Site-directed mutagenesis and single-channel currents define the ionic channel of the nicotinic acetylcholine receptor

icotinic acetylcholine receptor channel complexes (nAChR Site-directed mutagenesis and single-channel currents N channels) are concentrated at synapses throughout the CNS and PNS 1-a. The postsynaptic receptors bind ACh released from the presynaptic neuron. The receptors then undergo a conformational change that opens the ionic channels. Synaptic transmission is complete after cations pass through the channels and depolarize the postsynaptic cell. The purpose of the channels is to provide a permeable pathway through the membrane for cations. Recently, two groups have identified amino acids within the nAChR channel that interact with permeating cations 4'5. Site-directed mutations were introduced into the cDNAs of the nAChR subunits. The mRNAs of the mutated subunits were injected into Xenopus oocytes, and structurally altered channels were expressed. Single-channel currents from wild type and from altered channels revealed the relationship between structure and function. Imoto et al. 4 showed that the charged residues bracketing the uncharged transmembrane o~helix, M2, are important determinants of ionic permeation (Fig. 1). Leonard et al. 5 showed that polar residues within M2 can influence permeation and the residence time of an open channel blocker. Both studies indicate that M2 forms the lining of the pore.

Background Based on the amino acid sequences, many structural models have been proposed to explain how the nAChR subunits fold and arrange themselves around the central pore 1-a. The models predict that each subunit contributes one transmembrane a-helix to form the lining of the pore. There has been controversy about whether highly charged amphlpathic helices or uncharged helices provide the lining6. Results from various recent studies favor a pore lined by the uncharged helix, M2. Studies with chimeras between bovine and Torpedo 6-subunits showed that a-helix M2 and the amino acids connecting M2 and M3 influence ionic transport 7. In low divalent cation solutions, the bovine channel has a lower conducTINS, Vol. 12, No. 4, 1989

define the ionic channelof the nicotinic acetylcholine

receptor tance than the Torpedo channel. Torpedo nAChRs constructed with the M2 and connecting region from bovine nAChRs showed the lower conductance of the bovine channels. Likewise, bovine channels constructed with the Torpedo M2 and connecting region showed the higher conductance of the Torpedo channels. Some non-competitive antagonists bind within the pore and block the open channel. Two affinity labels of this intrachannel blocking site were found to bind to the M2 o~-helix8'9. The labelling experiments indicated that M2 is exposed within the pore. Permeability studies favor a pore lined by an uncharged transmembrane helix bracketed by net negative charge 1°. The permeation data indicated that the wide entrances of the nAChR channel contain net negative charge. Those negative amino acids attract permeant cations into the vestibules. The tapering transmembrane region is uncharged. Therefore, cations attracted into the vestibules rapidly pass through the narrower region of the pore without being slowed by a series of attractive and repulsive potentials that would be present if amphipathic helices lined the pore. M2 and the adjacent amino acids fulfill these requirements.

Results and interpretation of the mutagenesis experiments Figure 1 shows part of the amino acid sequences for subunits of Torpedo, bovine and mouse nAChRs. The amino acids labelled as columns 1 through 6 from the Torpedo nAChR were subjected to point mutations by Imoto et al. 4. When a negative or neutral amino acid in column 1 was changed to a positive amino acid, the conductance of the channel decreased especially for outward current. The single-channel currentvoltage relationship changed from linear for the wild type to inwardly rectifying for the column 1 mu-

tation. When a residue in column 4 was changed to a positive amino acid, the conductance of the channel again decreased. Positive charge introduced into column 4, however, caused outward rectification. When an amino acid in column 2 was changed to a positive charge, the conductance decrease was greater than after a similar substitution in columns 1 or 4. The column 2 mutations caused less current rectification. In fact, no current rectification was observed when the y-subunit Gin (Q) was changed to a positively charged Lys (K). At each of these three positions, columns 1, 2 or 4, the effects were greater as more amino acids in a column were changed at the same time. The conductance decrease was proportional to the charge changed, regardless of which amino acids were changed. There is a plausible explanation for these results with the mutated nAChR channels. The negative residues of column 1 are located at the cytoplasmic entrance to the pore. The net negative charge attracts cations into the cytoplasmic vestibule and when that negative charge is reduced, fewer cations are attracted. Then, fewer permeant cations are in a position to carry outward current, so the current-voltage relation shows inward rectification. The residues of column 4 are located in the external vestibule. When the negative charge of column 4 is reduced, fewer cations are attracted into the outer vestibule to carry inward current. This case results in outward rectificaton. The results obtained with the column 2 mutations require a slightly different explanation. The narrowest cross-section of the nAChR channel is very short n. Although the channel contains many binding sites for permeant cations, the narrowest region may provide (or be next to) the main binding site in the permeation pathway. Structural studies 12 and

© 1989. ElsevierSciencePublishersLtd. (UK) 0166 22361891502.00 -

John A. Dani

Departmentof Physiolo~/and MolecularBiophysics, BaylorCollegeof Medidne, One Baylor Plaza,Houston, TX 77030, USA.

