/3, 6

50 μM

Sieb, Milone, and Engel (1996)

Ca

271

34.3 MECHANISMS OF nAChRm MODULATION

TABLE 34.1

Selected Molecules That Modulate nAChRm Function—cont’d

Category

Molecules

Action(s)/binding site(s)

Potency

Reference(s)

Antibiotics

Gentamicin

NAM (NCI, CI)/1, 4

IC50 ¼ 25 μM

Amici, Eusebi, and Miledi (2005)

Penicillins

NAM (NCI, CI)/1, 6

IC50 ¼ 0.71 mM

Schlesinger, Krampfl, Haeseler, Dengler, and Bufler (2004)

Chlorpromazine

NAM (NCI)/4, 6

IC50 > 300 nM

Changeux and Edelstein (2005)

Fluoxetine

NAM (NCI)/3, 5

Tested concentration ¼ 2 μM

García-Colunga, Awad, and Miledi (1997)

Bupropion

NAM (NCI)/3, 4, 5, 6

IC50 ¼ 0.40–40.1 μM

Arias et al. (2009)

Imipramine

NAM (NCI)/5

Ki ¼ 0.85–3.8 μM

Sanghvi et al. (2008)

Propofol

NAM (NCI)/5, 6, 7, 9

IC50 ¼ 40–125 μM

Jayakar, Dailey, Eckenhoff, and Cohen (2013)

Isoflurane

NAM (NCI)/5, 6

Kd ¼ 0.36 mM

Arias and Bhumireddy (2005)

Ketamine

NAM (NCI, CI)/2, 5, 6

Kd ¼ 2 μM

Scheller et al. (1996)

Pentobarbital

NAM (NCI, CI)/1, 2, 6

Kd ¼ 15–30 μM

Krampfl, Schlesinger, Dengler, and Bufler (2000)

Procaine

NAM (NCI)/6, 8

Kd ¼ 690–790 μM

Arias and Bhumireddy (2005)

Lidocaine

NAM (NCI)/3, 4, 7, 9, 5, 6

IC50 ¼ 70 μM

Alberola-Die et al. (2011)

Proadifen

NAM (NCI, CI)/1, 3, 4

IC50 ¼ 19 μM

Spitzmaul, Gumilar, Dilger, and Bouzat (2009)

Adiphenine

NAM (NCI)/4, 5

IC50 ¼ 15 μM

Spitzmaul et al. (2009)

Anatoxin A

A/1

EC50 ¼ 50 nM

Wonnacott and Barik (2007)

Histrionicotoxins

NAM (NCI)/4, 6

Ki 0.1–1 μM

Changeux and Edelstein (2005)

D-Tubocurarine

CI/1

IC50 ¼ 50–100 nM

Arias (1997) and Wonnacott and Barik (2007)

α-Bungarotoxin

CI/1

Tested concentrations ¼ 0.01–10 nM

Wonnacott and Barik (2007)

α-Conotoxin

CI/1

Kd ¼ 0.1–1 nM

Wonnacott and Barik (2007)

Nicotine

A/1

ED20 ¼ 20 μM

Wonnacott and Barik (2007)

Epibatidine

A/1

Tested concentrations 2–300 μM

Prince and Sine (1998)

Strychnine

NCI/3

IC50 ¼ 7.3 μM

García-Colunga and Miledi (1999)

Mecamylamine

NCI/5, 6

1–10 μM up to 100 μM

Varanda et al. (1985)

Tetraethylammonium

A; NAM (CI)/1, 3, 6

IC50 ¼ 2–3 mM

Akk and Steinbach (2003)

Diethylamine

NAM/2, 3, 6, 7

IC50 ¼ 70 μM

Alberola-Die, FernándezBallester, González-Ros, Ivorra, and Morales (2016a)

2,6-Dimethylaniline

NAM/4, 9, 5, 6

IC50 ¼ 2.1 mM

Alberola-Die, FernándezBallester, González-Ros, Ivorra, and Morales (2016b)

Antipsychotics

General anesthetics

Local anesthetics

Toxins

Other amine/ammonium compounds

Some of the most representative modulators of nAChRm (grouped by their therapeutic action or structure) are listed. Their main effects, putative acting site(s), and potencies, according to the listed references, are indicated.

