- Email: [email protected]
Chapter 10 Structure and function of neuronal nicotinic acetylcholine receptors
J. Klein and K. Liiffelholz (Eds.)
Progress in Brain Research, Vol. 109 Q 19% Elsevier Science B.V. All rights reserved.
CHAPTER 10
Structure and function of neuronal nicotinic acetylcholine receptors Jon Lindstrom, Rene Anand, Vladimir Gerzanich, Xiao Peng, Fan Wang and Gregg Wells Department of Neuroscience. University of Pennsylvania Medical School. Philadelphia, PA 19104-6074 USA
Introduction Neuronal nicotinic acetylcholine receptors (AChRs) are part of a gene family which includes AChRs from skeletal muscle and part of a gene superfamily which includes glycine receptors, GABAA receptors, and 5HT3 receptors. Recent reviews of neuronal AChRs (Chapters 8 and 9) include those by Sargent (1993), Papke (1993), McGehee and Role (1995), and Lindstrom (1995;1996). Probably all of the receptors in this superfamily are formed from five homologous subunits oriented around a central ion channel like barrel staves, and the neuronal AChRs probably have shapes very similar to those of muscle type AChRs which have been determined for AChRs from Torpedo electric organ to a resolution of about 9 8, by electron crystallography (Unwin 1993, 1995). ‘The basic homologies in structure of receptors in this superfamily have been illustrated by two elegant experiments by Changeux and his co-workers. Homologies in overall domain relationships were demonstrated by showing that functional mosaics could be made in which the large N-terminal extracellular domain of a 7 AChRs (which contains the acetylcholine binding site) could be grafted just before the first transmembrane domain to the C-terminal part of 5HT3 receptors (which contains the ion channel and large cytoplasmic domain) (Eisile et al., 1993). This resulted in receptors with acetylcholine-gated cation channels having the ion selectivity of 5HT3 receptors. Close similarities in the overall struc-
tures of the ion channels were demonstrated by showing that changing only three amino acids in the sequence lining the cation-specific channel of excitatory a7 neuronal AChRs to amino acids typical of the anion-specific channels of inhibitory glycine or GABAA receptors changed the ion selectivity of the mutated a 7 AChR channels from cations to anions (Galzi et al., 1992). The AChR gene family can be thought of as having three branches. One branch consists of AChRs from skeletal muscles and fish electric organs which have the subunit composition (a1)#1$ in the fetal form or (al)#led in the mature form (reviewed in Changeux, 1990 and Karlin, 1991, 1993). The second branch consists of neuronal AChRs which, unlike muscle AChRs, do not bind the snake venom toxin a bungarotoxin (aBgt). They are formed from combinations of a2, a3, or a4 subunits with 82 and 84 subunits, and also sometimes with a5 subunits (Sargent, 1993; McGehee and Role, 1995). The predominant form of brain high affinity nicotine binding AChRs is formed from a4 and 82 subunits (Whiting and Lindstrom, 1988). The stoichiometry of these subunits is (a4),(82)3, at least when they are expressed in Xenopus oocytes (Anand et al., 1991; Cooper et al., 1991). Ganglionic postsynaptic AChRs contain a3 subunits. These are in combination with 84 and a5 subunits, and sometimes also with82 subunits (Conroy et al., 1992; Conroy and Berg, 1995). These subunits are in unknown stoichiometries, but probably something like (a3)$2#?4a5. Many neuronal AChRs appear to be
126
Concentralion of Nicotine (”M)
Fig. 1. Dose dependence of nicotine-induced upregulation. M10 cells are mouse fibroblasts permanently transfected with chicken a 4 and 82 subunits which exhibit the pharmacological properties of brain a462 AChRs and which function electrophysiologically (Whiting et al., 1991; Periera et al., 1994). Xenopus oocytes were injected with cRNA for a 4 and 82 RNAs. After 3 days in the indicated concentrations of nicotine, a462 AChRs were solubilized using Triton X-100, immunoisolated on microwells coated with mAb 290 to their 82 subunits and quantitated using 20 nM [3H]nicotine. Data are reproduced from Peng et al. (1994a).
located presynaptically, where their functional role may be to modulate the release of various transmitters rather than to form a critical postsynaptic link in transmission as they do at neuromuscular junctions (Henley et al., 1986; Swanson et al., 1987; Wonnacott, 1990; Clarke, 1995). Neuronal AChRs are known to be expressed very early in development. Specific developmental changes in subunit composition are likely but have not yet been determined in detail (Hamassaki-Britto et al., 1994; Cimino et al., 1995; Zoli et al., 1995). The third branch of the gene family consists of neuronal AChRs which bind aBgt. These are formed from a7, a8, or a9 subunits (Lindstrom, 1995, 1996; Elgoyhen et al., 1994). All of these can function as homomers when expressed from cRNAs in Xenopus oocytes. a 7 and a8 are also found together as heteromers in some native AChRs (Schoepfer et al., 1990; Keyser et al., 1993). It is unknown whether other unknown subunits are associated with these subunits in native AChRs, but they are not co-assembled with other known subunits (Anand et al., 1993a,b). Although a 7 is expressed as a homomer very efficiently, a 8 is not (Gerzanich et al., 1994), suggesting that at least a8
might normally be associated with other uncharacterized subunits. Preparations of aBgt binding proteins purified from brain have been found to contain several peptide components (Gotti et al., 1994). a7, a8, and a 9 AChRs all exhibit high conductance for Ca2+(Seguela et al., 1993; Elgoyhen et al., 1994; Gerzanich et al., 1994) which may allow them to take part in unusual synaptic mechanisms in which Ca2+acts as a second messenger to control other ion channels and other cellular functions (Fuchs and Murrow, 1992; Pugh and Berg, 1994). These AChRs also all exhibit very rapid desensitization, which has inhibited measuring their function in neurons until quite recently (Akondon et al., 1994). At least some a7 AChRs, and perhaps many of these AChRs, have been observed in extrasynaptic or perisynaptic positions (Jacob and Berg, 1983; Sargent and Wilson, 1995), again suggesting that some may participate in unusual synaptic mechanisms where their functional roles might involve sensing leak acetylcholine near a synapse or in a region of synapses, rather than classical postsynaptic function at a single synapse. There are two orphan subunits included in this gene family. a 6 and 83 subunit cDNAs have been identified which exhibit extensive homologies with other AChR subunits but which have not yet been reported to be components of native AChRs or to produce functional AChRs when co-expressed with other AChR subunit cDNAs (Sargent, 1993). In the following two sections some aspects of neuronal AChRs are illustrated in more detail. First, we briefly review some of our recent studies of the mechanism by which chronic exposure to nicotine affects a462 AChRs. Second, we briefly review some of the pharmacological properties of neuronal AChRs, especially the differences between a 7 and a8 AChRs and the usefulness of epibatidine (exo-2-(6-chloro-3-pyridyl)-7-azabicyclo-[2.2.l]-heptane) as a ligand for many types of neuronal AChRs.
Mechanism of upregulation of a462 AChRs by chronic exposure to nicotine Tobacco use or chronic exposure to nicotine
127 TABLE 1 Pharmacologicalproperties of a462 AChRs Upregulationa E 5 0 bM)
Function EC50 ( N M ) ~ Activation
Blocking
0.031 f 0.003 0.35 f 0.02 0.073 k 0.01 2.5 2 0.3
-
DMPP Carbamylcholine
0.17 f 0.02 0.21 f 0.04 3.0 f 0.3 15.0 f 4.0
Competitive antagonist Curare
None
-
Noncompetitive untagonist Mecamylamine
65.0 f 6.0
-
Agonists Cytisine Nicotine
0.00014 f O.ooOo3 0.0039 f 0.00021 0.0094 0.0002 0.36 2 0.013
-
*
-
2.7 f 0.5 0.37
Equilibrium bindingb KI 01M)
0.5
25.0
* 14.0
>lo00
aData from Peng et al. (1994a).
bData from Whiting et al. (1991).
causes an increase in brain a462 AChRs by about twofold (Schwartz and Kellar, 1985; Benwell et al., 1988; Flores et al., 1992). Wonnacott, 1990; Wonnacott et al., 1990) suggested that this increase in AChRs was an adaptive response of neurons to maintain nicotinic transmission despite the accumulation of desensitized AChRs due to chronic exposure to this agonist. We have found that agonist-induced upregulation of a462 AChRs is an intrinsic property of this protein which is exhibited when the cloned a462 AChRs are expressed in Xenopus oocytes or in a permanently transfected fibroblast cell line called M10, as shown in Fig. 1 (Peng et al., 1994a). This has now also been confirmed by others (Zhang et al., 1994). The EC5o for nicotine-induced upregulation of M, see Fig. 1) is pathologia4P2 AChRs ( 2 x cally significant because this concentration is equal to the mean steady state serum concentration of nicotine in smokers (Benowitz et al., 1990). The steady state concentration of nicotine in smokers is interesting because it is near the EC50 for activation of a4P2 AChRs by nicotine when applied M), but much greater than the acutely (3.5 x KD for binding nicotine by desensitized a462
M) (see Table 1 which is AChRs (3.9 x adapted from Peng et al., 1994a). Thus, at a steady state nicotine concentration of 2 x IW7M all a462 AChRs in a tobacco user should be desensitized, and the net behavioral effect should reflect inhibition of these AChRs. Chronic exposure to other agonists also causes upregulation in the amount of a462 AChRs, as shown in Fig. 2 (Peng et al., 1994a). The competitive antagonist curare prevents nicotine-induced upregulation. The rank order for efficacy in causing upregulation parallels the rank order of equilibrium binding affinity (presumably to the desensitized conformation) but not quite the rank order for activation (Table 1). The non-competitive antagonist mecamylamine can also causes upregulation and is synergistic with nicotine, as shown in Fig. 3 (Peng et al., 1994a). This parallels the effects of mecamylamine in brain (Collins et al., 1994). Blockage of function by mecamylamine is more effective in the presence of low concentrations of nicotine (Fig. 4). These data are consistent with the idea that mecamylamine is an open channel blocker which is able to bind better when the channel is opened by nicotine (Varanda et al., 1985; Martin et al., 1990).
n4P7 AChH Upregulation
7
111.)
l:'1 10."