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Fig. 1. Partial amino acid sequences for Torpedo californica, bovine and mouse nAChR subunits. The bovine o~- and fl-subunits and the mouse y-subunit are not included. The locations of the transmembrane regions M1, M2 and M3 are indicated. The amino acids connecting the transmembrane regions are labelled as part of the inner or the outer vestibule of the channel. Imoto et al. 4 used Torpedo nAChR subunits. Columns 1 through 6 are indicated. Positively charged residues are shaded light grey and negatively charged residues are shaded dark grey. Leonard et al. 5 used mouse nAChR subunits. Circles enclose the amino acids within A42 that were changed. (The amino acid sequences and alignments were taken from Refs 7, 21 and 22.)

analysis of an affinity label by a non-competitive blocker13, suggest that the narrowest region is located at the cytoplasmic end of M2. In that case, the negative charges of column 2 may be important determinants of the main cation binding site. Mutations near the binding site would be expected to alter dramatically the currents in both directions rather than producing rectification. Column 2 mutations gave similar results. Work by Imoto et al.4 supports this general interpretation. When 0.5 mM Mg2+ was added to the solution on one side of the membrane, the current carried to the other side of the membrane was decreased. Decreasing the negative charge at column 1 reduced the effect of Mg2÷ on outward current, and decreasing the negative charge in column 4 reduced the effect of Mg2+ on inward current. Magnesium could be exerting its effect by binding or by screening the negative charge in the vestibules. Binding or screening the charged residues in a vestibule reduces the ability of those negative charges to attract cations. Therefore, Mg2+ decreases the number of permeant cations in a position to pass through the pore. Mutations at the residues of column 1 or column 4 already decrease negative charge in the inner or outer vestibule, respec126

tively. In those cases, Mg2+ has fewer charges to affect, so the influence of Mg2+ on those mutated channels is reduced. This view of the channel accounts for the single-channel conductance differences seen in two earlier studies. Adult bovine nAChR channels have a larger conductance than fetal bovine channels. The channels differ because the fetal y-subunit is replaced in the adult by the esubunit 14. The difference in the charges bracketing M2 are consistent with the adult channel having a greater conductance. As can be seen in Fig. 1, the c-subunit of the adult is less positive in these regions than the y-subunit. In a study with 6-subunit chimeras, the region between M2 and M3 was found to regulate the difference in conductance between bovine and Torpedo channels 7. Between M2 and M3, the Torpedo 6-subunit has a negative residue and one fewer positive residue than the bovine 5-subunit (Fig. 1). This charge difference could explain the greater conductance of the Torpedo channel. The work of Leonard et al. 5 adds more evidence suggesting that M2 lines the pore. The circled amino acids from the mouse sequences in Fig. 1 were changed. The polar serines (S) in the oc- and 6-subunits were changed to non-polar alanines

(A). A nAChR with three Ser to Ala changes (one on each o~-subunit and one on the 6-subunit) displayed decreased outward-going currents. In addition, the Ser to Ala change decreased the residence time of a positively charged intrachannel blocker, QX-222. QX222 causes brief gaps or 'flickery' block in single-channel currents. The length of the gaps became shorter after a Ser was changed to an Ala. The decrease in the residence time was linearly correlated with the number of Ser to Ala changes. In keeping with this linear relationship, when the non-polar Phe (F) was changed to a polar Ser (S), the residence time of QX-222 increased. The results indicate that positively chargedQX-222 interacts with the negative dipole of the Ser residues of the M2 helix. Dipoles in this same region can influence permeation and decrease outward currents. Speculative structural model of the nAChR channel Figure 2 schematically represents a possible open channel structure of the nAChR. The tapering transmembrane region of the channel is lined by the M2 helix of each subunit. The narrowest region of the pore is located at the cytoplasmic end of M2. At that position, glutamic acids (E) and TINS, Vol. 12, No. 4, 1989

