- Email: [email protected]
Chapter 11: Functional diversity of neuronal nicotinic acetylcholine receptors
A.C. Cuello (Editor) Pmgress in Brain Research, Vol. 98 0 1993 Elsevier Science Publishers B.V. All rights reserved
113
CHAPTER 1 1
Functional diversity of neuronal nicotinic acetylcholine receptors Jim Patrick, Philippe SCquCla, Steven Vernino, Mariano Amador, Chuck Luetje and John A. Dani Division of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA
Introduction The neuronal nicotinic acetylcholine receptors are likely to be oligomeric proteins composed of five subunits (Cooper et a]., 1991). If these receptors were all homo-oligomers, there might be one unique receptor that corresponded to each pentameric arrangement of each of the available subunits. The situation is more complicated than that because most of the neuronal nicotinic receptors are heterooligomers. These hetero-oligomeric receptors contain both alpha and beta subunits. Because individual alpha subunits can participate in the formation of functional receptors with more than one kind of beta subunit, the number of possible receptors is larger. There are ten known members of the gene family that encodes the subunits of the neuronal nicotinic acetylcholine receptors. These genes and their products are called alpha2, alpha3, alpha4, alpha5, alpha6, alpha7, alpha8, beta2, beta3 and beta4 (see Boulter et al., 1986, 1987, 1990; Deneris et al., 1987, 1988; Goldman et al., 1987; Nef et al., 1987, 1988; Wada eta].; 1988; Duvoisin et al., 1989; Couturier et al., 1990; Lindstrom et a].; 1990; Luetje et al., 1990; Shoepfer et al., 1990; Lamar et al., 1992, and for the properties of these subunits in both rat and chick). These subunits are known to form at least eight different receptors; homo-oligomeric alpha7 and alpha 8 receptors and hetero-oligomeric receptors composed of alpha2heta2, alpha3lbeta2, alpha4heta2, alpha2lbeta4, alpha3lbeta4, and alpha4heta4. There are several reasons why this diversity is Likely to increase. First, there are members of the gene family (alpha5, alpha6, and beta3) whose functional roles in neuronal nicotinic receptors have not yet been demonstrated. Second, there may well be members of the gene family that have not yet been identified and finally, it seems likely that receptors containing more than one kind of alpha or more than one kind of beta subunit are allowed.
Several different acetylcholine-gated ion channels can be formed from the members of this gene family. We expect that the various subunits, each with different primary structures, will form oligomeric receptors having different properties. One question then is how do receptors with different subunit combinations differ from one another. We know, for example, that different combinations of subunits differ in their single channel properties (Papke et al., 1989) and in their responses to different agonists and antagonists (Luetje et al., 1990, 1991). They also differ in their permeability to cations and in their modulation by external cations (Vernino et al., 1991; Vernino and Dani, 1992; Mulle, 1992a,b). The extensive diversity of their cytoplasmic domains suggests additional diversity in their functional interactions with the cytoplasmic machinery. A second important question is what role do these different receptors play in the central nervous system and how does the central nervous system make use of this diversity of receptor types. The genes encoding these subunits are, in general, expressed in discrete but overlapping sets of brain nuclei (Wada et al., 1989, 1990; Miller and Patrick, 1992). While some brain nuclei can be characterized by the specific combination of neuronal nicotinic receptors that they express, other regions are less well defined. For example, almost all the known members of the gene family are expressed in the medial habenula. In this case, however, it is not known if a single cell expresses all these different genes or if specific combinations of subunits define cellular types in the habenula. In the cerebellum, however, the case is more extreme. All Purkinje cells express the beta2 gene and either alpha2, alpha3 or alpha4 (Wada et a]., 1989). Adjacent Purkinje cells may each express a beta2 subunit but differ in the alpha subunit gene expressed. This suggests a remarkable heterogeneity in receptor phenotype in what appears, and is usually treated as a homogeneous population of cells.
I I4 TABLE I
Receptor diversity Open time Conductance Ion selectivity Ligand selectivity Extracellular modulation Local cytoplasmic regulation Distribution on single neurons Distribution in central nervous system Developmental regulation An important question then is why are there so many different kinds of receptors. Table I is a partial list of the attributes of ligand-gated ion channels that might be determined by the primary structure of their constituent subunits. Receptors composed of structurally different subunits might be expected to exhibit different biophysical properties such as single channel conductance, open time or ion selectivity. They might also produce receptors that could be differentially distributed over the cell surface or that might be responsive to different cytoplasmic regulatory mechanisms. Additionally, the central nervous system may require neuronal nicotinic receptors with specific properties at different times during development and the diversity of genes might reflect the requirement that they be capable of independent expression during development. Our understanding of the role that these receptors play in development is meager at best. It is possible, however, that understanding the role of these receptors in the plasticity characteristic of the nervous system in the adult will help us formulate specific testable ideas about roles that they might play during development. The following paragraphs document several examples of the functional heterogeneity of the neuronal nicotinic receptors and present new properties that characterize these receptors. Specifically, we review data showing the pharmacological diversity of neuronal nicotinic acetylcholine receptors and show that these receptors differ from the muscle nicotinic acetylcholine receptors in their permeability to calcium and in their modulation by external calcium ions.
tor (Luetje and Patrick, 1991). The structures of the four agonists we tested are seen in Fig. 1, We examined the six possible alphaheta pairwise combinations of alpha2, alpha3, alpha4, beta2 and beta4 using the oocyte expression system. Xenopus oocytes were injected with RNAs encoding an alpha and a beta subunit and the expressed functional receptors were assayed using a twoelectrode voltage clamp. Because responses in individual oocytes show substantial variation, all responses were normalized to the current generated by 1 pM acetylcholine. The data in Fig. 2 show dose response curves for each of the ligands seen in Fig. 1 tested on each alphdbeta combination. The panels across the top correspond to receptors comprised of beta2 subunits and different alpha subunits and those across the bottom correspond to receptors comprised of a beta4 subunit and different alpha subunits. It is clear that the responses to ligands vary across the top panels with respect to the alpha subunits. There is about a 50-fold difference in the ratio of sensitivities of nicotine and acetylcholine between alpha2-containing receptors and alpha3containing receptors and suggests that the alpha subunits play an important role in determining the agonist specificity of these receptors. This is consistent with results obtained using antagonists. Neuronal neurotoxin blocks receptors containing an alpha3 subunit but has little effect on receptors in which the alpha subunit is alpha2 (Wada et al., 1988). Receptors containing alpha2 are almost 1000-fold less sensitive to the antagonist neuronal bungarotoxin than receptors containing the alpha3 subunit (Luetje et al., 1990). None of the receptors in the top panels are activated by cytosine. But, cytosine is the best of the four agonists on all of the beta4-containing receptors in the bottom panel. The fact that this abrupt change in rank order of potency occurs through the substitution of the beta4 subunit for the beta2
0
II H,C-C-O-CH~-CH,-N+-CH,
\
I
II
CH3
Acetylcholine
Nicotine
Cytisine
Dimethylphenylpiperazinium
Pharmacological diversity of neuronal nicotinic receptors Different combinations of subunits might produce receptors with different affinities for either agonists or antagonists. We tested this possibility for both agonists and antagonists and found that this ligand selectivity is determined by both the alpha and beta subunits that comprise any given recep-
(DMPP)
Fig. I . Structures of nicotinic acetylcholine receptor agonists.