Fig. 2. Comparison of dosdresponse relationships for a 4 s 2 upregulation in M10 cells and function in Xenopus oocytes. Expression of a462 AChR in M10 cells grown to confluence was induced by 1 pM dexamethasone for 3 days, then the indicated concentrations of ligands were added for 3 days and finally AChRs were quantitated in solid phase assays using [3HJnicotineto label AChRs immunoisolated through their 82 subunit as in Fig. 1. Voltage clamp measurements on Xenopus oocytes normalized the maximum currents to saturating agonist concentrations. Blockage by mecamylamine was measured at 1 pM nicotine. Data are reproduced from Peng et al. (1994a).
Ion flow through the cation channel of a462 AChRs does not appear to be necessary to trigger upregulation of these AChRs. This is shown by the observation that a channel blocker like mecamylamine can cause upregulation. It is also shown by the observation that the channel blocker chlorisondamine blocks AChR function but does not prevent nicotine-induced upregulation (El-Bizri and Clarke, 1994). The observation that the rank order of various ligands for equilibrium binding to desensitized a462 AChRs better parallels upregulation than does their order for activation (Table 1) is
also consistent with the idea that cation flow is not necessary to trigger upregulation but that induction of a particular conformation of the AChR may be. Nicotine-induced upregulation of a462 AChRs is not due to an increase in brain a4 or j32 mRNA (Marks et al., 1992). Similarly, nicotine-induced upregulation of a462 AChRs does not occur at the transcriptional level. Demonstration of AChR upregulation in Xenopus oocytes injected with fixed amount of a 4 and j32 mRNA is the best evidence that upregulation occurs post transcriptionally (Peng et al., 1994a). The mechanism of increase in a462 AChRs which is induced by nicotine involves a decrease in the rate of destruction of the AChRs in the surface membrane, as shown in Fig. 5 (Peng et al., 1994a). Nicotine, other agonists, and mecamylamine appear to induce a conformation (a type of desensitized conformation, perhaps) which is turned over more slowly than the resting conformation. This pathological change is an interesting contrast with myasthenia gravis, where chronic M
150
loo
-
50 -
0-
L I -
.z
Li
E 'I
E
1% ZE e
5
-
i
In
Nicotine Causes Upregulation
Compeiiiive Antagonist Prevents Upregulation
Noncompeiiiive Antagonist Causes Upregulation
Fig. 3. Effects of antagonists on upregulation of a4B2 AChRs in M10 cells. M10 cells were grown to confluence, AChR expression was induced with dexamethasone and then treated for 3 days with the indicated ligands before solid phase radioimmunoassay. Data are from Peng et al. (1994a).
129
+r ...
~i
IOpM ACh
J
IpA
2 min.
...
Nicotine 0.1pM +Mecaniylamine 3pM
were greatly reduced, even though the total number of AChRs was doubled, as shown in Fig. 6 (Peng et al., 1994a). Short term desensitization is normally reversed by washing over matters of minutes, but this reduction in response only recovered fully after 3 days, which is about the rate expected for synthesis of new AChRs. Native AChRs also exhibit downregulation of function after chronic exposure to nicotine (Marks et al., 1993). The results in oocytes suggest that long term exposure to high concentrations of nicotine can result in a permanently inactivated a4P2 AChR conformation. It is unknown whether this reflects the same nicotine-induced conformation that is turned over more slowly. The loss of functional a462 AChRs on chronic exposure to nicotine due to both reversible and permanent desensitization provides an explanation for tolerance. Thus, chronic tobacco users are tolerant of much higher nicotine concentrations than are nonusers because, even though the chronic users may have twice as many a462 AChRs, most of these excess AChRs can bind nicotine but not function in response to it.
Some pharmacological properties of neuronal AChRs Fig. 4. Mecamylamine blockage of a4P2 AChR function is more effective in the presence of nicotine. a462 AChRs were expressed in Xenopus oocytes. A continuous series of responses to a saturating concentration of acetylcholine is shown from top to bottom. A low concentration of nicotine causes only a small response and little or no blockage to a subsequent test response to acetylcholine. Mecamylamine caused some blockage to a test response to acetylcholine applied immediately after and some recovery was seen after further washing. Applying nicotine and mecamylamine together results in nearly complete blockage of a test response applied immediately after. Data are from Peng et al. (1994a).
exposure to autoantibodies causes a decrease in the amount of muscle AChRs by crosslinking them and thereby facilitating their endocytosis (Lindstrom et al., 1988). We found that after exposing oocytes expressing a462 AChRs to high concentrations of nicotine for 3 days their net responses to acetylcholine
aBgt binds virtually irreversibly to muscle type AChRs, but it has lower affinity for a7 AChRs, still lower affinity for a8 AChRs, and substantially lower affinity yet for a 9 AChRs, as shown in Table 2 (Keyser et al., 1993; Elgoyhen et al., 1994). The muscarinic antagonists atropine and glycinergic antagonist strychnine are nearly as potent as the nicotinic antagonist curare as antagonists of a7, a 8 and a 9 AChRs, as shown in Table 2. Despite the similarities in their N-terminal extracellular amino acid sequences, a7 and a8 AChRs differ in their pharmacological properties, with a7 having higher affinity for aBgt but lower affinity for small cholinergic ligands (Anand et al., 1993; Keyser et al., 1993; Gerzanich et al., 1994). This is reflected in the properties of their homomers. Pharmacological properties of a8 are known only in chick and a 9 only in rat. a 7 from chicks and humans are pharmacologically similar except for
130
Total AChRs
1
400
f Nicotine \\ \
lpM Dexamethasone Induction of a4p2 AChR Expression
DAY
35pM Cycloheximide Inhibition of Protein Synthesis SpM Nicotine Upregulation of a4p2 AChR
Surface AChRs 10°O
1
200
0
Induction of a 4 p 2 AChR Expression
Nicotine
K"
400
XpM Dexamethasone
/*\
/
1
\
\
A
\
'4.
2
3
4
5
6
\
* Control 7
DAYS 35pM Cycloheximide Inhibition of Protein Synthesis
5pM Nicotine Upregulation of a 4 p 2 AChR Fig. 5. Nicotine decreases the turnover rate of a482 AChRs in M I 0 cells. Cycloheximide was used to inhibit the synthesis of new a4B2 AChRs in dexamethasone-induced MI0 cells. In the experiment shown at the top, cycloheximide was added 1 day after dexamethasone induction. Then total a482 AChRs solubilized from the cells was measured by [3H]nicotine binding to immunoisolated AChRs in microwells, In the experiment shown in the lower panel, dexamethasone induction of a482 AChR synthesis was allowed to maximize over 4 days before nicotine was added to one set of cultures. In this case only surface AChRs were measured by binding of 12sI-labeledmAb290 to 82 subunits in intact cells. Data are from Peng et al. (1994a).
131
d c\l
a d t3 ( I
-
Fig. 6. Time course of recovery of a4B2 AChR function after chronic treatme t with nicotine of Xenopus oocytes. After measuring the initial response to 100pM acetylcholine, one group was incubated with 1 p M nicotine, another with lOpM nicotine and a third group was left untreated as a control. After 3 days the oocytes were washed and then responses to lOOpM acetylcholine were measured at the indicated times over the next 3 days. Data are from Peng et al. (1994a).
TABLE 2 Pharmacological properties of a 7 , a 8 and a 9 AChRs
a 7 chick
Antagonists aBgt
Curare Atropine Strychnine Agonists ACh Nicotine DMPP Epibatidine
0.0021e 7.3a
12oa 9.9a
16Oa
1.3a (agonist)'
83a (3.5% partial)c 2.2d agonist
a 7 human
0.001f 2of 1366
9.8'
5.gf
2.6f (agonisOf 4 . 4 (full agonistlf I.ld
a 8 chick
0.017e 5oa OSSa 2.P
0.031a 0.012a (agonist)= 0.3Y(full agonist)c 0.0012d
a 9 rat
Completely reversed in 10 min washb 0.3b
1.3b 0.02b
lob 30 (antagonist)b 5 % partial agonistb ?
aData from Anand et al. (1993a) for IC50 on binding of lzsIaBgt at 2 nM for native a7 and 20 nM for native a 8 AChR. bData from Elgoyhen et al. (1994) for I C 3 on function 10pM ACh. CDatafrom Gerzanich et al. (1994) for EC50 on a7 or a8 homomers. dData from Gerzanich et al. (1995) for EC of (-) epibatidine on a7 or a8 homomers on IC50 in their function. eData from Keyser et al. (1993) for KD Of '%a&$. fData from Peng et al, (1994b) for KD of '%Bgt for ICsn at 2 nM '"IaBgt for native a7 AChRs.
132
Epibatidine 0.3,3,30,300nM
U Acetylcholine
-1 1
-9 -1 -5 Concentration (log M)
I 200nA
10 sec.
‘Acetylcholine 3 0 ~ M Fig. 7. Activation by epibatidine of chicken a4B2 AChRs expressed in Xenopus oocytes. The top panel shows the responses to different concentrations of epibatidine and compares dosdresponse curves for epibatidine with those of nicotine and acetylcholine. The bottom panel compares the time courses of responses to saturating concentrations of epibatidine and nicotine. Data are from Gerzanich et al. (1995).