glutamines (Q) may be near to a binding site for permeant cations (see column 2 of Fig. 1). Because the narrowest region is very short, it is assumed that the M2 helices must spread outward as the open pore diameter increases. Spreading or movement of the helices may occur during the opening process, just as relative movement of the subunits has been shown to occur during desensitization 15. Since the M2 helices spread outward, M1 helices may become exposed to the pore at the interstices of the M2 helices. Net negative charge brackets the transmembrane region. These charged residues contribute to the cation selectivity of the channel. Along with other charged groups, they attract cations into the large entrances of the channel. Two ligand-gated chloride channels, the GABA receptor and the strychnine-binding glycine receptor, have excess positive charge at these positions bracketing their M2 regions 16'17. This general structure may be shared by the superfamily of ligand-gated receptor channels. The M1 helices are chosen to line the pore just behind the M2 helices for several reasons. One non-competitive antagonist, quinacrine, that is an intrachannel blocker, was found to affinity-label the M1 helix only when the channel was open ~8. This indicates that M1 is exposed to the lumen of the open channel. Like M2, M1 is highly conserved. A proline in the middle of M1 (see Fig. 1) is found in all sequences of the ligand-gated receptor channels: GABA receptor IO , glycine receptor 17, muscle nAChR (Fig. 1), neuronal nAChR 19, invertebrate nAChR 2°. Because of its cyclic structure, proline produces a kink in the a-helix structure. Apolar residues near the proline produce non-directional hydrophobic bonds, which makes this area more flexible. The structure of M1, its location next to the pore and the likelihood that it is the first transmembrane region after the ACh binding site all suggest that M1 may be involved in gating the channel.

Some unexplained results The results with some mutations are difficult to explain on the basis of the ideas discussed above. When a charge of the TotTINS, VoL 12, No. 4, 1989

Fig. 2. Speculative schematic representation of the open nAChR channel. The general shape of the channel is consistent with Ref. 12. The extracellular surface of the membrane is at the top of the figure. The M2 helix lines the pore, and the M 1 helix is exposed to the pore near the outer vestibule at the interstices of the M2 helices. Possible locations of charged amino acids are shown as circles containing pluses or minuses. The number and exact location of the charged residues are not intended to be precisely accurate. Detailed features such as glycosylation, phosphorylation and connections to the cytoskeleton are not represented.

pedo f~-, y- or 6-subunit of column The negative dipoles of the Ser 3, column 5 or column 6 was reversed, the conductance of the channel did not change 4. These residues are located at positions that are expected to influence ionic transport. If the cytoplasmic end of M2 is the narrowest region of the pore, then it is especially surprising that the positive residues of column 3 have no effect. These amino acids may have no effect because they are positioned away from the axis of the open pore. A test of the concentration dependence of conductance may have revealed the importance of these amino acids. Another surprising result is that the conductance of the mouse channel was affected only when the nAChR contained three Ser to Ala mutations 5. If the oc- or 6-subunit were changed separately, there was no effect on ionic permeation.

residues may make this area of the pore energetically favorable for cations. As the serines are replaced by alanines, the energetics of ionic transport become less favorable in this region. Only when all three serines have been removed, however, does the energy from the ion-channel interaction become rate limiting. Therefore, we should expect other areas of the open pore usually to limit ionic permeation. It is possible that the narrowest region just inward from the serines normally limits the rate of ionic transport.

Selected references 1 Anholt, R., Lindstrom, J. and Montal, M. (1985) in The Enzymesof Biological Membranes (VoL 3) (Martonosi, A. N., ed.), pp. 335-401, Plenum 2 McCarthy, M.P., Earnest, J.P., Young, E. F., Choe, S. and Stroud, 127

Acknowledgements I thank Drs S. Numa, T. Claudio and F. Hucho forproviding manuscriptsprior to publication. Work from my laboratory is supported by NIH grant NS21229.

R. M. (1986) Annu. Rev. Neurosci. 9, 383-413 3 Claudio, T. in Frontiers of Molecular

Biology, Molecular Neurobiology Volume (Glover, D. M. and Hames, B. D., eds), IRL Press (in press) 4 Imoto, K. et al. (1988) Nature 335, 645-648 5 Leonard, R. J., Labarca, C. G., Charnet, P., Davidson, N. and Lester, H. A. (1988) Science 242, 1578-1581 6 Guy, H.R. and Hucho, F. (1987) Trends Neurosci. 10, 318-321 7 Imoto, K. et al. (1986) Nature 324, 670-674 8 Giraudat, J., Dennis, M., Heidmann, T., Chang, J-Y. and Changeux, J-P.

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10 11 12 13

14

(1986) Proc. Natl Acad. Sci. USA 83, 2719-2723 Hucho, F., Oberthur, W. and Lottspeich, F. (1986) FEBS Lett. 205, 137-142 Dani, J. A. and Eisenman, G. (1987) J. Gen. PhysioL 89, 959-983 Dani, J. A. (1989) J. Neurosci. 9, 882-890 Toyoshima,-C:-and Unwin, N. (1988) Nature 336, 247-250 Hilgenfeld, R. and Hucho, F. (1988) in Transport through Membranes: Carriers Channels and Pumps (Fullrnan, A. et al., eds), pp. 359-367, D. Reidel Mishina, M. etal. (1986) Nature 321, 406-411

More thana Caz+ channel? Bruce Bean Department of Neurobiology, Harvard Medical School,220 LongwoodAvenue, Boston, MA 02115, USA.