I15
failure of cytosine to activate receptors containing the beta2 subunit is not a consequence of a lack of affinity of the ligand for the receptor. In fact, cytosine binds to receptors containing the beta2 subunit and prevents activation by acetylcholine (Luetje and Patrick, 1991).
subunit demonstrates that the beta subunits also contribute to ligand selectivity. This might occur through modification of a ligand binding site uniquely on the alpha subunit or through actual contributions of residues in the beta subunit to the actual composition of the ligand binding site. The -.
. . . . . . . .,
JJ): .. .,
. . . . .._, . . . . . . . .,
B. a2P4
A. a2P2
.-
Concentration, M
p)
i C.a3P2 5000 -
$.
4000
z d
I
Nic ACh
-
DMPP
0 1000
10.'
.9
B
2
6 4oo 0
Concentration, M
1
200
0
Concentration, M Fig. 2. Each neuronal nAChR subunit combination has unique pharmacological properties. Agonist-induced current responses of oocytes expressing a2P2 (A), a2p4 (B), a3P2 (0,a3w (D), a4p2 ( E ) or a4p4 (0 were measured under two electrode voltage clamp at a holding potential of -70 mV. Currents were elicited by bath application of various concentrations of acetylcholine (open circles), nicotine (closed circles), cytosine (triangles) and DMPP (squares). Responses are presented after normalization to the response of the same oocyte to 1 M acetylcholine (response to 1 M acetylcholine =loo). Each point represents the mean f SD of the responses of 3-8 separak oocytes.
I16
In addition to the determination of agonist selectivity, the beta4 subunit also modifies the interaction with neuronal bungarotoxin. As noted above, receptors containing the alpha3 subunit in combination with beta2 are blocked by low concentrations of neuronal bungarotoxin. However, replacement of the beta2 by the beta4 subunit renders these alpha3-containing receptors resistant to neuronal bungarotoxin. It is clear, therefore, that the beta subunits must be considered as partial determinants of the ligand binding site. Different alpha and beta subunits combine to make receptors that respond differently to the four ligands in Fig. 1. It is clear that both alpha and beta subunits contribute to the ligand selectivity of the receptors they form. It is not clear how these differences might influence the behavior of these receptors in the CNS but they do offer the investigator interested in cholinergic mechanisms in the brain a powerful tool with which to selectively activate or inhibit certain nicotinic cholinergic pathways.
Calcium permeability of neuronal nicotinic receptors The response of neuronal receptors in the oocyte differs from that of the muscle nicotinic receptors in that a greater portion of the current through nicotinic receptor channels is carried by Ca2+ ions. The oocyte contains an endogenous Ca2+-activated CI- channel. Because the reversal potential for CI- in the oocyte is about -25-30 mV, the chloride current appears as an inward cationic current at the normal holding potentials of -50 to -60 mV. Application of blockers of this endogenous Ca2+-activated CI- channel, such as niflumic acid or flufenamic acid reduces the current associated with activation of the neuronal receptors but has little effect on the current associated with activation of the muscle receptors (see Fig. 3). This result suggested that the neuronal nicotinic acetylcholine receptors differed from the muscle nicotinic acetylcholine receptors by being more permeable to calcium ions. We tested this possibility by examining the change in the reversal potential produced by increasing the external calcium concentration. As seen in Fig. 4 increasing the calcium concentration from 1 to 10 mM has only a small effect (1 mV) on the muscle receptor but has a more marked effect on the neuronal receptor comprised of an alpha3 and beta4 subunits (+7 mV). This result demonstrates that this neuronal receptor is more permeable to calcium relative to sodium than is the muscle receptor and suggests that a larger portion of the current through this receptor is carried by calcium ions. This idea is consistent with the observation that a larger portion of the current elicited by activation of this receptor in the oocyte disappears when the endogenous Ca2+-activated CIchannel is blocked by flufenamic and niflumic acid.
The alpha7 gene product forms a functional homooligomeric receptor in the Xenopus oocyte (Couturier et al., 1990). This ability to form a homo-oligomeric receptor distinguishes the alpha7 subunit from the other neuronal nicotinic receptor subunits. It is further distinguished by its pharmacology; the homo-oligomeric alpha7 receptor is blocked by strychnine with an EC,, of about 350nM. Strychnine is best known as an antagonist of the glycine receptor. The alpha7 homo-oligomer is also distinguished by the large contribution the endogenous Ca2+-activated CI- channel makes to the whole cell current in the oocyte. Blocking the endogenous Ca2+-activated CI- channel reduces the agonist induced alpha7 current by about 85% suggesting that the alpha7 current has a large Ca2+ component that subsequently activates the Ca2+-activated CI- channel. We tried to assess the contribution of this Ca2+ component by measuring the effect of increasing the external Ca2+ concentration on the reversal potential but were unable always to block the endogenous Ca2+-activated CI- current completely using flufenamic and niflumic acid. We therefore replaced the CI- with methanesulfonate, an impermeant anion. The results in Fig. 4 show the change in the reversal potential obtained in the presence of methane-
h '
' I
I
Fig. 3. Currents through neuronal nAChRs activate a Ca2+dependent C1- conductance. Muscle and a3p4 neuronal nAChRs were expressed in separate Xenopus oocytes. The currents induced by 10 pM Ach were measured in the presence and absence of 100 pM niflumic acid and 100 pM flufenamic acid, which inhibit the Ca2+-dependentCI-channel. The ACh-induced currents seen with oocytes expressing muscle nAChRs were unchanged by the CIchannel blockers, but the currents seen with neuronal nAChRs were smaller in the presence of blockers. The results indicate that Ca2+ carries enough current through neuronal nAChRs but not through muscle nAChRs to activate the Ca2+-dependent C1conductance. The external solution contained 18 mM CaCI2. The currents were measured in blockers, then the blockers were washed off.
1 I7
shift of the reversal potential observed under our experimental conditions, we can calculate that the PCalPNaratio for the rat alpha7 channel is around 20. This is higher than the PcJPNa ratio of 5 reported for the NMDA subtype of glutamate receptors (Mayer and Westbrook, 1987; Iino et al., 1988). Application of nicotine to chick ciliary ganglion neurons results in an a-BTX sensitive increase in cytoplasmic calcium (Vijayaraghavan et al., 1992). Our data suggest that at least a portion of this increase could result from the calcium flux through an alpha7-type receptor.
50 .
Modulation of neuronal nicotinic receptors by calcium ions There are additional interesting data in Fig. 3. Increasing the concentration of calcium in the external medium has
.loo0
20
.................................. 10
0
10
20
30
40
I
Muscle
Neuronal
0 18
18
50
Voltage ImV)
Fig. 4. Reversal potential shift comparisons between muscle, a3p4 and a7 cation channels expressed in Xenopus oocytes. Peak currents elicited by 2 mM acetylcholine (al&S or 20 mM nicotine (a3j34 and a7) were measured at different holding potentials and plotted as a function of membrane potential (voltage) in chloride-free Ringer containing 1 mM Ca2+ (solid circles) and 10 mM Ca2+ (open circles). To remove internal CI-, the oocytes were pre-incubated 224 h in chloride- and magnesiumfree methane sulfonate-based recording buffer before measurements. Reversal potential shift values (AErev) correspond to the mean f SD of the number ( n ) of measurements made in different oocytes. The current-voltage relationships illustrated are representative of the experiments performed for each combination of nicotinic receptor subunits. As expected from previous work, external Ca2+ decreases the amplitude of the response of muscle nicotinic acetylcholine receptors (Vijayaraghavan et al., 1992) but increases the response of neuronal nicotinic acetylcholine receptors (Miller and Patrick, 1992) and of a7 receptors.
sulfonate when the external calcium is increased from 1 to 10 mM. The observed shift of about 30 mV is larger than that obtained for either the muscle or any of the heterooligomeric neuronal nicotinic receptors examined thus far. We can use these data to place the apparent relative Ca2+ permeability of the alpha7 homo-oligomer within the context of the permeabilities of other known ligand gated ion channels. For example, a CdNa permeability ratio (Pca/PNa)of 0.2 was reported for the muscle nicotinic receptor (Adam et al., 1980; Vernino and Dani, 1992) and PCa/PNaof about 1.5 was reported for the neuronal nicotinic receptors present in chromaffin cells (Vernino and Dani, 1992) or in PC12 cells (Sands et al., 1990). From the
2.5-
5
c
10 s Neuronol
2.0-
L
D
e,
1.5 1.0
-
b 0.5
.