DMPP, which is virtually an antagonist on chick, however, a very potent full agonist on human a7 (Gerzanich et al., 1994; Peng et al., 1994b). It would be extremely useful to have a universal labeling reagent for AChRs. aBgt has long been an excellent ligand for muscle AChRs and a7 AChRs (Table 2). Nicotine has been a useful label for a462 AChRs (Table 1). Cholinergic labeling a 3 AChRs has been difficult because of their relatively low affinity for nicotine, and the failure of neuronal bungarotoxin to bind to detergent solubilized AChRs, so they have long been quantitated using 125ImAb35 (Smith et al., 1985). This mAb binds to the main immunogenic region on the extracellular surface of a 1 subunits (Saedi et al., 1990) and to homologous structures on chick a5 subunits (Conroy et al., 1992; Conroy and Berg,
1995) and human a 3 and a5 subunits (Wang et al., 1996). Epibatidine is now proving to be a very useful ligand for several subtypes of neuronal AChRs, including a 3 AChRs. Epibatidine was initially discovered in the skin of Ecuadoran poison frogs by John Daly (Spande et al., 1992). The frogs derive it from an unknown component of their diet in the wild (Daly, 1995). It has now been synthesized (e.g. Fletcher et al., 1994; reviewed in Broke, 1994). Initially it was discovered to be 200-fold more potent than morphine in preventing nociception. Later it was discovered to be a potent nicotinic agonist (Badio and Daly, 1994) and to elicit a variety of nicotinic effects (Sullivan et al., 1994). Epibatidine is the most potent neuronal AChR
133
Human a3P2 AChRs Expressed in Xenopus Oocytes
-
Epibatidine 3, 10,30,100 nM
2 0.6
sE 0.4
0.2
0 -10
-8
-6
-4
Concentration of Agonist (log M)
-2
Human a3P4 AChRs Expressed in Xenopus Oocytes Edbatidine 10.30.100.300 nM
10 sec.
Concentration of Agonist (log M)
Fig. 8. Activation by epibatidine of human a3j32 and a3j34 AChRs expressed in Xenopus oocytes. The top panel shows responses of a3j32 AChRs to various concentrations of epibatidine and compares dosdresponse curves for epibatidine, nicotine and acetylcholine. The bottom panel makes a similar comparison for a464 AChRs. Data are from Gerzanich et al. (1995).
agonist that we have studied on several types of cloned AChRs expressed in Xenopus oocytes, and r3H]epibatidine is an excellent labeling reagent which has proven especially useful for a3 AChRs (Gerzanich et al., 1995). Epibatidine is more potent than nicotine as an agonist for a462 AChRs, as shown in Fig. 7, or for a3p2 or a3p4 AChRs, as shown in Fig. 8. Note that Fig. 8 also illustrates the important concept that 82 and 8 4 play an important role in determining the affinity and efficacy of ligands (reviewed in detail by Papke, 1993). This is a consequence of the acetylcholine binding site being formed at the interface between a subunits
and adjacent structural subunits (Blount and Merlie, 1989). The extremely high affinity of r3H]epibatidine as a labeling reagent is illustrated for human a3p2 AChRs in Fig. 9. Epibatidine is still a more potent agonist than either nicotine or acetylcholine on both a7 and electric organ AChRs, but much less potent than on a 4 or a3 AChRs, as shown in Fig. 10. Thus, despite its potency as an agonist, epibatidine does not quite provide a universal ligand for AChRs. However, the AChRs for which it has relatively low affinity, like a1 and a7 AChRs, can be efficiently labeled with *=IaBgt. The affinity of
134 12 1 aJ
.E
10 i
**
16--
0
0
_-.
/A--
2 [3H]
a462 AChRs is highest for epibatidine, making it a better label than [3H]nicotine. But the very high affinity of [3H]epibatidine for a 3 AChRs provides a really useful breakthrough. Epibatidine is likely to become an important reagent for several types of neuronal AChRs. It is also likely to renew interest in an important role of nicotinic AChRs in nociception. Although epibatidine is not highly selective among neuronal AChR subtypes, which can be a pharmacological drawback, it is a small molecule structure that can provide remarkably high affinity for these AChRs.
I
i
I
R,,,=lxl0l5M
0
4
6
8
lo
Epibatidine (1O'OM)
Acknowledgements
Fig. 9. Binding of racemic [3H]epibatidine to human a3B2 AChRs expressed in oocytes. Physiological experiments of the types shown in Figs. 7 and 8 with + and - isomers of eoibatidine revealed sumrisinaly - - small differences in responses to the two isomers. Data are from Gerzanich et al. (1995).
The laboratory of Jon Lindstrom is supported by grants from the N m S (NS11323), The blokeless Tobacco Research Council, Inc., The Council for Tobacco Research - USA, Inc., and the Muscular Dystrophy Association.
Epibatidine 0.1,0.3, 1 , 3 pM
-(+)-
+Nicotine
w
Epibatidine
Concentration of Agonist
(log M)
Epibatidine 0.3,1,3, 10 pM
1
0.8
g 0.6 E
I
2 0.4
0.2
0
Concentration of Agonist (log M)
Fig. 10. Activation by epibatidine of human a7 homomers and muscle type Torpedo alplyd AChRs expressed in oocytes. Data are from Gerzanich et al. (1995).
135
References Alkondon, M., Reinhardt, S., Lobron C., Hermsen, B., Maelicke, A. and Albuquerque, E. (1994) Diversity of nicotinic acetylcholine receptors in rat hippocampal neurons. 11. The rundown and inward rectification of agonistelicited whole cell currents and identification of receptor subunits by in situ hybridization. J. Pharmacol. Exp. Ther., 27 1: 494-506. Anand, R., Conroy, W.G., Schoepfer, R., Whiting, P. and Lindstrom, J. (1991) Chicken neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes have a pentameric quaternary structure. J. Biol. Chem., 266: 1119211198. Anand, R., Peng, X., Ballesta, J. and Lindstrom, J. (1993a) Pharmacological characterization of a bungarotoxin sensitive AChRs immunoisolated from chick retina: contrasting properties of a 7 and a 8 subunit-containing subtypes. Mol. Pharmacol., 44: 1046-1050. Anand, R., Peng, X. and Lindstrom, J. (1993b) Homomeric and native a 7 acetylcholine receptors exhibit remarkably similar but nonidentical pharmacological properties suggesting that the native receptors is a heteromeric protein complex. FEES Letr., 327: 241-246. Badio, B. and Daly, J. (1994) Epibatidine, a potent analgesic and nicotinic agonist. Mol. P h a m c o l . , 45: 563-569. Benowitz, N., Porchet, H. and Jacob, P. (1990) Pharmacokinetics, metabolism and pharmacodynamics of nicotine. In: S. Wonnocott, M. Russell and I. Stolerman (Eds.), Nicotine Psychophannacology, Oxford Science Publications, Oxford, UK, pp. 112-157. Benwell, M., Balfour, D. and Anderson, J. (1988) Evidence that tobacco smoking increases the density of (-H3H] nicotine binding sites in human brain. J. Neurochem., 5 0 1243- 1247. Blount, P. and Merlie, J.P. (1989) Molecular basis of the two nonequivalent ligand binding sites of the muscle nicotinic acetylcholine receptor. Neuron, 3: 349-357. Broke, C. (1994) Synthetic approaches to epibatidine. Med. Chem. Res., 4: 4 4 W 8 . Changeux, J.P. (1990) Functional architecture and dynamics of the nicotinic acetylcholine receptor: an allosteric ligandgated ion channel. In: 1988-1989 Fidia Research Foundntion: Neuroscience Award Lechwes, Vol. 4, pp. 21168. Cimino, M., Marini, P., Colombo, S., Andena, M., Cattabeni, Fornasari, D. and Clemente, F. (1995) Expression of neuronal acetylcholine receptor a 4 and 8 2 subunits during postnatal development of the rat brain, J. Neural Tramm., 100: 77-92. Clarke, P. (1995) Nicotinic receptors and cholinergic transmission in the central nervous system, Ann. N. Y. Acad. Sci., 757: 73-83. Collins, A., Luo, Y.,Selvaag, S. and Marks, M. (1994) Sensitivity to nicotine and brain nicotinic receptors are altered by
chronic nicotine and mecamylamine infusion, J . Phannacol. Exp. Ther., 271: 125-133. Conroy, W. and Berg, D. (1995) Neurons can maintain multiple classes of nicotinic acetylcholine receptors distinI Biol. . Chem., guished by different subunit compositions. . 