272

34. PHARMACOLOGY OF MUSCLE-TYPE NICOTINIC RECEPTORS

FIG. 34.4 Mechanisms of nAChRm blockade. (A) Recordings illustrate different mechanisms of nAChRm blockade: (A1) Voltage-dependent (open-channel) blockade, usually elicited by charged molecules bound inside the channel pore. At
60 mV (brown trace indicates the imposed voltage potential), the blocker diminishes IACh (compare black and red recordings), but blockade vanishes at +40 mV. (A2) Noncompetitive blockers, at their IC50, reduce control IACh (black recording) to one-half (red recording); competitive (blue recording) and apparent-competitive (green recording) blockers decrease IACh depending on ACh concentration; a larger and complex IACh inhibition is elicited by molecules acting both as closed- and open-channel blockers when they are preapplied and then coapplied with the agonist (orange recording). (A3) Modifying the rate of nAChRm desensitization, IACh desensitization elicited by ACh (black recording) is markedly enhanced when ACh is coapplied with the modulator (purple recording). (B) Models of dose-response curves elicited by ACh either alone (black) or together with modulating compounds acting by different mechanisms: a noncompetitive way (red curve), for instance, when drugs bind into the channel pore; a competitive manner (blue curve) when molecules interact at the orthosteric site; an apparent-competitive mechanism (green curve) elicited by molecules acting by open- and closed-channel blockade (see text for details); finally, certain molecules interact with nAChRms at different sites, causing a large and complex inhibition (orange curve).

allosteric modulators (NAMs) or positive allosteric modulators (PAMs) whether they decrease or enhance nAChRm function, respectively. These modulators can act by binding to different nAChRm locations: (a) ECD acting modulators: these molecules bind to residues located at the ECD, and hence, they may act on resting-state nAChRm, triggering a conformational change that can either hinder or promote nAChRm opening (Fig. 34.3; site 3) or modify its desensitization (Fig. 34.3, site 4; Fig. 34.4A3; Table 34.1). NAMs acting at these

sites do not significantly shift the dose-response curves and typically behave as NCI, eliciting a closed-channel blockade (Fig. 34.4A2 and B). However, some of them show an “apparentcompetitive” pharmacological profile when coapplied with the agonist, slightly rightward shifting the dose-response curves (Fig. 34.4A2 and B). This is so when the NAM mainly evokes closed-channel blockade, but it also induces open-channel blockade. Thus, the higher the ACh concentration, the more nAChRms get open, and hence, the fewer remain in the resting

34.4 DIFFERENT THERAPEUTIC DRUGS MODULATE nAChRm FUNCTION

state and are prone to the blocker action outside the pore. Accordingly, an increase in ACh concentration will decrease the extent of inhibition, resembling a competitive mechanism of inhibition, as it happens for lidocaine (Alberola-Die, Martinez-Pinna, González-Ros, Ivorra, & Morales, 2011; Table 34.1). (b) TMD acting modulators: they are mostly hydrophobic molecules, which bind to residues located either at intrasubunit cavities among M1–M4 segments (Fig. 34.3, site 7) or at intersubunit crevices (Fig. 34.3, site 9; Table 34.1). Besides, these hydrophobic molecules can diffuse through the membrane and interact with amino acids located at the lipid-protein interface (Barrantes, 2004; Fig. 34.3, site 8). Furthermore, some TMD modulators can modify nAChRm desensitization by interacting with residues located into the ion channel, at specific positions near the extracellular side (Arias, 2010; Fig. 34.3, site 5). (c) ICD acting modulators: these molecules modify nAChRm function by binding to intracellular residues (Fig. 34.3, site 10). At least, some of them change the desensitization by acting through an indirect pathway, involving the generation of intracellular signaling molecules that phosphorylate ICD residues at the nAChRm (Ochoa, Chattopadhyay, & McNamee, 1989). Owing to the presence of different functional groups in the chemical structure of many ligands, not all the abovementioned mechanisms of action are mutually exclusive. Actually, a single molecule can bind to the nAChRm at distinct and even distant sites, giving rise to complex pharmacological profiles, which depend on both the way of application and the concentration of modulator used. Moreover, nAChRm modulators can attain their binding sites through two different and nonexclusive routes (Hille, 1977; Fig. 34.5): first, a hydrophilic pathway, used by polar ligands to interact with residues located at either the ECD or the channel pore (Fig. 34.5, red arrows), and second, a hydrophobic way, followed by nonpolar and lipophilic molecules to bind nAChRm residues at different TMD regions, including lipidprotein interface, and the ICD (Fig. 34.5, blue arrows). The path followed by each molecule to act on nAChRms mainly depends on the pKa of the modulator and the pH at the receptor environment, since both determine the molecule protonation. Many amphipathic molecules, as the local anesthetic lidocaine, are partially protonated at physiological pH, and so, they can follow both pathways to reach their binding sites.