!.
E
z
0.0
I
Muscle
0.3
1 [Co"]
3
10
30
(mM)
Fig. 5. Ca2+ modulates currents through nAChRs. ACh-induced currents are shown for Xenopus oocytes expressing muscle nAChR or a3P4 neuronal nAChR. The Ca2+ concentration of the test solution is given in mM beside each trace. For the muscle nAChRs, the currents overlap in 0.18 and 1.8 mM Ca2+. The holding potential was -40 mV, and the ACh concentration was 10 pM. The plot at the bottom shows the concentration-dependence of the Ca2+ effect. The ACh-induced currents normalized for each oocyte to the value in 1.8 mM Ca2+are plotted as a function of the external Ca2+ concentration. Increasing the external Ca2+ concentration decreases the ACh-induced currents through muscle nAChRs and increases the ACh-induced currents through neuronal nAChRs. The data points represent 3-15 trials. The enhancement by external Ca2' was observed with four neuronal ap-subunit combinations that we tested.
two consequences. It shifts the reversal potential to more positive potentials and it changes the magnitude of the whole cell currents. The data in Fig. 5 extend this observation over a range of Ca2+ concentrations. In the case of the muscle nicotinic receptor, increasing the external calcium decreases the whole-cell currents. In the case of the neuronal nicotinic receptor, increasing external calcium increases the whole-cell currents. The decrease in the whole-cell current seen with the muscle nicotinic acetylcholine receptor is understood to be due to a decrease in the single channel currents (Decker and Dani, 1990). Calcium can enter the muscle nicotinic receptor channel but does not permeate readily and thus reduces the monovalent cation current. Calcium has the same effect on the single channel currents of the neuronal nicotinic receptor. Increasing the external calcium ion concentrations produces a decrease in the single channel currents. It is not the case therefore that the larger whole cell current seen with the neuronal nicotinic receptor is a result of larger single channel currents (Vernino et al., 1991). The effect of calcium on the neuronal whole cell currents is independent of calcium permeation through the receptor channel and it seems to be the result of the interaction of calcium with the extracellular domain of the receptor. Neither magnesium nor barium produce a similar effect. Thus, modulation of the neuronal nicotinic receptors by calcium acting on the extracellular domain provides another example of the diversity in function that is associated with the receptors formed by the structurally different receptor subunits.
Conclusions Cholinergic function has been studied in the central and peripheral nervous systems for decades but the extent and diversity of nicotinic receptors in the central nervous system has only recently been appreciated. Clearly, many different neuronal nicotinic acetylcholine receptor genes are expressed in the central nervous system. In fact, any given neuron may express several different types of nicotinic receptors and this will complicate analysis of these receptors in neurons. Fortunately, the same molecular approaches that revealed this receptor gene family have also provided a means to study the individual receptors under well controlled conditions. It may be the case, however, that the oocyte gives false results because it lacks the cellular machinery required for proper assembly or modification of the receptors. Nonetheless, this preparation provides remarkable access to a controlled population of receptors. In the case of pharmacology, the muscle nicotinic acetylcholine receptor expressed in the oocyte has a pharmacological profile very
similar to that found in the cells from which the clones were derived (Luetje and Patrick, 1991). Likewise, the PC,/PNaratio for the muscle receptor expressed in the oocyte is very close to that determined in the cells from which the clones were derived (Adams et al, 1980; Decker and Dani, 1990; Vernino et al., 1991). The Pc,IP,, ratio determined for PC 12 cells is also close to that determined for the 014w receptor expressed in the oocyte (Sands and Barish, 1991). This result is less useful, however, because the composition of the neuronal nicotinic acetylcholine receptor expressed in PC12 cells is unknown. We have used the oocyte expression system to characterize several of the oligomeric receptors formed by the proteins produced by the nicotinic receptor gene family and find considerable diversity in their properties. The receptors differ remarkably in their responses to both agonists and antagonists and it is clear that both the alpha and beta subunits contribute to the ligand selectivity of these receptors. The neuronal nicotinic receptors have a higher relative permeability to calcium than does the muscle nicotinic receptor and the homo-oligomeric alpha7 receptor has a relative permeability to calcium that is greater than that of the NMDA type of glutamate receptor. This calcium permeability suggests that activation of these receptors can trigger calcium-dependent cytoplasmic mechanisms and may influence the behavior of the cell in unexpected ways. For example, the activation of nicotinic receptors in the medial septa1 nucleus (Wong and Gallagher, 1989) and in some Purkinje cells (Garza et al., 1987) appears to be inhibitory and may operate through a calcium-dependent mechanism, perhaps by gating a calcium-dependent potassium channel. Alternatively, calcium flux through these receptors may activate other calcium-dependent mechanisms that, for example, lead to changes in cell morphology, or gene expression. Our understanding of the ligand gated ion channels expressed in the central nervous system is rudimentary at best. We can reasonably extrapolate some properties from the muscle nicotinic acetylcholine receptor to the neuronal nicotinic acetylcholine receptors, but we are still left with major unanswered questions. Although the oocyte preparation has provided insights into allowable combinations of subunits, we still need to know what combinations exist in vivo. The fact that there are three members of the gene family that do not yet have identified functional roles suggests that there will be a greater diversity of receptors in vivo than found thus far in the oocyte. Consistent with this idea is the observation of nicotinic responses on rat neurons in culture that have pharmacologies unlike those seen thus far in the oocyte (Garza et al.; 1987). The diversity of neuronal nicotinic receptors may be increased substantially through formation of receptors containing more than one kind of alpha or beta subunit.
1 I9
Once we understand the true diversity of these receptors, we will still need to understand the role these receptors play in the central nervous system and how the central nervous system takes advantage of the diversity.