270: 4424-443 1. Conroy, W.. Vernallis, A. and Berg, D. (1992) The a 5 gene product assembles with multiple acetylcholine receptor subunits to form distinctive receptor subtypes in brain. Neuron, 9: 1-20. Cooper, E., Couturier, S. and Ballivet, M. (1991) Pentameric structure and subunit stoichiometry of a neuronal nicotinic acetylcholine receptor. Nature, 350: 235-238. Daly, J. (1995) The chemistry of poisons in amphibian skin. Proc. Natl. Acad. Sci. USA, 92: 9-13. Eisile, J.L., Bertrand, S., Galzi, J.L., Devillers-Thiery, A., Changeux, J.P. and Bertrand, D. (1993) Chimaeric nicotinic-serotonergic receptor combines distinct ligand binding and channel specificities. Nature, 366: 479409. El-Bizxi, H. and Clarke, P. (1994) Regulation of nicotinic receptors in rat brain following quasi-irreversible nicotinic blockade by chlorisondamine and chronic treatment with nicotine. Br. J. fhannacol., 113: 917-925. Elgoyhen, A., Johnson, D., Boulter, J., Vetter, D. and Heinemann, S. (1994) a9: an acetylcholine receptor with novel pharmacological properties expressed in rat cochlear hair cells. Cell, 79: 705-715. Fletcher, S., Baker, R., Chambers, M., Herbert, R., Hobbs, S., Thomas, S.,Vemer, H., Watt, A. and Ball, R. (1994) Total synthesis and determination of the absolute configuration of epibatidine. J. Org. Chem., 59: 1771-1778. Flores, C., Rogers, S.,Pabreza, J., Wolfe, B. and Kellar, K. (1992) A subtype of nicotinic cholinergic receptor in rat brain is composed of a 4 and 8 2 subunits and is upregulated by chronic nicotine treatment. Mol. Pharmacol., 41: 31-37. Fuchs, P. and Murrow, B. (1992) A novel cholinergic receptor mediates inhibition of chick cochlear hair cells. f r o c . R. SOC.London Ser B, 248: 35-40. Galzi, J.L., Devillers-Thiery, S., Hussy, N., Bertrand, S., Changeux, J.P. and Bertrand, D. (1992) Mutations in the channel domain of a neuronal nicotinic receptor convert ion selectivity from cationic to anionic. Nature, 359: 500505. Gerzanich, V., Anand, R. and Lindstrom, J. (1994) Homomers of a 8 subunits of nicotinic receptors functionally expressed in Xenopus oocytes exhibit similar channel but contrasting binding site properties compared to a 7 homomers. Mol. P h a m c o l . , 45: 212-220. Gerzanich, V., Peng, X., Wang, F., Wells, G., Anand, R., Fletcher, S. and Lindstrom, J. (1995) Comparative pharmacology of epibatidine - a potent agonist for neuronal nicotinic acetylcholine receptors. Mol. P h a m c o l . , 48: 774782. Gotti, C., Hanke, W., Maury, K., Moretti, M., Ballivet, M., Clemente, F. and Bertrand, D. (1994) Pharmacology and biophysical properties of a 7 and a 7 4 3 a bungarotoxin re-
136 ceptor subtypes immunopurified from the chick optic lobe. Eur. J. Neurosci., 6: 1281-1291. Hamassaki-Britto, D., Gardino, P., Hokoc, J., Keyser, K., Karten, H., Lindstrom, J. and Britto, L. (1994) Differential development of a bungarotoxin-sensitive and a bungarotoxin-insensitive nicotinic acetylcholine receptors in the chick retina. J. Comp. Neurol., 347: 161-170. Henley, J., Lindstrom, J. and Oswald, R. (1986) Acetylcholine receptor synthesis in retina and transport to the optic tectum in goldfish. Science, 232: 1627-1629. Jacob, .M. and Berg, D. (1983) The ultrastructural localization of a bungarotoxin binding sites in relation to synapses on chick ciliary ganglion neurons. J. Neurosci., 3: 260-271. Karlin, A. (1991) Explorations of the nicotinic acetylcholine receptor. Harvey Lectures Ser., 85: 71-107. Karlin, A. (1993) Structure of nicotinic acetylcholine receptors. Curr. Opin. Neurobiol., 3: 299-309. Keyser, K., Britto, L., Schoepfer, R., Whiting, P.,Cooper, J., Conroy, W., Brozozowska-Prechtl, A., Karten, H. and Lindstrom, J. (1993) Three subtypes of alpha bungarotoxinsensitive nicotinic acetylcholine receptors are expressed in chick retina. J. Neurosci., 13: 442-454. Lindstrom, J. (1995) Nicotinic acetylcholine receptors. In Alan North (Ed.), CRC Handbook of Receptors. CRC Press, pp. 153-175. Lindstrom, 1. (19%) Neuronal nicotinic acetylcholine receptors. In: T. Narahashi (Ed.),Ion Channels, Vol. IV, Plenum, New York, pp. 377-450. Lindstrom, J., Shelton, G.D. and Fujii, Y. (1988) Myasthenia gravis. Adv. Immunol., 42: 233-284. Marks, M., Pauly, J., Gross, D., Deneris, E., HermansBorgmeyer, I., Heinemann, S. and Collins, A (1992) Nicotine binding and nicotinic receptor subunit RNA after chronic nicotine treatment. J. Neurosci., 12: 27652784. Marks, M., Grady, S. and Collins, A. (1993) Downregulation of nicotinic receptor function after chronic nicotine infusion. J. Pharmacol. Exp. Ther., 266: 1268-1275. Martin, T., Suchocki, J., May, E. and Martin, B. (1990) Pharmacological evaluation of the antagonism of nicotines central effects by mecamylamine and pempidine. J. Phurm. Exp. Ther., 254: 45-5 1. McGehee, D. and Role, L. (1995) Physiological diversity of nicotinic acetylcholine receptors expressed by vertebrate neurons. Annu. Rev. Physwl., 57: 521-546. Papke, R. (1993) The kinetic properties of neuronal nicotinic receptor: genetic basis of functional diversity. Prog. Neurobiol., 41: 5 W 5 3 1 . Peng, X.,Gerzanich, V., Anand, R., Whiting, P. and Lindstrom, J. (1994a) Nicotine-induced upregulation of neuronal nicotinic receptors results from a decrease in the rate of turnover. Mol. Pharmacol., 46:523-530. Peng, X., Katz, M., Gerzanich, V., Anand, R. and Lindstrom, J. (1994b) Human a 7 acetylcholine receptor: cloning of the a 7 subunit from the SH-SY5Y cell line and determination of pharmacological properties of native receptors and func-
tional a 7 homomers expressed in Xenopus oocytes. Mol. Phannacol., 45: 546-554. Pereira, E., Alkondon, M., Reinhardt-Maelicke, S., Maelicke, A., Peng, X.,Lindstrom, J., Whiting, P. and Albuquerque, E. (1994) Physostigmine and galanthamine reveal the presence of the novel binding site on the a 4 8 2 subtype of neuronal nicotinic acetylcholine receptor stably expressed in fibroblast cells. J. Pharmacol. Erp. Ther., 270: 768-778. Pugh, P. and Berg, D. (1994) Neuronal acetylcholine receptors that bind a bungarotoxin mediate neurite retraction in a calcium-dependent manner. J. Neurosci., 14: 889-896. Saedi, M., Anand. R., Conroy, W.G. and Lindstrom, J. (1990) Determination of amino acids critical to the main immunogenic region of intact acetylcholine receptors by in vitro mutagenesis. FEBS Lett., 267: 55-59. Sargent, P. (1993) The diversity of neuronal nicotinic acetylcholine receptors. Annu. Rev. Neurosci., 16: 4 0 3 4 3 . Sargent, P. and Wilson, H. (1995) Distribution of nicotinic acetylcholine receptor subunit immunoreactivities on the surface of chick ciliary ganglion neurons. In: P. Clarke, M. Quick, F. Adlkofer and K. Thurau (Eds.), Eflects of Nicotine on Biological Systems II ,Birkhauser, Basel. Schoepfer, R., Conroy, W.G., Whiting, P., Gore, M. and Lindstrom, J. (1990) Brain alpha-bungarotoxin-binding protein cDNAs and mAbs reveal subtypes of this branch of the ligand-gated ion channel superfamily. Neuron, 5 : 3548. Schwartz, R. and Kellar, K. (1985) In vivo regulation of [3H] acetylcholine recognition sites in brain by nicotinic cholinergic drugs. J. Neurochem., 45: 427-433. Seguela, P., Wadiche, J., Dinelly-Miller, K., Dani, J. and Patrick, J. (1993) Molecular cloning, functional properties and distribution of rat brain a7: a nicotinic cation channel highly permeable to calcium. J. Neurosci., 13: 596-604. Smith, M., Stollberg, J., Lindstrom, J. and Berg, D.K. (1985) Characterization of a component in chick ciliary ganglia that cross-reacts with monoclonal antibodies to muscle and electric organ acetylcholine receptor. J. Neurosci., 5 : 27262731. Spande, T., Garraffo, M., Edwards, M., Yeh, H., Pannel, L. and Daly, J. (1992) Epibatidine: a novel (chloropyridyl) azabicyclo-heptane with potent analgesic activity from Ecuadoran poison frog. J. Am. Chem. Soc., 114: 34753478. Sullivan, J., Decker, M., Brioni, J., Donnelly-Roberts, D., Anderson, D., Bannon, A., Kang, C., Adams, P., PiattoniKaplan, M., Buckley, M., Gopalakrishnan, M., Williams, M. and Americ, S. (1994) (*)-Epibatidine elicits a diversity of in vitro and in vivo effects mediated by nicotinic acetylcholine receptor. J. Pharmacol. Erp. Ther., 271: 624-631. Swanson, L., Simmons, D., Whiting, P. and Lindstrom, J. (1987) Immunohistochemical localization of neuronal nicotinic receptors in the rodent central nervous system. J. Neurosci., 7: 3334-3342. Unwin, N. (1993) Nicotinic acetylcholine receptor at 9 A resolution. J. Mol. Biol., 229: 1101-1124.
137 Unwin, N. (1995) Acetylcholine receptor channel imaged in the open state. Nature, 373: 3 7 4 3 . Varanda, W., Aracava, A., Sherby, S., Van Meter, W., Eldefrawi, M. and Albuquerque, E. (1985) The acetylcholine receptor of the neuromuscular junction recognizes mecamylamine as a noncompetitive antagonist. Mol. Phrmacol., 28: 128-137. Wang, F., Ferzanich, V., Wells, F., Anand, R., Peng, X.,Keyser, K. and Lindstrom, J. (1996) Assembly of human neuronal nicotinic receptor a5 subunits with a3, 8 2 and 84 subunits. J. Biol. Chem. 271: 17656-17665. Whiting, P.J. and Lindstrom, J.M. (1988) Characterization of bovine and human neuronal nicotinic acetylcholine receptors using monoclonal antibodies. J. Neurosci., 8: 33953404. Whiting, P., Schoepfer, R., Lindstrom, J. and Priestley, T. (1991) Structural and pharmacological characterization of
the major brain nicotinic acetylcholine receptor subtype stably expressed in mouse fibroblasts. Mol. Pharmacol., 40: 463472. Wonnacott, S. (1990) The paradox of nicotinic acetylcholine receptor upregulation by nicotine. Trends Pharmacol. Sci., 11: 216-219. Wonnacott, S., Russell, M. and Stoleman, I. (1990) Nicotine Psychopharmacology: Molecular, Cellular and Behavioral Aspects, Oxford Science Publications, Oxford, UK. Zhang, X.,Gang, Z.H., Hellstrom-Lindahl, E. and Nordberg, A. (1994) Regulation of a462 nicotinic acetylcholine receptors in M I 0 cells following treatment with nicotinic agents. NeuroReport, 6: 3 13-3 17. Zoli, M., LeNovere, N., Hill, J. and Changeux, J.P. (1995) Developmental regulation of nicotinic ACh receptor mRNAs in the central and peripheral nervous systems. J. Neurosci., 15: 19 12-1 939.