273

FIG. 34.5 Scheme of hydrophilic and hydrophobic pathways of drug action. Many molecules modulating nAChRms are amphipathic; thus, charged and uncharged forms are present in the medium. Charged (protonated) molecules mostly reach their binding sites by the hydrophilic pathway (red arrows), whereas uncharged forms mainly follow the hydrophobic pathway (blue arrows). The proportion of each form is dependent on the molecule pKa and the medium pH; in the scheme, the balance “senses” the molecule pKa, rising the percentage of charged molecules as pKa increases.

34.4 DIFFERENT THERAPEUTIC DRUGS MODULATE nAChRm FUNCTION Regarding functional modulation of nAChRms, three characteristics of the NMJ should be considered: (i) The presence of two types of nAChRms differentially expressed in fetal and adult life. Junctional and extrajunctional nAChRms differ in both functional and pharmacological properties. Thus, the junctional subtype shows larger channel conductance and shorter mean-open time (Katz & Miledi, 1972; Neher & Sakmann, 1976) and, furthermore, faster desensitization (Morales & Sumikawa, 1992) than extrajunctional ones. Moreover, extrajunctional nAChRms are selectively blocked by αA-conotoxin OIVB, purified from Conus obscurus venom, since its affinity for the junctional subtype is almost two thousand times lower (Teichert et al., 2005). (ii) The large amplitude of epps, which confers a very high safety factor for signal transmission and makes the NMJ a singular synapsis. (iii) nAChRms are directly exposed to circulating compounds (including

274

34. PHARMACOLOGY OF MUSCLE-TYPE NICOTINIC RECEPTORS

toxins), in contrast to neuronal nAChRs, which are partially protected by either blood-brain or blood-nerve barriers. Notably, a large number of heterogeneous molecules, many amphipathic, interact with nAChRm modulating its function. Among them (Table 34.1), there are endogenous molecules (including hormones) and numerous compounds commonly used in clinical practice, as muscle relaxants, acetylcholinesterase inhibitors, antibiotics, antimalarials, antipsychotics, and local and general anesthetics, many of them acting in the micromolar range (Table 34.1). Besides, plenty of toxins, amine/ammonium compounds, and other molecules have powerful effects as either NAM or PAM on nAChRms. Even more, the action of certain molecules on nAChRms is markedly concentration-dependent, most likely because they act upon several modulating sites with different affinities and, therefore, they can elicit diverse, even antagonistic, effects.

34.5 FUTURE PERSPECTIVES Further studies on allosteric modulation of nAChRms are still required to better understand the precise mechanisms by which modulators act on nAChRms. This knowledge is relevant to reduce/prevent certain side effects triggered by different therapeutic molecules. For instance, dexamethasone is widely used to ameliorate patients suffering myasthenia gravis because of its immunosuppressive action; nevertheless, at first, this synthetic glucocorticoid might inhibit nAChRs, leading to worsening patient symptoms. Furthermore, these studies are fundamental to establish the bases to develop new therapeutic molecules, more proficient and with fewer side effects, to treat pathophysiological processes related with nAChRms dysfunctions.