References Adams, D.J., Dwyer, T.M. and Hille. B. (1980). The permeability of endplate channels to monovalent and divalent metal cations. J. Gen. Physiol., 75: 493-510. Boulter, J., Evans, K., Goldman, D., Martin, G.,Treco, D., Heinemann. S. and Patrick, J. (1986) Isolation of a cDNA clone coding for a possible neural nicotinic acetylcholine receptor alpha-subunit. Nature, 319: 368-374. Boulter, J., Connolly, J., Deneris, E., Goldman, D., Heinemann, S. and Patrick, J. (1987) Functional expression of two neuronal nicotinic acetylcholine receptors from cDNA clones identifies a gene family. Proc. Nutl. Acud. Sci. USA, 84: 7763-7767. Boulter, J., O'Shea-Greenfield, A,, Duvoisin, R., Connolly. J., Wada, E., Jensen, A,, Ballivet. M., Gardner. P.D., Deneris, E.. McKinnon. D., Heinemann, S . and Patrick, J. (1990) Alpha3, alpha5 and beta4: three members of the rat neuronal nicotinic acetylcholine receptor-related gene family form a gene cluster. J. B i d . Chem., 265: 44724482. Cooper, E., Couturier, S. and Ballivet, M., (1991) Pentameric structure and subunit stoichiometry of a neuronal nicotinic acetylcholine receptor. Nature, 350: 235-238. Couturier, S., Bertrand, D., Matter, J.-M., Hernandez, M-C., Bertrand, S., Millar, N., Valera, S . , Barkas, T. and Ballivet, M. ( I 990)A neuronal nicotinic acetylcholine receptor subunit (a7) is developmentally regulated and forms a homo-oligomeric channel blocked by a-BTX. Neuron, 5: 847-856. Decker, E.R. and Dani, J.A. (1990) Calcium permeability of the nicotinic acetylcholine receptor: the single-channel calcium influx is significant. 1. Neurosci., 10: 3413-3420. Deneris, E.S., Connolly, J.. Boulter, J., Patrick, J. and Heinemann, S. (1987) Identification of a gene that encodes a non-alpha subunit of neuronal nicotinic acetylcholine receptors. Neurnn, I : 45-54. Deneris, EX. Boulter, J., Swanson, L., Patrick, J. and Heinemann, S. (1988) p-3 a new member of the nicotinic acetylcholine receptor gene family is expressed in the brain. J . B i d . Chem.. 264: 6268-6272. Duvoisin, R.M., Deneris, E.S., Boulter, J., Patrick, J. and Heinemann S. (1989) The functional diversity of the neuronal nicotinic acetylcholine receptors is increased by a novel subunit: p4. Neuron, 3: 487-496. Garza, R., Bickford-Wimer, P.C.. Hoffer, B.J. and Freedman, R. (1987) Heterogeneity of nicotine actions in the rat cerebellum: an in vivo electrophysiologic study. Phurmacol. Exp. Ther., 240: 689-695. Goldman, D., Deneris, E., Luyten, W., Kohchar, A., Patrick, J. and Heinemann, S. (1987) Members of a nicotinic acetylcholine receptor gene family are expressed in different regions of the mammalim central nervous system. Cell, 48: 965-973. lino, M., Ozawa, S. and Tsuzuki, K. (1990) Permeation of calcium through excitatory amino acid receptor channels in cultured rat hippocampal neurons. J. Physiol., 424: 151-165.
Lamar, E. Dinely-Miller, K., Goldner, F. and Patrick, J (1992) A cDNA clone defines alpha6, a new member of the neuronal nicotinic receptor gene family. Unpublished. Lindstrom, J., Schoepfer, R.and Whiting, P., Molecular studies of the neuronal nicotinic acetylcholine receptor family. Molecular Neurobiology I: 281-337. Luetje, C.W. and Patrick, J. (1991) Both alpha and beta subunits contribute to the agonist sensitivity of neuronal nicotinic acetylcholine receptors. J . Neurosci., 11: 837-845. Luetje, C.W., Wada, K., Rogers, S., Abramson, S., Tsuji, K., Heinemann, S. and Patrick, J. (1990a) Neurotoxins distinguish between different neuronal nicotinic acetylcholine receptor subunit combinations. J. Neurochem., 55: 632-640. Luetje, C.W., Patrick, J. and Seguela, P. (1990b) Nicotine receptors in the mammalian brain. FASEB, 4: 2753-2760. Mayer, M.L. and Westbrook, G.L. (1987) Permeation and block of N-methyl-D-asparticacid receptor channels by divalent cations in mouse cultured central neurons. J. Physbl., 394: 501-527. Miller, K. and Patrick, J. (1992) Gene transcripts for the nicotinic acetylcholine receptor subunit, beta4, are distributed in multiple areas of the rat central nervous system. Bruin Res., 16: 339-344. Mulle, C., Choquet, D., Korn, H. and Changeux, J.-P.. (1992a) Calcium influx through nicotinic receptor in rat central neurons: relevance to cellular regulation. Neuron, 8: 135-143. Mulle, C., Lena. C. and Changeux, J.P. (1992b) Potentiation of nicotinic receptor response by external calcium in rat central neurons. Neuron, 8: 937- 945. Nef, P., Oneyser, C., Barkas, T. and Ballivet, M. (1987) In: A. Maelicke (Ed.), Nicotinic Acetylcholine Receptor Structure and Function, Springer, Berlin, pp. 417429. Nef, P., Oneyser, C., Alliod, C., Couturier, S. and Ballivet, M. (1988) Genes expressed in the brain define three distinct neuronal nicotinic acetylcholine receptors. EMBO J.. 7: 595-601. Papke, R.L., Boulter, J., Patrick, J. and Heinemann, S. (1989) Single channel currents of rat neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes. Neuron, 3: 589-596. Sands, S.B. and Barish, M.E. (1991) Calcium permeability of neuronal nicotinic acetylcholine receptor channels in PCI 2 cells. Brain Res., 560: 3 8 4 2 . Seguela, P., Wadiche, J., Miller, K.,Dani, J. and Patrick, J. (1992) Molecular cloning, functional expression and distribution of rat brain 1x7: a nicotinic cation channel highly permeable to calcium. J . Neurosci., 13: 596-604. Shoepfer, R., Conroy, W.G., Whiting, P., Gore, M and Lindstrom, J. (1990) Brain a-bungarotoxin binding protein cDNAs and MAbs reveal subtypes of this branch of the ligand-gated ion channel gene superfamily. Neuron, 5 : 35-48. Vernino. S. and Dani, J. (1992) Quantitative measurement of calcium flux through muscle and neuronal nicotinic acetylcholine receptors. J. Neurosci., submitted. Vernino, S., Amador, M., Luetje, C.W., Patrick. J. and Dani, J. (1991) Calcium modulation and high calcium permeability of neuronal nicotinic acetylcholine receptors. Neuron, 8: 127-1 34. Vijayaraghavan, S . , Pugh, P.C., Zhang, Z., Rathouz, M.M. and Berg, D.K. (1992) Nicotinic receptors that bind a-bungarotoxin on neurons raise intracellular free Ca2+. Neuron, 8: 353-362.
I20 Wada, E., Wada, K.. Boulter, E.. Deneris, E.S., Heinemann, S.. Patrick, J. and Swanson, L. (1989) The distribution of alpha2, alpha3, alpha4 and beta2 neuronal nicotinic receptor subunit mRNAs in the central nervous system. A hybridization histochemical study in the rat. J. Comp. Neurol., 284: 314-335. Wada, E., McKinnon, D., Heinemann, S., Patrick, J. and Swanson, L. W. (1990) The distribution of mRNA encoded by a new member of the neuronal nicotinic acetylcholine receptor gene family (alpha5) in the rat central nervous system. Brain Rex, 526: 45-53.
Wada, K., Ballivet. M., Boulter, J.. Connolly, J., Wada, E., Deneris, E.S., Swanson, L.W., Heinemann, S. and Patrick, J. (1988) Functional expression of a new pharmacological subtype of brain nicotinic acetylcholine receptor. Science, 240: 330-334. Wong, L.A. and Gallagher, J.P. (1989) A direct nicotinic receptormediated inhibition recorded intracellularly in vitro. Narure, 341: 439-442.