Progress in Brain Research, Vol. 109 Q 19% Elsevier Science B.V. All rights reserved.
CHAPTER 10
Structure and function of neuronal nicotinic acetylcholine receptors Jon Lindstrom, Rene Anand, Vladimir Gerzanich, Xiao Peng, Fan Wang and Gregg Wells Department of Neuroscience. University of Pennsylvania Medical School. Philadelphia, PA 19104-6074 USA
Introduction Neuronal nicotinic acetylcholine receptors (AChRs) are part of a gene family which includes AChRs from skeletal muscle and part of a gene superfamily which includes glycine receptors, GABAA receptors, and 5HT3 receptors. Recent reviews of neuronal AChRs (Chapters 8 and 9) include those by Sargent (1993), Papke (1993), McGehee and Role (1995), and Lindstrom (1995;1996). Probably all of the receptors in this superfamily are formed from five homologous subunits oriented around a central ion channel like barrel staves, and the neuronal AChRs probably have shapes very similar to those of muscle type AChRs which have been determined for AChRs from Torpedo electric organ to a resolution of about 9 8, by electron crystallography (Unwin 1993, 1995). ‘The basic homologies in structure of receptors in this superfamily have been illustrated by two elegant experiments by Changeux and his co-workers. Homologies in overall domain relationships were demonstrated by showing that functional mosaics could be made in which the large N-terminal extracellular domain of a 7 AChRs (which contains the acetylcholine binding site) could be grafted just before the first transmembrane domain to the C-terminal part of 5HT3 receptors (which contains the ion channel and large cytoplasmic domain) (Eisile et al., 1993). This resulted in receptors with acetylcholine-gated cation channels having the ion selectivity of 5HT3 receptors. Close similarities in the overall struc-
tures of the ion channels were demonstrated by showing that changing only three amino acids in the sequence lining the cation-specific channel of excitatory a7 neuronal AChRs to amino acids typical of the anion-specific channels of inhibitory glycine or GABAA receptors changed the ion selectivity of the mutated a 7 AChR channels from cations to anions (Galzi et al., 1992). The AChR gene family can be thought of as having three branches. One branch consists of AChRs from skeletal muscles and fish electric organs which have the subunit composition (a1)#1$ in the fetal form or (al)#led in the mature form (reviewed in Changeux, 1990 and Karlin, 1991, 1993). The second branch consists of neuronal AChRs which, unlike muscle AChRs, do not bind the snake venom toxin a bungarotoxin (aBgt). They are formed from combinations of a2, a3, or a4 subunits with 82 and 84 subunits, and also sometimes with a5 subunits (Sargent, 1993; McGehee and Role, 1995). The predominant form of brain high affinity nicotine binding AChRs is formed from a4 and 82 subunits (Whiting and Lindstrom, 1988). The stoichiometry of these subunits is (a4),(82)3, at least when they are expressed in Xenopus oocytes (Anand et al., 1991; Cooper et al., 1991). Ganglionic postsynaptic AChRs contain a3 subunits. These are in combination with 84 and a5 subunits, and sometimes also with82 subunits (Conroy et al., 1992; Conroy and Berg, 1995). These subunits are in unknown stoichiometries, but probably something like (a3)$2#?4a5. Many neuronal AChRs appear to be
126
Concentralion of Nicotine (”M)
Fig. 1. Dose dependence of nicotine-induced upregulation. M10 cells are mouse fibroblasts permanently transfected with chicken a 4 and 82 subunits which exhibit the pharmacological properties of brain a462 AChRs and which function electrophysiologically (Whiting et al., 1991; Periera et al., 1994). Xenopus oocytes were injected with cRNA for a 4 and 82 RNAs. After 3 days in the indicated concentrations of nicotine, a462 AChRs were solubilized using Triton X-100, immunoisolated on microwells coated with mAb 290 to their 82 subunits and quantitated using 20 nM [3H]nicotine. Data are reproduced from Peng et al. (1994a).
located presynaptically, where their functional role may be to modulate the release of various transmitters rather than to form a critical postsynaptic link in transmission as they do at neuromuscular junctions (Henley et al., 1986; Swanson et al., 1987; Wonnacott, 1990; Clarke, 1995). Neuronal AChRs are known to be expressed very early in development. Specific developmental changes in subunit composition are likely but have not yet been determined in detail (Hamassaki-Britto et al., 1994; Cimino et al., 1995; Zoli et al., 1995). The third branch of the gene family consists of neuronal AChRs which bind aBgt. These are formed from a7, a8, or a9 subunits (Lindstrom, 1995, 1996; Elgoyhen et al., 1994). All of these can function as homomers when expressed from cRNAs in Xenopus oocytes. a 7 and a8 are also found together as heteromers in some native AChRs (Schoepfer et al., 1990; Keyser et al., 1993). It is unknown whether other unknown subunits are associated with these subunits in native AChRs, but they are not co-assembled with other known subunits (Anand et al., 1993a,b). Although a 7 is expressed as a homomer very efficiently, a 8 is not (Gerzanich et al., 1994), suggesting that at least a8
might normally be associated with other uncharacterized subunits. Preparations of aBgt binding proteins purified from brain have been found to contain several peptide components (Gotti et al., 1994). a7, a8, and a 9 AChRs all exhibit high conductance for Ca2+(Seguela et al., 1993; Elgoyhen et al., 1994; Gerzanich et al., 1994) which may allow them to take part in unusual synaptic mechanisms in which Ca2+acts as a second messenger to control other ion channels and other cellular functions (Fuchs and Murrow, 1992; Pugh and Berg, 1994). These AChRs also all exhibit very rapid desensitization, which has inhibited measuring their function in neurons until quite recently (Akondon et al., 1994). At least some a7 AChRs, and perhaps many of these AChRs, have been observed in extrasynaptic or perisynaptic positions (Jacob and Berg, 1983; Sargent and Wilson, 1995), again suggesting that some may participate in unusual synaptic mechanisms where their functional roles might involve sensing leak acetylcholine near a synapse or in a region of synapses, rather than classical postsynaptic function at a single synapse. There are two orphan subunits included in this gene family. a 6 and 83 subunit cDNAs have been identified which exhibit extensive homologies with other AChR subunits but which have not yet been reported to be components of native AChRs or to produce functional AChRs when co-expressed with other AChR subunit cDNAs (Sargent, 1993). In the following two sections some aspects of neuronal AChRs are illustrated in more detail. First, we briefly review some of our recent studies of the mechanism by which chronic exposure to nicotine affects a462 AChRs. Second, we briefly review some of the pharmacological properties of neuronal AChRs, especially the differences between a 7 and a8 AChRs and the usefulness of epibatidine (exo-2-(6-chloro-3-pyridyl)-7-azabicyclo-[2.2.l]-heptane) as a ligand for many types of neuronal AChRs.
Mechanism of upregulation of a462 AChRs by chronic exposure to nicotine Tobacco use or chronic exposure to nicotine
127 TABLE 1 Pharmacologicalproperties of a462 AChRs Upregulationa E 5 0 bM)
Function EC50 ( N M ) ~ Activation
Blocking
0.031 f 0.003 0.35 f 0.02 0.073 k 0.01 2.5 2 0.3
-
DMPP Carbamylcholine
0.17 f 0.02 0.21 f 0.04 3.0 f 0.3 15.0 f 4.0
Competitive antagonist Curare
None
-
Noncompetitive untagonist Mecamylamine
65.0 f 6.0
-
Agonists Cytisine Nicotine
0.00014 f O.ooOo3 0.0039 f 0.00021 0.0094 0.0002 0.36 2 0.013
-
*
-
2.7 f 0.5 0.37
Equilibrium bindingb KI 01M)
0.5
25.0
* 14.0
>lo00
aData from Peng et al. (1994a).
bData from Whiting et al. (1991).
causes an increase in brain a462 AChRs by about twofold (Schwartz and Kellar, 1985; Benwell et al., 1988; Flores et al., 1992). Wonnacott, 1990; Wonnacott et al., 1990) suggested that this increase in AChRs was an adaptive response of neurons to maintain nicotinic transmission despite the accumulation of desensitized AChRs due to chronic exposure to this agonist. We have found that agonist-induced upregulation of a462 AChRs is an intrinsic property of this protein which is exhibited when the cloned a462 AChRs are expressed in Xenopus oocytes or in a permanently transfected fibroblast cell line called M10, as shown in Fig. 1 (Peng et al., 1994a). This has now also been confirmed by others (Zhang et al., 1994). The EC5o for nicotine-induced upregulation of M, see Fig. 1) is pathologia4P2 AChRs ( 2 x cally significant because this concentration is equal to the mean steady state serum concentration of nicotine in smokers (Benowitz et al., 1990). The steady state concentration of nicotine in smokers is interesting because it is near the EC50 for activation of a4P2 AChRs by nicotine when applied M), but much greater than the acutely (3.5 x KD for binding nicotine by desensitized a462
M) (see Table 1 which is AChRs (3.9 x adapted from Peng et al., 1994a). Thus, at a steady state nicotine concentration of 2 x IW7M all a462 AChRs in a tobacco user should be desensitized, and the net behavioral effect should reflect inhibition of these AChRs. Chronic exposure to other agonists also causes upregulation in the amount of a462 AChRs, as shown in Fig. 2 (Peng et al., 1994a). The competitive antagonist curare prevents nicotine-induced upregulation. The rank order for efficacy in causing upregulation parallels the rank order of equilibrium binding affinity (presumably to the desensitized conformation) but not quite the rank order for activation (Table 1). The non-competitive antagonist mecamylamine can also causes upregulation and is synergistic with nicotine, as shown in Fig. 3 (Peng et al., 1994a). This parallels the effects of mecamylamine in brain (Collins et al., 1994). Blockage of function by mecamylamine is more effective in the presence of low concentrations of nicotine (Fig. 4). These data are consistent with the idea that mecamylamine is an open channel blocker which is able to bind better when the channel is opened by nicotine (Varanda et al., 1985; Martin et al., 1990).
n4P7 AChH Upregulation
7
111.)
l:'1 10."