MINI-DICTIONARY OF TERMS Acetylcholine activated current (IACh) Ionic membrane current elicited by ACh, acting on nAChRms. Acetylcholinesterase Esterase located at the synaptic cleft that cleaves ACh. Allosteric modulator Molecule that binds outside the nAChRm orthosteric sites and modifies its function. They are classified as negative or positive allosteric modulators, depending if they decrease or enhance nAChRm performance, respectively. Closed-channel blocker Prevents the receptor from being activated by the agonist. Desensitization Reduction of nAChRm response when steadily exposed to agonist. Ligand-gated ion channel (LGIC) receptor Membrane ion channel activated by specific ligands, which is involved in fast synaptic transmission. Muscular-type nicotinic receptor (nAChRm) Heterologous pentameric (2α1, 1β1, 1ε, and 1δ subunits) membrane protein activated by ACh, present in adult neuromuscular junction (synaptic-type)

or in fetal, extrasynaptic, or denervated muscle (composed by γ instead of ε subunit). Myasthenia Muscle weakness that can be elicited by different nAChRm dysfunctions. Open-channel blocker Molecule that binds into the pore, plugging the open channel. Orthosteric binding site Loci where the agonist specifically binds to activate the receptor.

Key Facts of Desensitization • First described by Thesleff (1955) for nAChRm. • When steadily exposed to the agonist, nAChRm switches to a high-affinity ligand-binding state, which is nonconducting (closed state). • ACh and some partial agonists evoke nAChRm desensitization, this increasing with agonist concentration. • Not all LGICs desensitize. The rate of nAChR desensitization is largely dependent on the subunit composition. • There are, at least, two desensitization states of the nAChRm, with their own kinetic constants. • nAChRm recovery from desensitization might last seconds or even minutes after agonist withdrawal. • nAChRm modulators affect desensitization rates by acting at extracellular, transmembrane, or intracellular loci. • Some negative allosteric modulators increase nAChRm desensitization, whereas certain positive allosteric modulators decrease or even prevent it. • Some congenital myasthenic syndromes modify nAChRm desensitization.

Key Facts of nAChRm Disorders • Most nAChRm dysfunctions cause myasthenia, that is, muscle weakness. • Some toxins (α-bungarotoxin and α-conotoxin) or alkaloids (D-tubocurarine) act on nAChRm leading to severe myasthenia or even paralysis. • Myasthenia might be triggered by genetic alterations in one or several nAChRm subunits (often causing either slow- or fast-channel syndrome) or by autoimmune reduction in either the number of postsynaptic nAChRms (myasthenia gravis) or the amount of ACh released (Lambert-Eaton syndrome). • Muscle weakness associated with slow-channel syndrome (SCS) is caused by prolonged postsynaptic depolarization, elicited by delayed channel closure, decreased desensitization, or enhanced affinity for ACh. • Certain open-channel blockers of nAChRm (fluoxetine and quinidine) ameliorate myasthenia due to SCS, whereas cholinesterase inhibitors exacerbate it (Rodríguez Cruz, Palace, & Beeson, 2014).

REFERENCES

• Patients suffering myasthenia by fast-channel syndrome (FCS) have reduced end-plate potentials because of subunit mutations eliciting a slow channel opening, reduced open-channel probability, short open dwell-time, enhanced desensitization, or decreased ACh-binding affinity. • Anticholinesterase drugs (pyridostigmine) attenuate both FCS and myasthenia gravis symptoms. • The K+-channel blocker 3,4-diaminopyridine is useful for the treatment of myasthenia elicited by both FCS and Lambert-Eaton syndrome. Summary Points • Two subtypes of muscle nicotinic acetylcholine receptors (nAChRms) are differentially expressed throughout the life. • nAChRms are key elements for synaptic transmission at the neuromuscular junction. • A broad number of compounds, including endogenous molecules and widely used therapeutic drugs, interact with nAChRms. • nAChRms can be modulated by competitive, steric, and allosteric mechanisms. • A single molecule might modulate nAChRms by different mechanisms and by its binding to different loci. • Allosteric modulators can decrease or enhance nAChRm activity. • A better knowledge of nAChRm modulation will help to develop new therapeutic molecules addressed to treat diseases by nAChRm dysfunctions.

