The ion channel of the nicotinic acetylcholine receptor

The ion channel of the nicotinic acetylcl ine receptor H. RobertGuy and Ferdinand Hucho H. RobertGuy~ at the Laborato~of IHathemab'cal Biolo~),,Nabbnal Cancerln~tute, Nafionallnstitutesof

Health,Bethesd~MD 20892,USA,and FerdinandHuchoisat the lnstitut f~r Bioc~mie, Arbeibgruppe

Much attention has been given to a nwdel of the nicotinic acetylcholine rec@tor ion channel in which the lining is formed by five amphipathic o¢-helices (one from each of the five subunits) that are oriented so that very hydrophilic, charged side-chains on their polar faces extend into water inside the pore and hydrophobic side-chains on the opposite faces interact with other more hydrophobic transmembrane a-helices1"2. However, recent experiments indicate that this model may not be correct.

They postulated that the four hydrophobic segments still form transmembrane 0t-helices but that the pore is formed between five amphipathic a-helices (one from one subunit, MA in Fig. 1). The amphipathic helix model

The amphipathic a-helix model became widely accepted following several experimental findings. (1) Two groups 7'8 demonstrated that antibodies made to the carboxy termini of the subunits hound on the The channel of the nicotinic acetylcholine receptor cytoplasmic side of the membrane, indicating that an Neurochemie,Freie (AChR) is formed between five homologous suhunits odd number of segments in each subunit cross the Universit~t,Berlin, with the stoichiometry a21376a. Three laboratories that membrane. (2) Mishina et al. 9 used site-directed Thielallee63, 1000 sequenced one or more of these subunits from the mutagenesis to modify the mRNA sequence of the aBerlin33, FRG. electric organ of Torpedo noted that each subunit subunit, which with the other subunit was injected into contains four segments (M1-M4 in Fig. 1) comprised Xeno]ms oocytes. Properties of the modified channels of twenty or more predominantly hydrophobic amino were then measured by recording the membrane acid residues, which they proposed to be transmem- voltage response to agonists and the binding of snake brahe a-helices4-~. Soon thereafter two groups x'z neurotoxin. They found that deletions throughout the reasoned that, since the AChR has a large water-filled putative cytoplasmic domain had no effect, that delepore, amino acid side-chains on the walls of the pore tions in the putative apolar and amphipathic a-helices should be similar to those on the sturface of soluble eliminated the gating but affected agonist and antagonproteins, i.e. they should be primarily hydrophilic. ist binding only for M1, and that a deletion in the segment linking MA to M4 eliminated the gating but increased apparent agonist binding, suggesting that this segment interacts with the agonist binding site on the extracellular surface. These results were interpreted as supporting the amphipathic helix model; however, they obtained one result inconsistent with the model. AChRs in which much of the cytoplasmic domain and the amphipathic a-helix were deleted produced a voltage response about 3% of normal. Mishina et al. o speculated that a segment just before the deletion could form an amphipathic s-helix that replaced the normal one in the modified asubunit. (3) Brisson and Unwin 1° used electron microscopy to determine the general shape and volume of the AChR including the transmembrane region. They found that the cross-sectional area of the transmembrane region and volume of protein on each side of the membrane is more consistent with models that have five transmembrane a-helices. Support for the amphipathic ~Fig. 1. Models of segments postulated by different groups to cross the membrane. Model I is from helix model by these findings has 6 5 4 2 Noda et al. , Devillers- Thiery et al. , and Claudio et al. ; model 2 is from Guy, and Finer-Moore and been negated recently by another Stroudl; and model 3 is from Ratnam et al. Cylinders represent o~-helices and the zig-zag pattern represents a E-strand. In model 3 and the non-functional conformation of model 2, M4 could cross series of experiments. Lindstrom's the membrane twice as two E-strands or cross haft the membrane twice as two short o~-helices. In laboratory obtained results sugmodel 3 @ represents antibody binding sites 11. Results of mutagenesis experiments 9 are illustrated gesting that monoclonal antibodies on model 2; B indicates that a deletion in this area affects agonist and~or ol-bungarotoxin binding, P to a segment that the first two indicates that permeability response is eliminated, and N indicates no effects on the agonist binding models assigned to the extraceUusite or permeability response. lar regions appear to bind on the 31 8