Fig. 2. Comparison of dosdresponse relationships for a 4 s 2 upregulation in M10 cells and function in Xenopus oocytes. Expression of a462 AChR in M10 cells grown to confluence was induced by 1 pM dexamethasone for 3 days, then the indicated concentrations of ligands were added for 3 days and finally AChRs were quantitated in solid phase assays using [3HJnicotineto label AChRs immunoisolated through their 82 subunit as in Fig. 1. Voltage clamp measurements on Xenopus oocytes normalized the maximum currents to saturating agonist concentrations. Blockage by mecamylamine was measured at 1 pM nicotine. Data are reproduced from Peng et al. (1994a).
Ion flow through the cation channel of a462 AChRs does not appear to be necessary to trigger upregulation of these AChRs. This is shown by the observation that a channel blocker like mecamylamine can cause upregulation. It is also shown by the observation that the channel blocker chlorisondamine blocks AChR function but does not prevent nicotine-induced upregulation (El-Bizri and Clarke, 1994). The observation that the rank order of various ligands for equilibrium binding to desensitized a462 AChRs better parallels upregulation than does their order for activation (Table 1) is
also consistent with the idea that cation flow is not necessary to trigger upregulation but that induction of a particular conformation of the AChR may be. Nicotine-induced upregulation of a462 AChRs is not due to an increase in brain a4 or j32 mRNA (Marks et al., 1992). Similarly, nicotine-induced upregulation of a462 AChRs does not occur at the transcriptional level. Demonstration of AChR upregulation in Xenopus oocytes injected with fixed amount of a 4 and j32 mRNA is the best evidence that upregulation occurs post transcriptionally (Peng et al., 1994a). The mechanism of increase in a462 AChRs which is induced by nicotine involves a decrease in the rate of destruction of the AChRs in the surface membrane, as shown in Fig. 5 (Peng et al., 1994a). Nicotine, other agonists, and mecamylamine appear to induce a conformation (a type of desensitized conformation, perhaps) which is turned over more slowly than the resting conformation. This pathological change is an interesting contrast with myasthenia gravis, where chronic M
150
loo
-
50 -
0-
L I -
.z
Li
E 'I
E
1% ZE e
5
-
i
In
Nicotine Causes Upregulation
Compeiiiive Antagonist Prevents Upregulation
Noncompeiiiive Antagonist Causes Upregulation
Fig. 3. Effects of antagonists on upregulation of a4B2 AChRs in M10 cells. M10 cells were grown to confluence, AChR expression was induced with dexamethasone and then treated for 3 days with the indicated ligands before solid phase radioimmunoassay. Data are from Peng et al. (1994a).
129
+r ...
~i
IOpM ACh
J
IpA
2 min.
...
Nicotine 0.1pM +Mecaniylamine 3pM
were greatly reduced, even though the total number of AChRs was doubled, as shown in Fig. 6 (Peng et al., 1994a). Short term desensitization is normally reversed by washing over matters of minutes, but this reduction in response only recovered fully after 3 days, which is about the rate expected for synthesis of new AChRs. Native AChRs also exhibit downregulation of function after chronic exposure to nicotine (Marks et al., 1993). The results in oocytes suggest that long term exposure to high concentrations of nicotine can result in a permanently inactivated a4P2 AChR conformation. It is unknown whether this reflects the same nicotine-induced conformation that is turned over more slowly. The loss of functional a462 AChRs on chronic exposure to nicotine due to both reversible and permanent desensitization provides an explanation for tolerance. Thus, chronic tobacco users are tolerant of much higher nicotine concentrations than are nonusers because, even though the chronic users may have twice as many a462 AChRs, most of these excess AChRs can bind nicotine but not function in response to it.
Some pharmacological properties of neuronal AChRs Fig. 4. Mecamylamine blockage of a4P2 AChR function is more effective in the presence of nicotine. a462 AChRs were expressed in Xenopus oocytes. A continuous series of responses to a saturating concentration of acetylcholine is shown from top to bottom. A low concentration of nicotine causes only a small response and little or no blockage to a subsequent test response to acetylcholine. Mecamylamine caused some blockage to a test response to acetylcholine applied immediately after and some recovery was seen after further washing. Applying nicotine and mecamylamine together results in nearly complete blockage of a test response applied immediately after. Data are from Peng et al. (1994a).
exposure to autoantibodies causes a decrease in the amount of muscle AChRs by crosslinking them and thereby facilitating their endocytosis (Lindstrom et al., 1988). We found that after exposing oocytes expressing a462 AChRs to high concentrations of nicotine for 3 days their net responses to acetylcholine
aBgt binds virtually irreversibly to muscle type AChRs, but it has lower affinity for a7 AChRs, still lower affinity for a8 AChRs, and substantially lower affinity yet for a 9 AChRs, as shown in Table 2 (Keyser et al., 1993; Elgoyhen et al., 1994). The muscarinic antagonists atropine and glycinergic antagonist strychnine are nearly as potent as the nicotinic antagonist curare as antagonists of a7, a 8 and a 9 AChRs, as shown in Table 2. Despite the similarities in their N-terminal extracellular amino acid sequences, a7 and a8 AChRs differ in their pharmacological properties, with a7 having higher affinity for aBgt but lower affinity for small cholinergic ligands (Anand et al., 1993; Keyser et al., 1993; Gerzanich et al., 1994). This is reflected in the properties of their homomers. Pharmacological properties of a8 are known only in chick and a 9 only in rat. a 7 from chicks and humans are pharmacologically similar except for
130
Total AChRs
1
400
f Nicotine \\ \
lpM Dexamethasone Induction of a4p2 AChR Expression
DAY
35pM Cycloheximide Inhibition of Protein Synthesis SpM Nicotine Upregulation of a4p2 AChR
Surface AChRs 10°O
1
200
0
Induction of a 4 p 2 AChR Expression
Nicotine
K"
400
XpM Dexamethasone
/*\
/
1
\
\
A
\
'4.
2
3
4
5
6
\
* Control 7
DAYS 35pM Cycloheximide Inhibition of Protein Synthesis
5pM Nicotine Upregulation of a 4 p 2 AChR Fig. 5. Nicotine decreases the turnover rate of a482 AChRs in M I 0 cells. Cycloheximide was used to inhibit the synthesis of new a4B2 AChRs in dexamethasone-induced MI0 cells. In the experiment shown at the top, cycloheximide was added 1 day after dexamethasone induction. Then total a482 AChRs solubilized from the cells was measured by [3H]nicotine binding to immunoisolated AChRs in microwells, In the experiment shown in the lower panel, dexamethasone induction of a482 AChR synthesis was allowed to maximize over 4 days before nicotine was added to one set of cultures. In this case only surface AChRs were measured by binding of 12sI-labeledmAb290 to 82 subunits in intact cells. Data are from Peng et al. (1994a).
131
d c\l
a d t3 ( I
-
Fig. 6. Time course of recovery of a4B2 AChR function after chronic treatme t with nicotine of Xenopus oocytes. After measuring the initial response to 100pM acetylcholine, one group was incubated with 1 p M nicotine, another with lOpM nicotine and a third group was left untreated as a control. After 3 days the oocytes were washed and then responses to lOOpM acetylcholine were measured at the indicated times over the next 3 days. Data are from Peng et al. (1994a).
TABLE 2 Pharmacological properties of a 7 , a 8 and a 9 AChRs
a 7 chick
Antagonists aBgt
Curare Atropine Strychnine Agonists ACh Nicotine DMPP Epibatidine
0.0021e 7.3a
12oa 9.9a
16Oa
1.3a (agonist)'
83a (3.5% partial)c 2.2d agonist
a 7 human
0.001f 2of 1366
9.8'
5.gf
2.6f (agonisOf 4 . 4 (full agonistlf I.ld
a 8 chick
0.017e 5oa OSSa 2.P
0.031a 0.012a (agonist)= 0.3Y(full agonist)c 0.0012d
a 9 rat
Completely reversed in 10 min washb 0.3b
1.3b 0.02b
lob 30 (antagonist)b 5 % partial agonistb ?
aData from Anand et al. (1993a) for IC50 on binding of lzsIaBgt at 2 nM for native a7 and 20 nM for native a 8 AChR. bData from Elgoyhen et al. (1994) for I C 3 on function 10pM ACh. CDatafrom Gerzanich et al. (1994) for EC50 on a7 or a8 homomers. dData from Gerzanich et al. (1995) for EC of (-) epibatidine on a7 or a8 homomers on IC50 in their function. eData from Keyser et al. (1993) for KD Of '%a&$. fData from Peng et al, (1994b) for KD of '%Bgt for ICsn at 2 nM '"IaBgt for native a7 AChRs.
132
Epibatidine 0.3,3,30,300nM
U Acetylcholine
-1 1
-9 -1 -5 Concentration (log M)
I 200nA
10 sec.
‘Acetylcholine 3 0 ~ M Fig. 7. Activation by epibatidine of chicken a4B2 AChRs expressed in Xenopus oocytes. The top panel shows the responses to different concentrations of epibatidine and compares dosdresponse curves for epibatidine with those of nicotine and acetylcholine. The bottom panel compares the time courses of responses to saturating concentrations of epibatidine and nicotine. Data are from Gerzanich et al. (1995).