113
CHAPTER 1 1
Functional diversity of neuronal nicotinic acetylcholine receptors Jim Patrick, Philippe SCquCla, Steven Vernino, Mariano Amador, Chuck Luetje and John A. Dani Division of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA
Introduction The neuronal nicotinic acetylcholine receptors are likely to be oligomeric proteins composed of five subunits (Cooper et a]., 1991). If these receptors were all homo-oligomers, there might be one unique receptor that corresponded to each pentameric arrangement of each of the available subunits. The situation is more complicated than that because most of the neuronal nicotinic receptors are heterooligomers. These hetero-oligomeric receptors contain both alpha and beta subunits. Because individual alpha subunits can participate in the formation of functional receptors with more than one kind of beta subunit, the number of possible receptors is larger. There are ten known members of the gene family that encodes the subunits of the neuronal nicotinic acetylcholine receptors. These genes and their products are called alpha2, alpha3, alpha4, alpha5, alpha6, alpha7, alpha8, beta2, beta3 and beta4 (see Boulter et al., 1986, 1987, 1990; Deneris et al., 1987, 1988; Goldman et al., 1987; Nef et al., 1987, 1988; Wada eta].; 1988; Duvoisin et al., 1989; Couturier et al., 1990; Lindstrom et a].; 1990; Luetje et al., 1990; Shoepfer et al., 1990; Lamar et al., 1992, and for the properties of these subunits in both rat and chick). These subunits are known to form at least eight different receptors; homo-oligomeric alpha7 and alpha 8 receptors and hetero-oligomeric receptors composed of alpha2heta2, alpha3lbeta2, alpha4heta2, alpha2lbeta4, alpha3lbeta4, and alpha4heta4. There are several reasons why this diversity is Likely to increase. First, there are members of the gene family (alpha5, alpha6, and beta3) whose functional roles in neuronal nicotinic receptors have not yet been demonstrated. Second, there may well be members of the gene family that have not yet been identified and finally, it seems likely that receptors containing more than one kind of alpha or more than one kind of beta subunit are allowed.
Several different acetylcholine-gated ion channels can be formed from the members of this gene family. We expect that the various subunits, each with different primary structures, will form oligomeric receptors having different properties. One question then is how do receptors with different subunit combinations differ from one another. We know, for example, that different combinations of subunits differ in their single channel properties (Papke et al., 1989) and in their responses to different agonists and antagonists (Luetje et al., 1990, 1991). They also differ in their permeability to cations and in their modulation by external cations (Vernino et al., 1991; Vernino and Dani, 1992; Mulle, 1992a,b). The extensive diversity of their cytoplasmic domains suggests additional diversity in their functional interactions with the cytoplasmic machinery. A second important question is what role do these different receptors play in the central nervous system and how does the central nervous system make use of this diversity of receptor types. The genes encoding these subunits are, in general, expressed in discrete but overlapping sets of brain nuclei (Wada et al., 1989, 1990; Miller and Patrick, 1992). While some brain nuclei can be characterized by the specific combination of neuronal nicotinic receptors that they express, other regions are less well defined. For example, almost all the known members of the gene family are expressed in the medial habenula. In this case, however, it is not known if a single cell expresses all these different genes or if specific combinations of subunits define cellular types in the habenula. In the cerebellum, however, the case is more extreme. All Purkinje cells express the beta2 gene and either alpha2, alpha3 or alpha4 (Wada et a]., 1989). Adjacent Purkinje cells may each express a beta2 subunit but differ in the alpha subunit gene expressed. This suggests a remarkable heterogeneity in receptor phenotype in what appears, and is usually treated as a homogeneous population of cells.
I I4 TABLE I
Receptor diversity Open time Conductance Ion selectivity Ligand selectivity Extracellular modulation Local cytoplasmic regulation Distribution on single neurons Distribution in central nervous system Developmental regulation An important question then is why are there so many different kinds of receptors. Table I is a partial list of the attributes of ligand-gated ion channels that might be determined by the primary structure of their constituent subunits. Receptors composed of structurally different subunits might be expected to exhibit different biophysical properties such as single channel conductance, open time or ion selectivity. They might also produce receptors that could be differentially distributed over the cell surface or that might be responsive to different cytoplasmic regulatory mechanisms. Additionally, the central nervous system may require neuronal nicotinic receptors with specific properties at different times during development and the diversity of genes might reflect the requirement that they be capable of independent expression during development. Our understanding of the role that these receptors play in development is meager at best. It is possible, however, that understanding the role of these receptors in the plasticity characteristic of the nervous system in the adult will help us formulate specific testable ideas about roles that they might play during development. The following paragraphs document several examples of the functional heterogeneity of the neuronal nicotinic receptors and present new properties that characterize these receptors. Specifically, we review data showing the pharmacological diversity of neuronal nicotinic acetylcholine receptors and show that these receptors differ from the muscle nicotinic acetylcholine receptors in their permeability to calcium and in their modulation by external calcium ions.
tor (Luetje and Patrick, 1991). The structures of the four agonists we tested are seen in Fig. 1, We examined the six possible alphaheta pairwise combinations of alpha2, alpha3, alpha4, beta2 and beta4 using the oocyte expression system. Xenopus oocytes were injected with RNAs encoding an alpha and a beta subunit and the expressed functional receptors were assayed using a twoelectrode voltage clamp. Because responses in individual oocytes show substantial variation, all responses were normalized to the current generated by 1 pM acetylcholine. The data in Fig. 2 show dose response curves for each of the ligands seen in Fig. 1 tested on each alphdbeta combination. The panels across the top correspond to receptors comprised of beta2 subunits and different alpha subunits and those across the bottom correspond to receptors comprised of a beta4 subunit and different alpha subunits. It is clear that the responses to ligands vary across the top panels with respect to the alpha subunits. There is about a 50-fold difference in the ratio of sensitivities of nicotine and acetylcholine between alpha2-containing receptors and alpha3containing receptors and suggests that the alpha subunits play an important role in determining the agonist specificity of these receptors. This is consistent with results obtained using antagonists. Neuronal neurotoxin blocks receptors containing an alpha3 subunit but has little effect on receptors in which the alpha subunit is alpha2 (Wada et al., 1988). Receptors containing alpha2 are almost 1000-fold less sensitive to the antagonist neuronal bungarotoxin than receptors containing the alpha3 subunit (Luetje et al., 1990). None of the receptors in the top panels are activated by cytosine. But, cytosine is the best of the four agonists on all of the beta4-containing receptors in the bottom panel. The fact that this abrupt change in rank order of potency occurs through the substitution of the beta4 subunit for the beta2
0
II H,C-C-O-CH~-CH,-N+-CH,
\
I
II
CH3
Acetylcholine
Nicotine
Cytisine
Dimethylphenylpiperazinium
Pharmacological diversity of neuronal nicotinic receptors Different combinations of subunits might produce receptors with different affinities for either agonists or antagonists. We tested this possibility for both agonists and antagonists and found that this ligand selectivity is determined by both the alpha and beta subunits that comprise any given recep-
(DMPP)
Fig. I . Structures of nicotinic acetylcholine receptor agonists.