Acknowledgment R.C. held a predoctoral fellowship from Universidad de Alicante (FPUUA36).

References Akk, G., & Steinbach, J. H. (2003). Activation and block of mouse muscle-type nicotinic receptors by tetraethylammonium. The Journal of Physiology, 551(1), 155–168. https://doi.org/10.1113/ jphysiol.2003.043885. Alberola-Die, A., Fernández-Ballester, G., González-Ros, J. M., Ivorra, I., & Morales, A. (2016a). Muscle-type nicotinic receptor blockade by diethylamine, the hydrophilic moiety of lidocaine. Frontiers in Molecular Neuroscience, 9, 12. https://doi.org/10.3389/fnmol.2016.00012. Alberola-Die, A., Fernández-Ballester, G., González-Ros, J. M., Ivorra, I., & Morales, A. (2016b). Muscle-type nicotinic receptor modulation by 2,6-dimethylaniline, a molecule resembling the hydrophobic moiety of lidocaine. Frontiers in Molecular Neuroscience, 9, 127. https://doi. org/10.3389/fnmol.2016.00127. Alberola-Die, A., Martinez-Pinna, J., González-Ros, J. M., Ivorra, I., & Morales, A. (2011). Multiple inhibitory actions of lidocaine on Torpedo nicotinic acetylcholine receptors transplanted to Xenopus oocytes. Journal of Neurochemistry, 117(6), 1009–1019. https://doi. org/10.1111/j.1471-4159.2011.07271.x. Albuquerque, E. X., Pereira, E. F., Alkondon, M., & Rogers, S. W. (2009). Mammalian nicotinic acetylcholine receptors: from structure to