DMPP, which is virtually an antagonist on chick, however, a very potent full agonist on human a7 (Gerzanich et al., 1994; Peng et al., 1994b). It would be extremely useful to have a universal labeling reagent for AChRs. aBgt has long been an excellent ligand for muscle AChRs and a7 AChRs (Table 2). Nicotine has been a useful label for a462 AChRs (Table 1). Cholinergic labeling a 3 AChRs has been difficult because of their relatively low affinity for nicotine, and the failure of neuronal bungarotoxin to bind to detergent solubilized AChRs, so they have long been quantitated using 125ImAb35 (Smith et al., 1985). This mAb binds to the main immunogenic region on the extracellular surface of a 1 subunits (Saedi et al., 1990) and to homologous structures on chick a5 subunits (Conroy et al., 1992; Conroy and Berg,
1995) and human a 3 and a5 subunits (Wang et al., 1996). Epibatidine is now proving to be a very useful ligand for several subtypes of neuronal AChRs, including a 3 AChRs. Epibatidine was initially discovered in the skin of Ecuadoran poison frogs by John Daly (Spande et al., 1992). The frogs derive it from an unknown component of their diet in the wild (Daly, 1995). It has now been synthesized (e.g. Fletcher et al., 1994; reviewed in Broke, 1994). Initially it was discovered to be 200-fold more potent than morphine in preventing nociception. Later it was discovered to be a potent nicotinic agonist (Badio and Daly, 1994) and to elicit a variety of nicotinic effects (Sullivan et al., 1994). Epibatidine is the most potent neuronal AChR
133
Human a3P2 AChRs Expressed in Xenopus Oocytes
-
Epibatidine 3, 10,30,100 nM
2 0.6
sE 0.4
0.2
0 -10
-8
-6
-4
Concentration of Agonist (log M)
-2
Human a3P4 AChRs Expressed in Xenopus Oocytes Edbatidine 10.30.100.300 nM
10 sec.
Concentration of Agonist (log M)
Fig. 8. Activation by epibatidine of human a3j32 and a3j34 AChRs expressed in Xenopus oocytes. The top panel shows responses of a3j32 AChRs to various concentrations of epibatidine and compares dosdresponse curves for epibatidine, nicotine and acetylcholine. The bottom panel makes a similar comparison for a464 AChRs. Data are from Gerzanich et al. (1995).
agonist that we have studied on several types of cloned AChRs expressed in Xenopus oocytes, and r3H]epibatidine is an excellent labeling reagent which has proven especially useful for a3 AChRs (Gerzanich et al., 1995). Epibatidine is more potent than nicotine as an agonist for a462 AChRs, as shown in Fig. 7, or for a3p2 or a3p4 AChRs, as shown in Fig. 8. Note that Fig. 8 also illustrates the important concept that 82 and 8 4 play an important role in determining the affinity and efficacy of ligands (reviewed in detail by Papke, 1993). This is a consequence of the acetylcholine binding site being formed at the interface between a subunits
and adjacent structural subunits (Blount and Merlie, 1989). The extremely high affinity of r3H]epibatidine as a labeling reagent is illustrated for human a3p2 AChRs in Fig. 9. Epibatidine is still a more potent agonist than either nicotine or acetylcholine on both a7 and electric organ AChRs, but much less potent than on a 4 or a3 AChRs, as shown in Fig. 10. Thus, despite its potency as an agonist, epibatidine does not quite provide a universal ligand for AChRs. However, the AChRs for which it has relatively low affinity, like a1 and a7 AChRs, can be efficiently labeled with *=IaBgt. The affinity of
134 12 1 aJ
.E
10 i
**
16--
0
0
_-.
/A--
2 [3H]
a462 AChRs is highest for epibatidine, making it a better label than [3H]nicotine. But the very high affinity of [3H]epibatidine for a 3 AChRs provides a really useful breakthrough. Epibatidine is likely to become an important reagent for several types of neuronal AChRs. It is also likely to renew interest in an important role of nicotinic AChRs in nociception. Although epibatidine is not highly selective among neuronal AChR subtypes, which can be a pharmacological drawback, it is a small molecule structure that can provide remarkably high affinity for these AChRs.
I
i
I
R,,,=lxl0l5M
0
4
6
8
lo
Epibatidine (1O'OM)
Acknowledgements
Fig. 9. Binding of racemic [3H]epibatidine to human a3B2 AChRs expressed in oocytes. Physiological experiments of the types shown in Figs. 7 and 8 with + and - isomers of eoibatidine revealed sumrisinaly - - small differences in responses to the two isomers. Data are from Gerzanich et al. (1995).
The laboratory of Jon Lindstrom is supported by grants from the N m S (NS11323), The blokeless Tobacco Research Council, Inc., The Council for Tobacco Research - USA, Inc., and the Muscular Dystrophy Association.
Epibatidine 0.1,0.3, 1 , 3 pM
-(+)-
+Nicotine
w
Epibatidine
Concentration of Agonist
(log M)
Epibatidine 0.3,1,3, 10 pM
1
0.8
g 0.6 E
I
2 0.4
0.2
0
Concentration of Agonist (log M)
Fig. 10. Activation by epibatidine of human a7 homomers and muscle type Torpedo alplyd AChRs expressed in oocytes. Data are from Gerzanich et al. (1995).
135
References Alkondon, M., Reinhardt, S., Lobron C., Hermsen, B., Maelicke, A. and Albuquerque, E. (1994) Diversity of nicotinic acetylcholine receptors in rat hippocampal neurons. 11. The rundown and inward rectification of agonistelicited whole cell currents and identification of receptor subunits by in situ hybridization. J. Pharmacol. Exp. Ther., 27 1: 494-506. Anand, R., Conroy, W.G., Schoepfer, R., Whiting, P. and Lindstrom, J. (1991) Chicken neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes have a pentameric quaternary structure. J. Biol. Chem., 266: 1119211198. Anand, R., Peng, X., Ballesta, J. and Lindstrom, J. (1993a) Pharmacological characterization of a bungarotoxin sensitive AChRs immunoisolated from chick retina: contrasting properties of a 7 and a 8 subunit-containing subtypes. Mol. Pharmacol., 44: 1046-1050. Anand, R., Peng, X. and Lindstrom, J. (1993b) Homomeric and native a 7 acetylcholine receptors exhibit remarkably similar but nonidentical pharmacological properties suggesting that the native receptors is a heteromeric protein complex. FEES Letr., 327: 241-246. Badio, B. and Daly, J. (1994) Epibatidine, a potent analgesic and nicotinic agonist. Mol. P h a m c o l . , 45: 563-569. Benowitz, N., Porchet, H. and Jacob, P. (1990) Pharmacokinetics, metabolism and pharmacodynamics of nicotine. In: S. Wonnocott, M. Russell and I. Stolerman (Eds.), Nicotine Psychophannacology, Oxford Science Publications, Oxford, UK, pp. 112-157. Benwell, M., Balfour, D. and Anderson, J. (1988) Evidence that tobacco smoking increases the density of (-H3H] nicotine binding sites in human brain. J. Neurochem., 5 0 1243- 1247. Blount, P. and Merlie, J.P. (1989) Molecular basis of the two nonequivalent ligand binding sites of the muscle nicotinic acetylcholine receptor. Neuron, 3: 349-357. Broke, C. (1994) Synthetic approaches to epibatidine. Med. Chem. Res., 4: 4 4 W 8 . Changeux, J.P. (1990) Functional architecture and dynamics of the nicotinic acetylcholine receptor: an allosteric ligandgated ion channel. In: 1988-1989 Fidia Research Foundntion: Neuroscience Award Lechwes, Vol. 4, pp. 21168. Cimino, M., Marini, P., Colombo, S., Andena, M., Cattabeni, Fornasari, D. and Clemente, F. (1995) Expression of neuronal acetylcholine receptor a 4 and 8 2 subunits during postnatal development of the rat brain, J. Neural Tramm., 100: 77-92. Clarke, P. (1995) Nicotinic receptors and cholinergic transmission in the central nervous system, Ann. N. Y. Acad. Sci., 757: 73-83. Collins, A., Luo, Y.,Selvaag, S. and Marks, M. (1994) Sensitivity to nicotine and brain nicotinic receptors are altered by
chronic nicotine and mecamylamine infusion, J . Phannacol. Exp. Ther., 271: 125-133. Conroy, W. and Berg, D. (1995) Neurons can maintain multiple classes of nicotinic acetylcholine receptors distinI Biol. . Chem., guished by different subunit compositions. . 