I15
failure of cytosine to activate receptors containing the beta2 subunit is not a consequence of a lack of affinity of the ligand for the receptor. In fact, cytosine binds to receptors containing the beta2 subunit and prevents activation by acetylcholine (Luetje and Patrick, 1991).
subunit demonstrates that the beta subunits also contribute to ligand selectivity. This might occur through modification of a ligand binding site uniquely on the alpha subunit or through actual contributions of residues in the beta subunit to the actual composition of the ligand binding site. The -.
. . . . . . . .,
JJ): .. .,
. . . . .._, . . . . . . . .,
B. a2P4
A. a2P2
.-
Concentration, M
p)
i C.a3P2 5000 -
$.
4000
z d
I
Nic ACh
-
DMPP
0 1000
10.'
.9
B
2
6 4oo 0
Concentration, M
1
200
0
Concentration, M Fig. 2. Each neuronal nAChR subunit combination has unique pharmacological properties. Agonist-induced current responses of oocytes expressing a2P2 (A), a2p4 (B), a3P2 (0,a3w (D), a4p2 ( E ) or a4p4 (0 were measured under two electrode voltage clamp at a holding potential of -70 mV. Currents were elicited by bath application of various concentrations of acetylcholine (open circles), nicotine (closed circles), cytosine (triangles) and DMPP (squares). Responses are presented after normalization to the response of the same oocyte to 1 M acetylcholine (response to 1 M acetylcholine =loo). Each point represents the mean f SD of the responses of 3-8 separak oocytes.
I16
In addition to the determination of agonist selectivity, the beta4 subunit also modifies the interaction with neuronal bungarotoxin. As noted above, receptors containing the alpha3 subunit in combination with beta2 are blocked by low concentrations of neuronal bungarotoxin. However, replacement of the beta2 by the beta4 subunit renders these alpha3-containing receptors resistant to neuronal bungarotoxin. It is clear, therefore, that the beta subunits must be considered as partial determinants of the ligand binding site. Different alpha and beta subunits combine to make receptors that respond differently to the four ligands in Fig. 1. It is clear that both alpha and beta subunits contribute to the ligand selectivity of the receptors they form. It is not clear how these differences might influence the behavior of these receptors in the CNS but they do offer the investigator interested in cholinergic mechanisms in the brain a powerful tool with which to selectively activate or inhibit certain nicotinic cholinergic pathways.
Calcium permeability of neuronal nicotinic receptors The response of neuronal receptors in the oocyte differs from that of the muscle nicotinic receptors in that a greater portion of the current through nicotinic receptor channels is carried by Ca2+ ions. The oocyte contains an endogenous Ca2+-activated CI- channel. Because the reversal potential for CI- in the oocyte is about -25-30 mV, the chloride current appears as an inward cationic current at the normal holding potentials of -50 to -60 mV. Application of blockers of this endogenous Ca2+-activated CI- channel, such as niflumic acid or flufenamic acid reduces the current associated with activation of the neuronal receptors but has little effect on the current associated with activation of the muscle receptors (see Fig. 3). This result suggested that the neuronal nicotinic acetylcholine receptors differed from the muscle nicotinic acetylcholine receptors by being more permeable to calcium ions. We tested this possibility by examining the change in the reversal potential produced by increasing the external calcium concentration. As seen in Fig. 4 increasing the calcium concentration from 1 to 10 mM has only a small effect (1 mV) on the muscle receptor but has a more marked effect on the neuronal receptor comprised of an alpha3 and beta4 subunits (+7 mV). This result demonstrates that this neuronal receptor is more permeable to calcium relative to sodium than is the muscle receptor and suggests that a larger portion of the current through this receptor is carried by calcium ions. This idea is consistent with the observation that a larger portion of the current elicited by activation of this receptor in the oocyte disappears when the endogenous Ca2+-activated CIchannel is blocked by flufenamic and niflumic acid.
The alpha7 gene product forms a functional homooligomeric receptor in the Xenopus oocyte (Couturier et al., 1990). This ability to form a homo-oligomeric receptor distinguishes the alpha7 subunit from the other neuronal nicotinic receptor subunits. It is further distinguished by its pharmacology; the homo-oligomeric alpha7 receptor is blocked by strychnine with an EC,, of about 350nM. Strychnine is best known as an antagonist of the glycine receptor. The alpha7 homo-oligomer is also distinguished by the large contribution the endogenous Ca2+-activated CI- channel makes to the whole cell current in the oocyte. Blocking the endogenous Ca2+-activated CI- channel reduces the agonist induced alpha7 current by about 85% suggesting that the alpha7 current has a large Ca2+ component that subsequently activates the Ca2+-activated CI- channel. We tried to assess the contribution of this Ca2+ component by measuring the effect of increasing the external Ca2+ concentration on the reversal potential but were unable always to block the endogenous Ca2+-activated CI- current completely using flufenamic and niflumic acid. We therefore replaced the CI- with methanesulfonate, an impermeant anion. The results in Fig. 4 show the change in the reversal potential obtained in the presence of methane-
h '
' I
I
Fig. 3. Currents through neuronal nAChRs activate a Ca2+dependent C1- conductance. Muscle and a3p4 neuronal nAChRs were expressed in separate Xenopus oocytes. The currents induced by 10 pM Ach were measured in the presence and absence of 100 pM niflumic acid and 100 pM flufenamic acid, which inhibit the Ca2+-dependentCI-channel. The ACh-induced currents seen with oocytes expressing muscle nAChRs were unchanged by the CIchannel blockers, but the currents seen with neuronal nAChRs were smaller in the presence of blockers. The results indicate that Ca2+ carries enough current through neuronal nAChRs but not through muscle nAChRs to activate the Ca2+-dependent C1conductance. The external solution contained 18 mM CaCI2. The currents were measured in blockers, then the blockers were washed off.
1 I7
shift of the reversal potential observed under our experimental conditions, we can calculate that the PCalPNaratio for the rat alpha7 channel is around 20. This is higher than the PcJPNa ratio of 5 reported for the NMDA subtype of glutamate receptors (Mayer and Westbrook, 1987; Iino et al., 1988). Application of nicotine to chick ciliary ganglion neurons results in an a-BTX sensitive increase in cytoplasmic calcium (Vijayaraghavan et al., 1992). Our data suggest that at least a portion of this increase could result from the calcium flux through an alpha7-type receptor.
50 .
Modulation of neuronal nicotinic receptors by calcium ions There are additional interesting data in Fig. 3. Increasing the concentration of calcium in the external medium has
.loo0
20
.................................. 10
0
10
20
30
40
I
Muscle
Neuronal
0 18
18
50
Voltage ImV)
Fig. 4. Reversal potential shift comparisons between muscle, a3p4 and a7 cation channels expressed in Xenopus oocytes. Peak currents elicited by 2 mM acetylcholine (al&S or 20 mM nicotine (a3j34 and a7) were measured at different holding potentials and plotted as a function of membrane potential (voltage) in chloride-free Ringer containing 1 mM Ca2+ (solid circles) and 10 mM Ca2+ (open circles). To remove internal CI-, the oocytes were pre-incubated 224 h in chloride- and magnesiumfree methane sulfonate-based recording buffer before measurements. Reversal potential shift values (AErev) correspond to the mean f SD of the number ( n ) of measurements made in different oocytes. The current-voltage relationships illustrated are representative of the experiments performed for each combination of nicotinic receptor subunits. As expected from previous work, external Ca2+ decreases the amplitude of the response of muscle nicotinic acetylcholine receptors (Vijayaraghavan et al., 1992) but increases the response of neuronal nicotinic acetylcholine receptors (Miller and Patrick, 1992) and of a7 receptors.
sulfonate when the external calcium is increased from 1 to 10 mM. The observed shift of about 30 mV is larger than that obtained for either the muscle or any of the heterooligomeric neuronal nicotinic receptors examined thus far. We can use these data to place the apparent relative Ca2+ permeability of the alpha7 homo-oligomer within the context of the permeabilities of other known ligand gated ion channels. For example, a CdNa permeability ratio (Pca/PNa)of 0.2 was reported for the muscle nicotinic receptor (Adam et al., 1980; Vernino and Dani, 1992) and PCa/PNaof about 1.5 was reported for the neuronal nicotinic receptors present in chromaffin cells (Vernino and Dani, 1992) or in PC12 cells (Sands et al., 1990). From the
2.5-
5
c
10 s Neuronol
2.0-
L
D
e,
1.5 1.0
-
b 0.5
.