275

function. Physiological Reviews, 89(1), 73–120. https://doi.org/ 10.1152/physrev.00015.2008. Amici, M., Eusebi, F., & Miledi, R. (2005). Effects of the antibiotic gentamicin on nicotinic acetylcholine receptors. Neuropharmacology, 49(5), 627–637. https://doi.org/10.1016/j.neuropharm.2005.04.015. Arias, H. R. (1997). Topology of ligand binding sites on the nicotinic acetylcholine receptor. Brain Research Reviews, 25(2), 133–191. https:// doi.org/10.1016/S0165-0173(97)00020-9. Arias, H. R. (2010). Positive and negative modulation of nicotinic receptors. Advances in Protein Chemistry and Structural Biology, 80, 153–203. https://doi.org/10.1016/B978-0-12-381264-3.00005-9. Arias, H. R., & Bhumireddy, P. (2005). Anesthetics as chemical tools to study the structure and function of nicotinic acetylcholine receptors. Current Protein & Peptide Science, 6(5), 451–472. https://doi.org/ 10.2174/138920305774329331. Arias, H. R., Gumilar, F., Rosenberg, A., Targowska-Duda, K. M., Feuerbach, D., Jozwiak, K., et al. (2009). Interaction of bupropion with muscle-type nicotinic acetylcholine receptors in different conformational states. Biochemistry, 48(21), 4506–4518. https://doi. org/10.1021/bi802206k. Barrantes, F. J. (2004). Structural basis for lipid modulation of nicotinic acetylcholine receptor function. Brain Research Reviews, 47(1–3), 71–95. https://doi.org/10.1016/j.brainresrev.2004.06.008. Bouzat, C., & Sine, S. M. (2017). Nicotinic acetylcholine receptors at the single-channel level. British Journal of Pharmacology. https://doi.org/ 10.1111/bph.13770. Changeux, J. P. (2012). The nicotinic acetylcholine receptor: the founding father of the pentameric ligand-gated ion channel superfamily. The Journal of Biological Chemistry, 287(48), 40207–40215. https://doi. org/10.1074/jbc.R112.407668. Changeux, J. P., & Edelstein, S. J. (2005). Nicotinic acetylcholine receptors: From molecular biology to cognition. New York: Odile Jacob Publishing Corporation. Chatzidaki, A., & Millar, N. S. (2015). Allosteric modulation of nicotinic acetylcholine receptors. Biochemical Pharmacology, 97(4), 408–417. https://doi.org/10.1016/j.bcp.2015.07.028. Dale, H. H., Feldberg, W., & Vogt, M. (1936). Release of acetylcholine at voluntary motor nerve endings. The Journal of Physiology. 86, https:// doi.org/10.1113/jphysiol.1936.sp003371. Del Castillo, J., & Katz, B. (1955). On the localization of acetylcholine receptors. The Journal of Physiology, 128(1), 157–181. https://doi. org/10.1113/jphysiol.1955.sp005297. Eccles, J. C., Katz, B., & Kuffler, S. W. (1941). Nature of the “end-plate potential” in curarized muscle. Journal of Neurophysiology, 4(5), 362–387. García-Colunga, J., Awad, J. N., & Miledi, R. (1997). Blockage of muscle and neuronal nicotinic acetylcholine receptors by fluoxetine (Prozac). Proceedings of the National Academy of Sciences of the United States of America, 94(5), 2041–2044. García-Colunga, J., & Miledi, R. (1999). Modulation of nicotinic acetylcholine receptors by strychnine. Proceedings of the National Academy of Sciences of the United States of America, 96(7), 4113–4118. https:// doi.org/10.1073/pnas.96.7.4113. García-Colunga, J., Vázquez-Gómez, E., & Miledi, R. (2004). Combined actions of zinc and fluoxetine on nicotinic acetylcholine receptors. The Pharmacogenomics Journal, 4(6), 388–393. https://doi.org/ 10.1038/sj.tpj.6500275. Hamouda, A. K., Kimm, T., & Cohen, J. B. (2013). Physostigmine and galanthamine bind in the presence of agonist at the canonical and non-canonical subunit interfaces of a nicotinic acetylcholine receptor. The Journal of Neuroscience, 33(2), 485–494. https://doi.org/10.1523/ JNEUROSCI.3483-12.2013. Hille, H. R. (1977). Local anesthetics: hydrophilic and hydrophobic pathways for the drug-receptor reaction. The Journal of General Physiology, 69(4), 497–515. https://doi.org/10.1085/jgp.69.4.497. Hoffman, P. W., Ravindran, A., & Huganir, R. L. (1994). Role of phosphorylation in desensitization of acetylcholine receptors expressed in Xenopus oocytes. The Journal of Neuroscience, 14(7), 4185–4195.