270: 4424-443 1. Conroy, W.. Vernallis, A. and Berg, D. (1992) The a 5 gene product assembles with multiple acetylcholine receptor subunits to form distinctive receptor subtypes in brain. Neuron, 9: 1-20. Cooper, E., Couturier, S. and Ballivet, M. (1991) Pentameric structure and subunit stoichiometry of a neuronal nicotinic acetylcholine receptor. Nature, 350: 235-238. Daly, J. (1995) The chemistry of poisons in amphibian skin. Proc. Natl. Acad. Sci. USA, 92: 9-13. Eisile, J.L., Bertrand, S., Galzi, J.L., Devillers-Thiery, A., Changeux, J.P. and Bertrand, D. (1993) Chimaeric nicotinic-serotonergic receptor combines distinct ligand binding and channel specificities. Nature, 366: 479409. El-Bizxi, H. and Clarke, P. (1994) Regulation of nicotinic receptors in rat brain following quasi-irreversible nicotinic blockade by chlorisondamine and chronic treatment with nicotine. Br. J. fhannacol., 113: 917-925. Elgoyhen, A., Johnson, D., Boulter, J., Vetter, D. and Heinemann, S. (1994) a9: an acetylcholine receptor with novel pharmacological properties expressed in rat cochlear hair cells. Cell, 79: 705-715. Fletcher, S., Baker, R., Chambers, M., Herbert, R., Hobbs, S., Thomas, S.,Vemer, H., Watt, A. and Ball, R. (1994) Total synthesis and determination of the absolute configuration of epibatidine. J. Org. Chem., 59: 1771-1778. Flores, C., Rogers, S.,Pabreza, J., Wolfe, B. and Kellar, K. (1992) A subtype of nicotinic cholinergic receptor in rat brain is composed of a 4 and 8 2 subunits and is upregulated by chronic nicotine treatment. Mol. Pharmacol., 41: 31-37. Fuchs, P. and Murrow, B. (1992) A novel cholinergic receptor mediates inhibition of chick cochlear hair cells. f r o c . R. SOC.London Ser B, 248: 35-40. Galzi, J.L., Devillers-Thiery, S., Hussy, N., Bertrand, S., Changeux, J.P. and Bertrand, D. (1992) Mutations in the channel domain of a neuronal nicotinic receptor convert ion selectivity from cationic to anionic. Nature, 359: 500505. Gerzanich, V., Anand, R. and Lindstrom, J. (1994) Homomers of a 8 subunits of nicotinic receptors functionally expressed in Xenopus oocytes exhibit similar channel but contrasting binding site properties compared to a 7 homomers. Mol. P h a m c o l . , 45: 212-220. Gerzanich, V., Peng, X., Wang, F., Wells, G., Anand, R., Fletcher, S. and Lindstrom, J. (1995) Comparative pharmacology of epibatidine - a potent agonist for neuronal nicotinic acetylcholine receptors. Mol. P h a m c o l . , 48: 774782. Gotti, C., Hanke, W., Maury, K., Moretti, M., Ballivet, M., Clemente, F. and Bertrand, D. (1994) Pharmacology and biophysical properties of a 7 and a 7 4 3 a bungarotoxin re-
136 ceptor subtypes immunopurified from the chick optic lobe. Eur. J. Neurosci., 6: 1281-1291. Hamassaki-Britto, D., Gardino, P., Hokoc, J., Keyser, K., Karten, H., Lindstrom, J. and Britto, L. (1994) Differential development of a bungarotoxin-sensitive and a bungarotoxin-insensitive nicotinic acetylcholine receptors in the chick retina. J. Comp. Neurol., 347: 161-170. Henley, J., Lindstrom, J. and Oswald, R. (1986) Acetylcholine receptor synthesis in retina and transport to the optic tectum in goldfish. Science, 232: 1627-1629. Jacob, .M. and Berg, D. (1983) The ultrastructural localization of a bungarotoxin binding sites in relation to synapses on chick ciliary ganglion neurons. J. Neurosci., 3: 260-271. Karlin, A. (1991) Explorations of the nicotinic acetylcholine receptor. Harvey Lectures Ser., 85: 71-107. Karlin, A. (1993) Structure of nicotinic acetylcholine receptors. Curr. Opin. Neurobiol., 3: 299-309. Keyser, K., Britto, L., Schoepfer, R., Whiting, P.,Cooper, J., Conroy, W., Brozozowska-Prechtl, A., Karten, H. and Lindstrom, J. (1993) Three subtypes of alpha bungarotoxinsensitive nicotinic acetylcholine receptors are expressed in chick retina. J. Neurosci., 13: 442-454. Lindstrom, J. (1995) Nicotinic acetylcholine receptors. In Alan North (Ed.), CRC Handbook of Receptors. CRC Press, pp. 153-175. Lindstrom, 1. (19%) Neuronal nicotinic acetylcholine receptors. In: T. Narahashi (Ed.),Ion Channels, Vol. IV, Plenum, New York, pp. 377-450. Lindstrom, J., Shelton, G.D. and Fujii, Y. (1988) Myasthenia gravis. Adv. Immunol., 42: 233-284. Marks, M., Pauly, J., Gross, D., Deneris, E., HermansBorgmeyer, I., Heinemann, S. and Collins, A (1992) Nicotine binding and nicotinic receptor subunit RNA after chronic nicotine treatment. J. Neurosci., 12: 27652784. Marks, M., Grady, S. and Collins, A. (1993) Downregulation of nicotinic receptor function after chronic nicotine infusion. J. Pharmacol. Exp. Ther., 266: 1268-1275. Martin, T., Suchocki, J., May, E. and Martin, B. (1990) Pharmacological evaluation of the antagonism of nicotines central effects by mecamylamine and pempidine. J. Phurm. Exp. Ther., 254: 45-5 1. McGehee, D. and Role, L. (1995) Physiological diversity of nicotinic acetylcholine receptors expressed by vertebrate neurons. Annu. Rev. Physwl., 57: 521-546. Papke, R. (1993) The kinetic properties of neuronal nicotinic receptor: genetic basis of functional diversity. Prog. Neurobiol., 41: 5 W 5 3 1 . Peng, X.,Gerzanich, V., Anand, R., Whiting, P. and Lindstrom, J. (1994a) Nicotine-induced upregulation of neuronal nicotinic receptors results from a decrease in the rate of turnover. Mol. Pharmacol., 46:523-530. Peng, X., Katz, M., Gerzanich, V., Anand, R. and Lindstrom, J. (1994b) Human a 7 acetylcholine receptor: cloning of the a 7 subunit from the SH-SY5Y cell line and determination of pharmacological properties of native receptors and func-
tional a 7 homomers expressed in Xenopus oocytes. Mol. Phannacol., 45: 546-554. Pereira, E., Alkondon, M., Reinhardt-Maelicke, S., Maelicke, A., Peng, X.,Lindstrom, J., Whiting, P. and Albuquerque, E. (1994) Physostigmine and galanthamine reveal the presence of the novel binding site on the a 4 8 2 subtype of neuronal nicotinic acetylcholine receptor stably expressed in fibroblast cells. J. Pharmacol. Erp. Ther., 270: 768-778. Pugh, P. and Berg, D. (1994) Neuronal acetylcholine receptors that bind a bungarotoxin mediate neurite retraction in a calcium-dependent manner. J. Neurosci., 14: 889-896. Saedi, M., Anand. R., Conroy, W.G. and Lindstrom, J. (1990) Determination of amino acids critical to the main immunogenic region of intact acetylcholine receptors by in vitro mutagenesis. FEBS Lett., 267: 55-59. Sargent, P. (1993) The diversity of neuronal nicotinic acetylcholine receptors. Annu. Rev. Neurosci., 16: 4 0 3 4 3 . Sargent, P. and Wilson, H. (1995) Distribution of nicotinic acetylcholine receptor subunit immunoreactivities on the surface of chick ciliary ganglion neurons. In: P. Clarke, M. Quick, F. Adlkofer and K. Thurau (Eds.), Eflects of Nicotine on Biological Systems II ,Birkhauser, Basel. Schoepfer, R., Conroy, W.G., Whiting, P., Gore, M. and Lindstrom, J. (1990) Brain alpha-bungarotoxin-binding protein cDNAs and mAbs reveal subtypes of this branch of the ligand-gated ion channel superfamily. Neuron, 5 : 3548. Schwartz, R. and Kellar, K. (1985) In vivo regulation of [3H] acetylcholine recognition sites in brain by nicotinic cholinergic drugs. J. Neurochem., 45: 427-433. Seguela, P., Wadiche, J., Dinelly-Miller, K., Dani, J. and Patrick, J. (1993) Molecular cloning, functional properties and distribution of rat brain a7: a nicotinic cation channel highly permeable to calcium. J. Neurosci., 13: 596-604. Smith, M., Stollberg, J., Lindstrom, J. and Berg, D.K. (1985) Characterization of a component in chick ciliary ganglia that cross-reacts with monoclonal antibodies to muscle and electric organ acetylcholine receptor. J. Neurosci., 5 : 27262731. Spande, T., Garraffo, M., Edwards, M., Yeh, H., Pannel, L. and Daly, J. (1992) Epibatidine: a novel (chloropyridyl) azabicyclo-heptane with potent analgesic activity from Ecuadoran poison frog. J. Am. Chem. Soc., 114: 34753478. Sullivan, J., Decker, M., Brioni, J., Donnelly-Roberts, D., Anderson, D., Bannon, A., Kang, C., Adams, P., PiattoniKaplan, M., Buckley, M., Gopalakrishnan, M., Williams, M. and Americ, S. (1994) (*)-Epibatidine elicits a diversity of in vitro and in vivo effects mediated by nicotinic acetylcholine receptor. J. Pharmacol. Erp. Ther., 271: 624-631. Swanson, L., Simmons, D., Whiting, P. and Lindstrom, J. (1987) Immunohistochemical localization of neuronal nicotinic receptors in the rodent central nervous system. J. Neurosci., 7: 3334-3342. Unwin, N. (1993) Nicotinic acetylcholine receptor at 9 A resolution. J. Mol. Biol., 229: 1101-1124.
137 Unwin, N. (1995) Acetylcholine receptor channel imaged in the open state. Nature, 373: 3 7 4 3 . Varanda, W., Aracava, A., Sherby, S., Van Meter, W., Eldefrawi, M. and Albuquerque, E. (1985) The acetylcholine receptor of the neuromuscular junction recognizes mecamylamine as a noncompetitive antagonist. Mol. Phrmacol., 28: 128-137. Wang, F., Ferzanich, V., Wells, F., Anand, R., Peng, X.,Keyser, K. and Lindstrom, J. (1996) Assembly of human neuronal nicotinic receptor a5 subunits with a3, 8 2 and 84 subunits. J. Biol. Chem. 271: 17656-17665. Whiting, P.J. and Lindstrom, J.M. (1988) Characterization of bovine and human neuronal nicotinic acetylcholine receptors using monoclonal antibodies. J. Neurosci., 8: 33953404. Whiting, P., Schoepfer, R., Lindstrom, J. and Priestley, T. (1991) Structural and pharmacological characterization of
the major brain nicotinic acetylcholine receptor subtype stably expressed in mouse fibroblasts. Mol. Pharmacol., 40: 463472. Wonnacott, S. (1990) The paradox of nicotinic acetylcholine receptor upregulation by nicotine. Trends Pharmacol. Sci., 11: 216-219. Wonnacott, S., Russell, M. and Stoleman, I. (1990) Nicotine Psychopharmacology: Molecular, Cellular and Behavioral Aspects, Oxford Science Publications, Oxford, UK. Zhang, X.,Gang, Z.H., Hellstrom-Lindahl, E. and Nordberg, A. (1994) Regulation of a462 nicotinic acetylcholine receptors in M I 0 cells following treatment with nicotinic agents. NeuroReport, 6: 3 13-3 17. Zoli, M., LeNovere, N., Hill, J. and Changeux, J.P. (1995) Developmental regulation of nicotinic ACh receptor mRNAs in the central and peripheral nervous systems. J. Neurosci., 15: 19 12-1 939.