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cytoplasmic side of the membrane, and antibodies to MA and the segment linking MA to M4 bind to the cytoplasmic side n. These findings led them to position MA outside the membrane (on the cytoplasmic side) and to propose the model in Fig. 1C, in which the first transmembrane segment, M6, is a G-strand and the pore forms between a different set of amphipathic a-helices, M7s. The first antibody result was especially surprising because the antibody binding segment begins only eight residues from the only N-glycosylation site, which must be on the outside. This antibody binding site was postulated to be in the cytoplasm because it became accessible to antibodies only when the membrane was permeabilized by a detergent and because other antibodies thought to bind on the cytoplasmic side inhibited its binding. These results could be misleading if the detergent drastically altered the protein structure. Experiments indicating that antibodies to MA and the link between MA and M4 bind in the cytoplasm included a much more convincing technique developed by La Rochelle et al. iz, in which large gold beads are attached to an antibody known to bind on the extraceUdar side and small gold beads are bound to the probe antibody made to a specific segment. It can then be determined clearly using electron microscopy whether the antibodies bind to the same or opposite sides of the membrane. Even with this method, misleading results could be obtained if the antibodies do not bind to the functional conformation but do bind to AChRs in a non-functional conformation for which the transmembrane folding pattern differs from that of the functional conformation. Finer-Moore and Stroud 1 postdated that a structure like that shown in Fig. 1D exists prior to aggregation of the subunits and that MA and M4 cross the membrane only when the final aggregation occurs. The antibody labelling is not quantitative and the fraction of AChRs to which they bind has not been determined. Thus, if any AChR subunits are present that have not aggregated or that have deaggregated, or if insertion of MA and M4 is reversible, then one would expect antibodies to bind to MA on the cytoplasmic side of the membrane. Also, McNamee's laboratory ~3observed the agonist-induced flux in lipid vesicles required the presence of cholesterol and negatively charged lipids and that addition of cholesterol caused an increase of a-helix content and addition of negatively charged lipids caused an increase in G-sheet content. Thus, the nature of the lipid bilayer may affect the AChR's structure, and perturbing the lipid bilayer may create non-functional AChRs in which the transmembrane protein is less ordered. The membrane is always lysed in antibody experiments to make the cytoplasmic surface accessible to the antibodies. If this process alters the topology of some AChRs, then the antibody results are difficult to interpret. Additional doubt about results of antibody experiments has been cast by experiments indicating that the disulfide bridge that links b-subunits of different Torpedo AChR monomers is on the extracellular side of the membrane 14'1~. Studies suggest that the cysteine residues that form this bridge are near the carboxy terminus of the 6-subunit. If so, the original four helix model could be correct. Helix-9. model

Recent findings from Changeux's 16 and Hucho's 17 TINS, VoL 10, No. 8, 1987

Fig. 2. Model of the ion channel of the nicotinic acetylcholine receptor. (A) Longitudinal section. (B) Crosssection at the level of the site labelled by TPMP. (C) Alternative model in which TPMP can bind to M2s by fitting between IVlAs. The channel has a wide en trance of about 30 ,~ diameter ~°and narrows clown to less than 6.4 ,~, the diameter of the lar&est permeating cation 22. The reaction site of the photolabel TPMP is formed by homologous amino acids of the membrane-spannin& helix M2 of the five receptor subunits and may be part of the pore.