276

34. PHARMACOLOGY OF MUSCLE-TYPE NICOTINIC RECEPTORS

Jayakar, S. S., Dailey, W. P., Eckenhoff, R. G., & Cohen, J. B. (2013). Identification of propofol binding sites in a nicotinic acetylcholine receptor with a photoreactive propofol analog. The Journal of Biological Chemistry, 288(9), 6178–6189. https://doi.org/10.1074/ jbc.M112.435909. Jonsson, M., Dabrowski, M., Gurley, D. A., Larsson, O., Johnson, E. C., Fredholm, B. B., et al. (2006). Activation and inhibition of human muscular and neuronal nicotinic acetylcholine receptors by succinylcholine. Anesthesiology, 104, 724–733. Kalamida, D., Poulas, K., Avramopoulou, V., Fostieri, E., Lagoumintzis, G., Lazaridis, K., et al. (2007). Muscle and neuronal nicotinic acetylcholine receptors. Structure, function and pathogenicity. The FEBS Journal, 274(15), 3799–3845. https://doi.org/10.1111/ j.1742-4658.2007.05935.x. Kaldany, R. R., & Karlin, A. (1983). Reaction of quinacrine mustard with the acetylcholine receptor from Torpedo californica. The Journal of Biological Chemistry, 258(10), 6232–6242. Katz, B., & Miledi, R. (1972). The statistical nature of the acetylcholine potential and its molecular components. The Journal of Physiology, 224(3), 665–699. https://doi.org/10.1113/jphysiol.1972.sp009918. Ke, L., & Lukas, R. J. (1996). Effects of steroid exposure on ligand binding and functional activities of diverse nicotinic acetylcholine receptor subtypes. Journal of Neurochemistry, 67(3), 1100–1112. https://doi. org/10.1046/j.1471-4159.1996.67031100.x. Krampfl, K., Schlesinger, F., Dengler, R., & Bufler, J. (2000). Pentobarbital has curare-like effects on adult-type nicotinic acetylcholine receptor channel currents. Anesthesia and Analgesia, 90(4), 970–974. Langley, J. N. (1905). On the reaction of cells and of nerve-endings to certain poisons, chiefly as regards the reaction of striated muscle to nicotine and to curari. The Journal of Physiology, 33(4–5), 374–413. https://doi.org/10.1113/jphysiol.1905.sp001128. Lee, C. Y., & Tseng, L. F. (1966). Distribution of Bungarus multicinctus venom following envenomation. Toxicon, 3(4), 281–290. https:// doi.org/10.1016/0041-0101(66)90076-6. Liu, M., & Dilger, J. P. (2009). Site selectivity of competitive antagonists for the mouse adult muscle nicotinic acetylcholine receptor. Molecular Pharmacology, 75(1), 166–173. https://doi.org/10.1124/ mol.108.051060. Miledi, R. (1960). Junctional and extra-junctional acetylcholine receptors in skeletal muscle fibres. The Journal of Physiology, 151(1), 24–30. https://doi.org/10.1113/jphysiol.1960.sp006417. Miledi, R., Molinoff, P., & Potter, L. T. (1971). Isolation of the cholinergic receptor protein of Torpedo electric tissue. Nature, 229(5286), 554–557. https://doi.org/10.1038/229554a0. Morales, A., Aleu, J., Ivorra, I., Ferragut, J. A., González-Ros, J. M., & Miledi, R. (1995). Incorporation of reconstituted acetylcholine receptors from Torpedo into the Xenopus oocyte membrane. Proceedings of the National Academy of Sciences of the United States of America, 92(18), 8468–8472. Morales, A., & Sumikawa, K. (1992). Desensitization of junctional and extrajunctional nicotinic ACh receptors expressed in Xenopus oocytes. Brain Research Molecular Brain Research, 16(3–4), 323–329. https://doi.org/10.1016/0169-328X(92)90242-4. Neher, E., & Sakmann, B. (1976). Noise analysis of drug induced voltage clamp currents in denervated frog muscle fibres. The Journal of Physiology, 258(3), 705–729. https://doi.org/10.1113/jphysiol.1976. sp011442. Neher, E., & Steinbach, J. H. (1978). Local anaesthetics transiently block currents through single acetylcholine-receptor channels. The Journal of Physiology, 277, 153–176. https://doi.org/10.1113/jphysiol.1978. sp012267. Nelson, N., Anholt, R., Lindstrom, J., & Montal, M. (1980). Reconstitution of purified acetylcholine receptors with functional ion channels in planar lipid bilayers. Proceedings of the National Academy of Sciences of the United States of America, 77(5), 3057–3061.