!.
E
z
0.0
I
Muscle
0.3
1 [Co"]
3
10
30
(mM)
Fig. 5. Ca2+ modulates currents through nAChRs. ACh-induced currents are shown for Xenopus oocytes expressing muscle nAChR or a3P4 neuronal nAChR. The Ca2+ concentration of the test solution is given in mM beside each trace. For the muscle nAChRs, the currents overlap in 0.18 and 1.8 mM Ca2+. The holding potential was -40 mV, and the ACh concentration was 10 pM. The plot at the bottom shows the concentration-dependence of the Ca2+ effect. The ACh-induced currents normalized for each oocyte to the value in 1.8 mM Ca2+are plotted as a function of the external Ca2+ concentration. Increasing the external Ca2+ concentration decreases the ACh-induced currents through muscle nAChRs and increases the ACh-induced currents through neuronal nAChRs. The data points represent 3-15 trials. The enhancement by external Ca2' was observed with four neuronal ap-subunit combinations that we tested.
two consequences. It shifts the reversal potential to more positive potentials and it changes the magnitude of the whole cell currents. The data in Fig. 5 extend this observation over a range of Ca2+ concentrations. In the case of the muscle nicotinic receptor, increasing the external calcium decreases the whole-cell currents. In the case of the neuronal nicotinic receptor, increasing external calcium increases the whole-cell currents. The decrease in the whole-cell current seen with the muscle nicotinic acetylcholine receptor is understood to be due to a decrease in the single channel currents (Decker and Dani, 1990). Calcium can enter the muscle nicotinic receptor channel but does not permeate readily and thus reduces the monovalent cation current. Calcium has the same effect on the single channel currents of the neuronal nicotinic receptor. Increasing the external calcium ion concentrations produces a decrease in the single channel currents. It is not the case therefore that the larger whole cell current seen with the neuronal nicotinic receptor is a result of larger single channel currents (Vernino et al., 1991). The effect of calcium on the neuronal whole cell currents is independent of calcium permeation through the receptor channel and it seems to be the result of the interaction of calcium with the extracellular domain of the receptor. Neither magnesium nor barium produce a similar effect. Thus, modulation of the neuronal nicotinic receptors by calcium acting on the extracellular domain provides another example of the diversity in function that is associated with the receptors formed by the structurally different receptor subunits.
Conclusions Cholinergic function has been studied in the central and peripheral nervous systems for decades but the extent and diversity of nicotinic receptors in the central nervous system has only recently been appreciated. Clearly, many different neuronal nicotinic acetylcholine receptor genes are expressed in the central nervous system. In fact, any given neuron may express several different types of nicotinic receptors and this will complicate analysis of these receptors in neurons. Fortunately, the same molecular approaches that revealed this receptor gene family have also provided a means to study the individual receptors under well controlled conditions. It may be the case, however, that the oocyte gives false results because it lacks the cellular machinery required for proper assembly or modification of the receptors. Nonetheless, this preparation provides remarkable access to a controlled population of receptors. In the case of pharmacology, the muscle nicotinic acetylcholine receptor expressed in the oocyte has a pharmacological profile very
similar to that found in the cells from which the clones were derived (Luetje and Patrick, 1991). Likewise, the PC,/PNaratio for the muscle receptor expressed in the oocyte is very close to that determined in the cells from which the clones were derived (Adams et al, 1980; Decker and Dani, 1990; Vernino et al., 1991). The Pc,IP,, ratio determined for PC 12 cells is also close to that determined for the 014w receptor expressed in the oocyte (Sands and Barish, 1991). This result is less useful, however, because the composition of the neuronal nicotinic acetylcholine receptor expressed in PC12 cells is unknown. We have used the oocyte expression system to characterize several of the oligomeric receptors formed by the proteins produced by the nicotinic receptor gene family and find considerable diversity in their properties. The receptors differ remarkably in their responses to both agonists and antagonists and it is clear that both the alpha and beta subunits contribute to the ligand selectivity of these receptors. The neuronal nicotinic receptors have a higher relative permeability to calcium than does the muscle nicotinic receptor and the homo-oligomeric alpha7 receptor has a relative permeability to calcium that is greater than that of the NMDA type of glutamate receptor. This calcium permeability suggests that activation of these receptors can trigger calcium-dependent cytoplasmic mechanisms and may influence the behavior of the cell in unexpected ways. For example, the activation of nicotinic receptors in the medial septa1 nucleus (Wong and Gallagher, 1989) and in some Purkinje cells (Garza et al., 1987) appears to be inhibitory and may operate through a calcium-dependent mechanism, perhaps by gating a calcium-dependent potassium channel. Alternatively, calcium flux through these receptors may activate other calcium-dependent mechanisms that, for example, lead to changes in cell morphology, or gene expression. Our understanding of the ligand gated ion channels expressed in the central nervous system is rudimentary at best. We can reasonably extrapolate some properties from the muscle nicotinic acetylcholine receptor to the neuronal nicotinic acetylcholine receptors, but we are still left with major unanswered questions. Although the oocyte preparation has provided insights into allowable combinations of subunits, we still need to know what combinations exist in vivo. The fact that there are three members of the gene family that do not yet have identified functional roles suggests that there will be a greater diversity of receptors in vivo than found thus far in the oocyte. Consistent with this idea is the observation of nicotinic responses on rat neurons in culture that have pharmacologies unlike those seen thus far in the oocyte (Garza et al.; 1987). The diversity of neuronal nicotinic receptors may be increased substantially through formation of receptors containing more than one kind of alpha or beta subunit.
1 I9
Once we understand the true diversity of these receptors, we will still need to understand the role these receptors play in the central nervous system and how the central nervous system takes advantage of the diversity.