laboratories support a model in which the channel lining is formed by one of the hydrophobic segments, M2. Using the non-competitive antagonist chlorpromazine as a photoaffmity label, a binding site for this putative channel-blocking reagent has been localized in position 262 of the 6-subunit t6. The same site was identified as the reaction site of the channel blocker triphenylmethylphosphonium (TPMP). Because the latter reagent has been shown to react with homologous amino acids in M2 of the oc- and 13-subunits as well, it was concluded that the binding site is at a region where the reagent can be in contact simultaneously with all five subunits 17. The narrow portion of the pore is the only such region in the AChR. The obvious explanation is that M2 forms the pore lining. A less obvious explana319

Fig. 3. Helix net representation of putative transmembrane segments. Single letter code is used. Residues for the o~-subunitsare in the upper left quadrant of each circle, those for fl-subunits are in the upper right, 7 in lower left, and ~ in lower right. Letters that overlap more than one quadrant indicate that the amino acid residue is found in both subunits. A black region indicates that more than one amino acid residue type has been reported in vertebrate AChRs at this position. A white letter indicates that only one substitution for the most commonly observed residue has been reported. The serine (S) residue on A42 that binds TPMP and chlorpromazine is indicated by an arrow. Sequences come from Torpedo, calf, rat, mouse, human and chicken, and from electric organ, muscle, and nerve tissue la. tion is that both M2 and MA form the pore lining and that the positive portion of the reagents is in the channel while the hydrophobic portion fits between the MA helices where it can bind to M2. Models in which the pore lining is formed only by MA segments have the problem that if lysine side chains are placed so their positively charged amino groups can form salt bridges with carboxyl groups and/or be hydrated by water inside the pore, and MA helices are arranged in an optimal way according to 'ridges into grooves' packing, then the pore is too small to allow passage of large organic cations known to go through it is. This problem is eliminated if MA helices are placed farther apart and M2 forms part of the lining (Fig. 2). Recently, a crucial role for M2 in the formation of the channel was suggested on the basis of site-directed mutagenesis experiments. Imoto et aL 19 have shown by constructing a chimeric 8-subunit from calf and Torpedo receptors that the rate of ion transport through the open channel is influenced by M2 and the adjacent bend portion between segments M2 and M3. The charged amino acid side chains may be especially important. Steric vs. allosteric blocking The binding site for channel blocking agents has not been resolved completely. Karlin's group 2° have found that another possible channel blocking agent, quinacrine azide, can be covalently attached somewhere on M1 of the oc-subunits. Their results indicate that quinacrine azide labels preferentially when the channel is transiently in an open or fast-onset desensitized state. The experiments with chlorpromazine and TPMP were performed under conditions in which most receptors are in a closed or slow-onset desensitized conformation. Both chiorpromazine and TPMP bind reversibly and react covalently with a desensitized conformation. It is not yet clear whether separate sites exist or whether the quinacrine azide site overlaps with the site for chiorpromazine and TPMP. Originally, non-competitive antagonists of the nicotinic acetylcholine receptor were introduced as allosteric inhibitors of the ion channel and not as steric blockers 21. Channel models based on labelling experiments with these compounds assume that they bind to a site within the channel. It should be pointed out that this is not a contradiction. For example, TPMP could