Noda, M., Takahasi, H., Tanabe, T., Toyosato, M., Furutani, Y., Hirose, T., et al. (1982). Primary structure of α-subunit precursor of Torpedo californica acetylcholine receptor deduced from cDNA 793–797. https://doi.org/ sequence. Nature, 299(5886), 10.1038/299793a0. Ochoa, E. L., Chattopadhyay, A., & McNamee, M. G. (1989). Desensitization of the nicotinic acetylcholine receptor. Molecular mechanisms and effect of modulators. Cellular and Molecular Neurobiology, 9(2), 141–178. https://doi.org/10.1007/BF00713026. Olivera-Bravo, S., Ivorra, I., & Morales, A. (2007). Diverse inhibitory actions of quaternary ammonium cholinesterase inhibitors on Torpedo nicotinic ACh receptors transplanted to Xenopus oocytes. British Journal of Pharmacology, 151(8), 1280–1292. https://doi.org/10.1038/ sj.bjp.0707329. Prince, R. J., Pennington, R. A., & Sine, S. M. (2002). Mechanism of tacrine block at adult human muscle nicotinic acetylcholine receptors. The Journal of General Physiology, 120(3), 369–393. https://doi.org/ 10.1085/jgp.20028583. Prince, R. J., & Sine, S. M. (1998). Epibatidine activates muscle acetylcholine receptors with unique site selectivity. Biophysical Journal, 75(4), 1817–1827. https://doi.org/10.1016/S0006-3495(98)77623-4. Rodríguez Cruz, P. M., Palace, J., & Beeson, D. J. (2014). Inherited disorders of the neuromuscular junction: an update. Journal of Neurology, 261(11), 2234–2243. https://doi.org/10.1007/s00415-014-7520-7. Sanghvi, M., Hamouda, A. K., Jozwiak, K., Blanton, M. P., Trudell, J. R., & Arias, H. R. (2008). Identifying the binding site(s) for antidepressants on the Torpedo nicotinic acetylcholine receptor: [3H]2azidoimipramine photolabeling and molecular dynamics studies. Biochimica et Biophysica Acta, 1778(12), 2690–2699. https://doi.org/ 10.1016/j.bbamem.2008.08.019. Scheller, M., Bufler, J., Hertle, I., Schneck, H. J., Franke, C., & Kochs, E. (1996). Ketamine blocks currents through mammalian nicotinic acetylcholine receptor channels by interaction with both the open and the closed state. Anesthesia and Analgesia, 83(4), 830–836. Schlesinger, F., Krampfl, K., Haeseler, G., Dengler, R., & Bufler, J. (2004). Competitive and open channel block of recombinant nAChR channels by different antibiotics. Neuromuscular Disorders, 14(5), 307–312. https://doi.org/10.1016/j.nmd.2004.01.005. Sieb, J. P., Milone, M., & Engel, A. G. (1996). Effects of the quinoline derivatives quinine, quinidine, and chloroquine on neuromuscular transmission. Brain Research, 712(2), 179–189. https://doi.org/ 10.1016/0006-8993(95)01349-0. Spitzmaul, G., Gumilar, F., Dilger, J. P., & Bouzat, C. (2009). The local anaesthetics proadifen and adiphenine inhibit nicotinic receptors by different molecular mechanisms. British Journal of Pharmacology, 157(5), 804–817. https://doi.org/10.1111/j.1476-5381.2009.00214.x. Teichert, R. W., Rivier, J., Torres, J., Dykert, J., Miller, C., & Olivera, B. M. (2005). A uniquely selective inhibitor of the mammalian fetal neuromuscular nicotinic acetylcholine receptor. The Journal of Neuroscience, 25(3), 732–736. https://doi.org/10.1523/JNEUROSCI.406504.2005. Thesleff, S. (1955). The mode of neuromuscular block caused by acetylcholine, nicotine, decamethonium and succinylcholine. Acta Physiologica Scandinavica, 34(2–3), 218–231. https://doi.org/10.1111/ j.1748-1716.1955.tb01242.x. Unwin, N. (2005). Refined structure of the nicotinic acetylcholine receptor at 4 Å resolution. Journal of Molecular Biology, 346(4), 967–989. https://doi.org/10.1016/j.jmb.2004.12.031. Varanda, W. A., Aracava, Y., Sherby, S. M., VanMeter, W. G., Eldefrawi, M. E., & Albuquerque, E. X. (1985). The acetylcholine receptor of the neuromuscular junction recognizes mecamylamine as a noncompetitive antagonist. Molecular Pharmacology, 28(2), 128–137. Wonnacott, S., & Barik, J. (2007). Nicotinic ACh receptors. Tocris Reviews, 28, Tocris Cookson.