References Adams, D.J., Dwyer, T.M. and Hille. B. (1980). The permeability of endplate channels to monovalent and divalent metal cations. J. Gen. Physiol., 75: 493-510. Boulter, J., Evans, K., Goldman, D., Martin, G.,Treco, D., Heinemann. S. and Patrick, J. (1986) Isolation of a cDNA clone coding for a possible neural nicotinic acetylcholine receptor alpha-subunit. Nature, 319: 368-374. Boulter, J., Connolly, J., Deneris, E., Goldman, D., Heinemann, S. and Patrick, J. (1987) Functional expression of two neuronal nicotinic acetylcholine receptors from cDNA clones identifies a gene family. Proc. Nutl. Acud. Sci. USA, 84: 7763-7767. Boulter, J., O'Shea-Greenfield, A,, Duvoisin, R., Connolly. J., Wada, E., Jensen, A,, Ballivet. M., Gardner. P.D., Deneris, E.. McKinnon. D., Heinemann, S . and Patrick, J. (1990) Alpha3, alpha5 and beta4: three members of the rat neuronal nicotinic acetylcholine receptor-related gene family form a gene cluster. J. B i d . Chem., 265: 44724482. Cooper, E., Couturier, S. and Ballivet, M., (1991) Pentameric structure and subunit stoichiometry of a neuronal nicotinic acetylcholine receptor. Nature, 350: 235-238. Couturier, S., Bertrand, D., Matter, J.-M., Hernandez, M-C., Bertrand, S., Millar, N., Valera, S . , Barkas, T. and Ballivet, M. ( I 990)A neuronal nicotinic acetylcholine receptor subunit (a7) is developmentally regulated and forms a homo-oligomeric channel blocked by a-BTX. Neuron, 5: 847-856. Decker, E.R. and Dani, J.A. (1990) Calcium permeability of the nicotinic acetylcholine receptor: the single-channel calcium influx is significant. 1. Neurosci., 10: 3413-3420. Deneris, E.S., Connolly, J.. Boulter, J., Patrick, J. and Heinemann, S. (1987) Identification of a gene that encodes a non-alpha subunit of neuronal nicotinic acetylcholine receptors. Neurnn, I : 45-54. Deneris, EX. Boulter, J., Swanson, L., Patrick, J. and Heinemann, S. (1988) p-3 a new member of the nicotinic acetylcholine receptor gene family is expressed in the brain. J . B i d . Chem.. 264: 6268-6272. Duvoisin, R.M., Deneris, E.S., Boulter, J., Patrick, J. and Heinemann S. (1989) The functional diversity of the neuronal nicotinic acetylcholine receptors is increased by a novel subunit: p4. Neuron, 3: 487-496. Garza, R., Bickford-Wimer, P.C.. Hoffer, B.J. and Freedman, R. (1987) Heterogeneity of nicotine actions in the rat cerebellum: an in vivo electrophysiologic study. Phurmacol. Exp. Ther., 240: 689-695. Goldman, D., Deneris, E., Luyten, W., Kohchar, A., Patrick, J. and Heinemann, S. (1987) Members of a nicotinic acetylcholine receptor gene family are expressed in different regions of the mammalim central nervous system. Cell, 48: 965-973. lino, M., Ozawa, S. and Tsuzuki, K. (1990) Permeation of calcium through excitatory amino acid receptor channels in cultured rat hippocampal neurons. J. Physiol., 424: 151-165.
Lamar, E. Dinely-Miller, K., Goldner, F. and Patrick, J (1992) A cDNA clone defines alpha6, a new member of the neuronal nicotinic receptor gene family. Unpublished. Lindstrom, J., Schoepfer, R.and Whiting, P., Molecular studies of the neuronal nicotinic acetylcholine receptor family. Molecular Neurobiology I: 281-337. Luetje, C.W. and Patrick, J. (1991) Both alpha and beta subunits contribute to the agonist sensitivity of neuronal nicotinic acetylcholine receptors. J . Neurosci., 11: 837-845. Luetje, C.W., Wada, K., Rogers, S., Abramson, S., Tsuji, K., Heinemann, S. and Patrick, J. (1990a) Neurotoxins distinguish between different neuronal nicotinic acetylcholine receptor subunit combinations. J. Neurochem., 55: 632-640. Luetje, C.W., Patrick, J. and Seguela, P. (1990b) Nicotine receptors in the mammalian brain. FASEB, 4: 2753-2760. Mayer, M.L. and Westbrook, G.L. (1987) Permeation and block of N-methyl-D-asparticacid receptor channels by divalent cations in mouse cultured central neurons. J. Physbl., 394: 501-527. Miller, K. and Patrick, J. (1992) Gene transcripts for the nicotinic acetylcholine receptor subunit, beta4, are distributed in multiple areas of the rat central nervous system. Bruin Res., 16: 339-344. Mulle, C., Choquet, D., Korn, H. and Changeux, J.-P.. (1992a) Calcium influx through nicotinic receptor in rat central neurons: relevance to cellular regulation. Neuron, 8: 135-143. Mulle, C., Lena. C. and Changeux, J.P. (1992b) Potentiation of nicotinic receptor response by external calcium in rat central neurons. Neuron, 8: 937- 945. Nef, P., Oneyser, C., Barkas, T. and Ballivet, M. (1987) In: A. Maelicke (Ed.), Nicotinic Acetylcholine Receptor Structure and Function, Springer, Berlin, pp. 417429. Nef, P., Oneyser, C., Alliod, C., Couturier, S. and Ballivet, M. (1988) Genes expressed in the brain define three distinct neuronal nicotinic acetylcholine receptors. EMBO J.. 7: 595-601. Papke, R.L., Boulter, J., Patrick, J. and Heinemann, S. (1989) Single channel currents of rat neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes. Neuron, 3: 589-596. Sands, S.B. and Barish, M.E. (1991) Calcium permeability of neuronal nicotinic acetylcholine receptor channels in PCI 2 cells. Brain Res., 560: 3 8 4 2 . Seguela, P., Wadiche, J., Miller, K.,Dani, J. and Patrick, J. (1992) Molecular cloning, functional expression and distribution of rat brain 1x7: a nicotinic cation channel highly permeable to calcium. J . Neurosci., 13: 596-604. Shoepfer, R., Conroy, W.G., Whiting, P., Gore, M and Lindstrom, J. (1990) Brain a-bungarotoxin binding protein cDNAs and MAbs reveal subtypes of this branch of the ligand-gated ion channel gene superfamily. Neuron, 5 : 35-48. Vernino. S. and Dani, J. (1992) Quantitative measurement of calcium flux through muscle and neuronal nicotinic acetylcholine receptors. J. Neurosci., submitted. Vernino, S., Amador, M., Luetje, C.W., Patrick. J. and Dani, J. (1991) Calcium modulation and high calcium permeability of neuronal nicotinic acetylcholine receptors. Neuron, 8: 127-1 34. Vijayaraghavan, S . , Pugh, P.C., Zhang, Z., Rathouz, M.M. and Berg, D.K. (1992) Nicotinic receptors that bind a-bungarotoxin on neurons raise intracellular free Ca2+. Neuron, 8: 353-362.
I20 Wada, E., Wada, K.. Boulter, E.. Deneris, E.S., Heinemann, S.. Patrick, J. and Swanson, L. (1989) The distribution of alpha2, alpha3, alpha4 and beta2 neuronal nicotinic receptor subunit mRNAs in the central nervous system. A hybridization histochemical study in the rat. J. Comp. Neurol., 284: 314-335. Wada, E., McKinnon, D., Heinemann, S., Patrick, J. and Swanson, L. W. (1990) The distribution of mRNA encoded by a new member of the neuronal nicotinic acetylcholine receptor gene family (alpha5) in the rat central nervous system. Brain Rex, 526: 45-53.
Wada, K., Ballivet. M., Boulter, J.. Connolly, J., Wada, E., Deneris, E.S., Swanson, L.W., Heinemann, S. and Patrick, J. (1988) Functional expression of a new pharmacological subtype of brain nicotinic acetylcholine receptor. Science, 240: 330-334. Wong, L.A. and Gallagher, J.P. (1989) A direct nicotinic receptormediated inhibition recorded intracellularly in vitro. Narure, 341: 439-442.