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well block stericaUy the ion flow through the channel by binding to a site within the channel entrance and simultaneously cause allosteric effects through conformational changes of the receptor protein. If one believes that pore lining segments are functionally important regions that should be highly conserved, then MA is not likely to form the lining. Fig. 3 shows that the sequence of M2 is conserved better than other segments among vertebrate species 18. If one favors an amphipathic a-helix for lining segments, M7 is conserved better than MA, especially for negatively charged side chains that could contribute to the ion selectivity. However, these M7 glutamates (E in Fig. 3) are not conserved well in Drosophilaz2. The AChR has a large, relatively non-selective pore that allows passage of almost all cations with diameters less than 6 or 7A(Ref. 23). It is thus conceivable that, like side chains on the surface of a soluble protein, the side chains that extend into the channel are not well conserved. We thus see that arguments can be made for and against the three models considered here and that none of the models are consistent with the 'most likely' interpretation of all experimental findings. Studies of the secondary structure of AChRs reconstituted in lipid vesicles indicate that the AChR has from 17-38% (_+4%) (upper value includes 14% disordered o0 o~helix and at least as much [3-sheet structure 24'25. It is possible that some transmembrane segments are [3strands and that none of the models are correct. Final proof will come from X-ray analysis of the crystal structure, which probably will not be available for some time. In the mean time, at least the question of transmembrane folding - i.e. which parts of the polypeptide chains are extracellular, which are intracellular, and which are intramembrane - could be addressed by more simple means, like localization through microsequencing phosphorylation sites (intracellular), glycosylation sites (extraceUular), sites in contact with lipids (intramembrane). Besides topological studies of this kind, the fine structure of the channel will be mapped by introducing point mutations through site-directed mutagenesis. Until ambiguities described here are resolved, one should not accept any of these models and certainly should not develop models of other membrane channels based on those of the AChR. TINS, VoL 10, No. 8, 1987

A good model, like a good wine, should improve with age. Although it is too soon to be sure, the amphipathic a-helix model seems to be turning sour.

Selected references 1 Finer-Moore, J. and Stroud, R. M. (1984) Proc. NatlAcad. Sci. USA 81,155-159 2 Guy, H. R. (1984) 8iophy. J. 45, 249-261 3 Reynolds, J. and Karlin, A. (1978) Biochemistry 17, 20352038 4 Claudio, T., Ballivet, M., Patrick, J. and Heineman, S. (1983) Proc. Natl Acad. Sci. USA 80, 1111-1115 5 Devillers-Thiery, A., Giraudat, J., Bentaboulet, M. and Changeux, J-P. (1983) Proc. NatlAcad. Sci. USA 80, 20672071 6 Noda, M., Takahashi, H., Tanabe, T., Toyosato, M., Furutani, Y., Hirose, T., Asai. M., Inayama, S., Miyata, T. and Numa, S. (1982) Nature 299, 793-797 7 Lindstrom, J., Criado, M., Hochschwender, S., Fox, J. L. and Sarin, V. (1984) Nature 311,573-575 8 Young, E. F., Ralston, E,, Blake, J., Ramachandran, J., Hall, Z. W. and Stroud, R. M. (1985) Proc. Natl Acad. Sci. USA 82, 626-630 9 Mishina, M., Tobimatsu, T., Imoto, K., Tanaka, K., Fujita, Y., Fukuda, K., Kurasaki, M., Takahashi, H., Morimoto, Y., Hirose, T., Inayama, S., Takahashi, T., Kuno, M. and Numa, S. (1985) Nature 313,364-369 10 Brisson, A. and Unwin, P. N. T. (1985) Nature 315, 474-477 11 Lindstrom, J. (1986) Trends Neurosci. 9, 401-407 12 La Rochelle, W., Wray, B., Sealock, R. and Froehner, S. (1985) J. Cell. Biol. 100, 684-691