128

he central role played by voltage-dependent Caz÷ chanT nels in processes such as synaptic transmission, smooth and cardiac muscle contraction, and secretion has made them interesting to a wide variety of biologists. So it was natural that after successfully cloning the acetylcholine receptor and Na ÷ channels, Shosaku Numa's group turned part of their considerable energies to the Ca2+ channel. The starting point for this effort was the purification and partial amino acid sequencing of a presumed Ca 2÷ channel protein from skeletal muscle, identified by its high-affinity binding site for dihydropyridine (DHP) drugs. This family of drugs, which includes nifedipine and nitrendipine, is valuable clinically for treating hypertension and angina; the drugs act by blocking Ca"z+ channels in vascular smooth muscle and thereby relaxing arterioles. High-affinity binding sites for the drugs, generally assumed to be Ca2+ channels, are found in smooth muscle, cardiac muscle and brain. However, the highest density of DHP binding sites is in skeletal muscle, specifically localized in the transverse tubules (t-tubules), a system of infolded surface membrane reaching into the interior of the muscle fiber. The DHP receptor from skeletal muscle is a complex consisting of four polypeptides of molecular masses 175 kDa, 170 kDa, 52 kDa and 32 kDa 1. The DHP binding site is located on the 170 kDa peptide (called the cq-subunit), and it is this peptide that was sequenced via molecular cloning by Tsutomo

Tanabe and his colleagues in Numa's laboratory2 (see also Ref. 3). (In fact, at the time the cloning effort began, the distinction between the 170 kDa and the 175 kDa peptides had not yet been realized, and Numa reported at a recent meeting that his group first cloned and sequenced the 175 kDa peptide. The sequence of this peptide has recently been reported by another group3; it has no homology with channel-forming peptides and its function is unknown.) The DHP-binding peptide has a high degree of homology with the several types of voltage-dependent Na + channel that have been cloned4-6. Like the Na + channel, the DHP receptor contains four internal repeats, each of which contains six probable membranespanning segments. One of the segments in each repeat is closely homologous to the so-called $4 segment in Na + channels, hypothesized to be the voltage-sensor of the Na ÷ channel, which is also found in the voltage-dependent K ÷ channel encoded by the shaker locus of Drosophila'. It therefore seems reasonable to think that the DHP-binding peptide could form a voltage-dependent Ca2+ channel. However, although the mRNA for a rat brain Na + channel and the shaker K ÷ channel have been shown to produce functional channels when injected into Xenopus oocytes8'9, this has so far not been shown for the DHP receptor (and one guesses it is not for lack of trying). It may be that Xenopus oocytes simply happen to be a poor expression system for the protein, perhaps lacking the machinery for

© 1989, ElsevierScience Publishers Ltd, (UK) 0166- 2236/89/$02.00

15 Unwin, N., Toyoshima, C. and Kubalek, E. (1988)J. Cell Biol. 107, 1123-1138 16 Schofield, P. R. et a/. (1987) Nature 328, 221-227 17 Grenningloh, G. et al. (1987) Nature 328, 215-220 18 Karlin, A., Kao, P. N. and Dipaola, M. (1986) Trends Pharmacol. Sci. 7, 304-308 19 Boulter, J. eta/. (1986) Nature 319, 368-374 20 Hermans-Borgmeyer, I. et al. (1986) EMBO J. 5, 1503-1508 21 Noda, M. et al. (1983) Nature 302, 528-532 22 Takai, T. et al. (1985) Nature 315, 761-764

proper post-translational processing. (At a minimum, proteolytic processing seems likely, because the amino acid sequence deduced from the cDNA has a predicted molecular mass of 212 kDa, larger than the 170 kDa DHP-binding peptide purified from muscle.) More interestingly, it may be that other subunits of the DHP-binding complex are needed to form a functional Ca2+ channel. Another intriguing possibility is that the DHP receptor does not primarily function as a Ca2÷ channel even when normally present in skeletal muscle. The possibility of another function for this protein was first raised by electrophysiological experiments aimed at understanding one of the central problems of muscle physiology, excitation-contraction coupling (E-C coupling). Somehow, depolarization of the t-tubule membrane by an action potential causes release of Ca 2+ from the internal stores of the sarcoplasmic reticulum; the mechanism of this coupling is a long-standing puzzle. An important clue came in 1973, when Martin Schneider and Knox Chandler, studying the electrical capacitance of skeletal muscle membranes, discovered that depolarization of the muscle fiber membrane (including the t-tubule system) produced movement of electrical charges within the membrane 1°. Because the voltagedependence of the intramembrane charge movement was similar to that of contraction, they hypothesized that the charge movement arises from molecular rearrangement of intramembrane molecules that act as voltage sensors controlling E-C coupling. Subsequent work has tended to support this interpretation. The identity of the TINS, Vol. 12, No. 4, 1989