13 McNamee, M. G., Fong, T. M., Jones, O. T. and Earnest, J. P. (1986) in Nicotinic Acetylcholine Receptor Structure and Function (NATOASIServices, Vol. H3) (Maelicke, A., ed.), pp. 147-157, Springer-Vedag 14 McCrea, P., Popot, J-L. and Engleman, D. (1986) Biophys. J. 49, 355a 15 Dunn, S. M. J., Conti-Tronconi, B.M. and Raftery, M.A. (1986) Biochem. Biophys. Res. Commun. 139, 830-837 16 Giraudat, J., Dennis, M., Heidman, T., Chang, J.Y. and Changeux, J.-P. (1986) Proc. NatlAcad. Sci. USA 83, 27192723 17 Hucho, F., Oberth0r, W. and Lottspeich, F. (1986) FEBSLett. 205, 137-142 18 Guy, H. R. (1986) in Nicotinic Acetylcholine Receptor Structure and Function (NATO ASI Series, Vol. H3) (Maelicke, A., ed.), pp. 447-463, Springer-Vedag 19 Imoto, K,, Methfessel, C., Sakmann, B., Mishina, M., Mori, Y., Konno, T., Fukuda, K., Kurasaki, M., Bujo, H., Fujita, Y. and Numa, S., (1986) Nature 324, 670-674 20 Kadin, A., Kao, P. N. and DiPaola, M. (1986) Trends Pharmacol. Sci. 7, 304-308 21 Changeux, J-P. (1981) Harvey Lect. 75, 85-254 22 Gundelfinger, E.D., Hermans-Borgmeyer, I., Zopf, D., Sawruk, E. and Betz, H. (1986) in Nicotinic Acetylcholine Receptor Structure and Function (NATO ASI Series, Vol. H3) (Maelicke, A., ed.), pp. 437-446, Springer-Verlag 23 Dwyer, T. M., Adams, D. J. and Hille, B. (1980) J. Gen. Physiol. 75, 469-492 24 Yager, P., Chang, E. L., Williams, R.W. and Dalziel, A. W. (1984) Biophys. J. 45, 26-28 25 Mielke, D. L., Kaldany, R-R., Karlin, A. and Wallace, B.A. (1985) Ann. N. Y. Acad. Sci. 463, 392-395

Structureof cytoplasmas revealedby modernelectron microscopytechniques Paul Bridgman An accurate depiction of cytoplasmic structure is critical for understanding the basic properties of cells, such as maintenance of shape, polarity, motility and intracellular transport. In addition, macromolecular structural associations between cytoplasmic components, such as the cytoskeleton and the ground substance (the medium in which cellular conrl)onents are suspended), may be importantfor defining the environmental backgroundfor metabolic processes. Central to this is the question of whether cytoplasmic enzymes involved in metabolic processes are freely soluble and randomly distributed within the cytoplasm of a living cell or are in a bound and therefore structured state. The co,reaosition and organization of the ground substance and how it relates to the cytoskeleton has been the focus of a number of ultrastrucrural studies of ceU cytoplasm in the last ten years. These studies have now stimulated further investigation of cytoplasmic structure using a variety of electron microscopic techniques.

tion electron microscopy showed the cytoplasm of most cells to be packed with various membrane-bound organelles. Filling the space between organelles was an amorphous or granular material generally called the ground substance, and contiguous with this were identifiable materials such as ribosomes and glycogen granules. These observations were also supported by early freeze-etch studies that avoided the use of chemical fixation or heavy metal stains2. Later studies showed the cytoplasm also contained a cytoskeleton made up of three main systems of filaments: microtubules, intermediate filaments and actin filaments. Cytoskeletal organization and composition has since been a major focus of cell biology3,4.

PaulS#downanisat theDepartmentof Anatomyand Neurobiotogy, Washington Univels#y~ o o l of Medidne,560South EudidAvenue,St Louis,M063110, USA.

The cytoplasmic ground substance and the microtrabecular lattice It had been assumed by many electron microscopists that the ground substance represented the soluble Consensus on the basic organization and structure of proteins that were readily extractable through biocell cytoplasm has been difficult to reach from the time chemical methods. Implicit in this assumption was that biologists first began to study the properties of living in the living cell these proteins were randomly districells responsible for shape and motility. The possibility buted in solution. This model was challenged in the of accurately describing cytoplasmic structure first 1970s by the work of Keith Porter and colleagues using became feasible with the arrival of electron micro- a new means of viewing whole cells in the electron scopy. However, many early observations made by microscope5'6. Their method involved growing culelectron microscopy lacked accurate detail, since spe- tured cells directly on coated electron microscope cimen preparation techniques had not been refined. grids and then processing them for electron microDouble fixation with aldehydes and osmiumI vastly scopy by fixation, dehydration and critical-point-dryimproved specimen preparation. Subsequent thin sec- ing. Whole-mounts gave dramatic three-dimensional

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