Structure of the Pore-forming Transmembrane Domain of a Ligand-gated Ion Channel

The structure of the pore-forming transmembrane domain of the nicotinic acetylcholine receptor from Torpedohas been investigated by infrared spectroscopy. Treatment of affinity-purified receptor with either Pronase or proteinase K digests the extramembranous domains (roughly 75% of the protein mass), leaving hydrophobic membrane-imbedded peptides 3–6 kDa in size that are resistant to peptide 1H/2H exchange. Infrared spectra of the transmembrane domain preparations exhibit relatively sharp and symmetric amide I and amide II band contours centered near 1655 and 1545 cm−1, respectively, in both1H2O and 2H2O. The amide I band is very similar to the amide I bands observed in the spectra of α-helical proteins, such as myoglobin and bacteriorhodopsin, that lack β structure and exhibit much less β-sheet character than is observed in proteins with as little as 20% β sheet. Curve-fitting estimates 75–80% α-helical character, with the remaining peptides likely adopting extended and/or turn structures at the bilayer surface. Infrared dichroism spectra are consistent with transmembrane α-helices oriented perpendicular to the bilayer surface. The evidence strongly suggests that the transmembrane domain of the nicotinic receptor, the most intensively studied ligand-gated ion channel, is composed of five bundles of four transmembrane α-helices. The structure of the pore-forming transmembrane domain of the nicotinic acetylcholine receptor from Torpedohas been investigated by infrared spectroscopy. Treatment of affinity-purified receptor with either Pronase or proteinase K digests the extramembranous domains (roughly 75% of the protein mass), leaving hydrophobic membrane-imbedded peptides 3–6 kDa in size that are resistant to peptide 1H/2H exchange. Infrared spectra of the transmembrane domain preparations exhibit relatively sharp and symmetric amide I and amide II band contours centered near 1655 and 1545 cm−1, respectively, in both1H2O and 2H2O. The amide I band is very similar to the amide I bands observed in the spectra of α-helical proteins, such as myoglobin and bacteriorhodopsin, that lack β structure and exhibit much less β-sheet character than is observed in proteins with as little as 20% β sheet. Curve-fitting estimates 75–80% α-helical character, with the remaining peptides likely adopting extended and/or turn structures at the bilayer surface. Infrared dichroism spectra are consistent with transmembrane α-helices oriented perpendicular to the bilayer surface. The evidence strongly suggests that the transmembrane domain of the nicotinic receptor, the most intensively studied ligand-gated ion channel, is composed of five bundles of four transmembrane α-helices. nicotinic acetylcholine receptor Fourier transform infrared 3-(trifluoromethyl)-3-m-([125I]iodophenyl)diazirine dichroic ratio polyacrylamide gel electrophoresis N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine The nicotinic acetylcholine receptor (nAChR)1 fromTorpedo is an abundant neurotransmitter-gated ion channel that is used extensively as a structural model for homologous ligand-gated channels located throughout the central and peripheral nervous systems (1Smith G.B. Olsen R.W. Trends Pharmacol. Sci. 1995; 16: 162-168Abstract Full Text PDF PubMed Scopus (451) Google Scholar, 2Jackson M.B. Yakel J.L. Annu. Rev. Physiol. 1995; 57: 447-468Crossref PubMed Scopus (189) Google Scholar, 3Kuhse J. Betz H. Kirsch J. Curr. Opin. Neurobiol. 1995; 5: 318-323Crossref PubMed Scopus (188) Google Scholar, 4Changeux J.P. Edelstein S.J. Neuron. 1998; 21: 959-980Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar). The nAChR is composed of four subunits organized pseudo-symmetrically as an α2βγδ pentamer around a central ion channel pore. The ion channel transiently gates open in response to the binding of two molecules of acetylcholine, leading to the flux of cations across the postsynaptic membrane. Prolonged exposure to acetylcholine converts the nAChR into a nonconducting desensitized state. Each of the four subunits contains a large extracellular N-terminal domain, four 25–30-amino acid-long hydrophobic segments designated M1 to M4, and a cytoplasmic loop between the M3 and M4 transmembrane segments. The N-terminal domain of each α-subunit contains one binding site for acetylcholine. The four transmembrane segments form the ion channel pore, with M2 from each subunit directly lining the ion channel. The intracellular loop between M3 and M4 may serve as a regulatory domain in that several residues can be phosphorylated, leading to altered rates of desensitization. The structure of the transmembrane domain of the nAChR has been the subject of considerable recent investigation. All four transmembrane segments were originally assigned α-helical secondary structures based on hydrophobicity analysis (5Noda M. Takahashi H. Tanabe T. Toyosato M. Kikyotani S. Furutani Y. Hirose T. Takashima H. Inayama S. Miyata T. Numa S. Nature. 1983; 302: 528-532Crossref PubMed Scopus (533) Google Scholar, 6Claudio T. Ballivet M. Patrick J. Heinemann S. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 1111-1115Crossref PubMed Scopus (276) Google Scholar). The resulting four-transmembrane-α-helices-per-subunit model is supported by a large body of biochemical and biophysical data, including the α-helical-labeling pattern of the exposed transmembrane surfaces by both ion channel-specific and lipid-soluble photoactivatable probes (7Revah F. Galzi J.L. Giraudat J. Haumont P.Y. Lederer F. Changeux J.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4675-4679Crossref PubMed Scopus (151) Google Scholar, 8Blanton M.P. Cohen J.B. Biochemistry. 1994; 33: 2859-2872Crossref PubMed Scopus (211) Google Scholar, 9Blanton M.P. Cohen J.B. Biochemistry. 1992; 31: 3738-3750Crossref PubMed Scopus (137) Google Scholar, 10Tamamizu S. Guzman G.R. Santiago J. Rojas L.V. McNamee M.G. Lasalde-Dominicci J.A. Biochemistry. 2000; 39: 4666-4673Crossref PubMed Scopus (60) Google Scholar), the predominantly α-helical secondary structure of the exchange-resistant and likely integral membrane “core” nAChR peptide hydrogens (11Baenziger J.E. Méthot N. J. Biol. Chem. 1995; 270: 29129-29137Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 12Méthot N. Baenziger J.E. Biochemistry. 1998; 37: 14815-14822Crossref PubMed Scopus (31) Google Scholar), and the predominantly α-helical secondary structures of isolated and reconstituted transmembrane segments (13Corbin J. Méthot N. Wang H.H. Baenziger J.E. Blanton M.P. J. Biol. Chem. 1998; 273: 771-777Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 14Opella S.J. Marassi F.M. Gesell J.J. Valente A.P. Kim Y. Oblatt-Montal M. Montal M. Nat. Struct. Biol. 1999; 6: 374-379Crossref PubMed Scopus (299) Google Scholar, 15Lugovskoy A.A. Maslennikov I.V. Utkin Y.N. Tsetlin V.I. Cohen J.B. Arseniev A.S. Eur. J. Biochem. 1998; 255: 455-461Crossref PubMed Scopus (45) Google Scholar, 16Pashkov V.S. Maslennikov I.V. Tchikin L.D. Efremov R.G. Ivanov V.T. Arseniev A.S. FEBS Lett. 1999; 457: 117-121Crossref PubMed Scopus (29) Google Scholar). A 9 Å resolution structure of the nAChR exhibits five rods of electron density lining the ion channel pore, consistent with α-helical transmembrane M2 pore-lining segments (17Unwin N. J. Mol. Biol. 1993; 229: 1101-1124Crossref PubMed Scopus (716) Google Scholar). In contrast, the cryoelectron microscopic studies reveal a diffuse pattern of electron density at the periphery of the receptor transmembrane domain that has been interpreted in terms of M1, M3, and M4 from each subunit, contributing to a ring of transmembrane β-strands surrounding an inner core of channel-lining M2 α-helices (17Unwin N. J. Mol. Biol. 1993; 229: 1101-1124Crossref PubMed Scopus (716) Google Scholar). FTIR studies of proteolytically degraded native nAChR membranes and molecular modeling are both consistent with a mixture of α-helices and β-strands forming the transmembrane domain (18Gorne-Tschelnokow U. Strecker A. Kaduk C. Naumann D. Hucho F. EMBO J. 1994; 13: 338-341Crossref PubMed Scopus (100) Google Scholar, 19Hucho F. Gorne-Tschelnokow U. Strecker A. Trends Biochem. Sci. 1994; 19: 383-387Abstract Full Text PDF PubMed Scopus (42) Google Scholar, 20Ortells M.O. Lunt G.G. Protein Eng. 1996; 9: 51-59Crossref PubMed Scopus (41) Google Scholar). A mixed secondary structure has also been suggested for the transmembrane domain of the glycine receptor (21Leite J.F. Amoscato A.A. Cascio M. J. Biol. Chem. 2000; 275: 13683-13689Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 22Cascio M. Shenkel S. Grodzicki R.L. Sigworth F.J. Fox R.O. J. Biol. Chem. 2001; 276: 20981-20988Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The mixed α-helix/β-sheet structure suggested by Unwin (17Unwin N. J. Mol. Biol. 1993; 229: 1101-1124Crossref PubMed Scopus (716) Google Scholar) represents a new motif for the transmembrane domains of integral membrane proteins in general and for neurotransmitter-gated ion channels in particular. The proposed transmembrane structure has important implications for the mechanisms of channel gating and desensitization as well as for the mechanisms of channel regulation by endogenous factors such as receptor phosphorylation. The transmembrane model also questions the general validity of interpreting hydrophobicity plots in terms of transmembrane α-helices. Here, we examine the secondary structure of the transmembrane domain of affinity-purified nAChR. In contrast to recent proteolytic studies on native membranes (18Gorne-Tschelnokow U. Strecker A. Kaduk C. Naumann D. Hucho F. EMBO J. 1994; 13: 338-341Crossref PubMed Scopus (100) Google Scholar), our analysis shows that all nAChR transmembrane segments adopt α-helical secondary structures. Our results strongly support an nAChR transmembrane domain composed of five bundles of four-transmembrane α-helices. The nAChR from frozen Torpedo californica electric tissue (Marinus, Inc., Long Beach, CA) was affinity-purified on a bromoacetylcholine bromide-derivatized Bio-Rad Affi-Gel 201 column and reconstituted into membranes composed of 3:1:1 egg phosphatidylcholine/dioleoylphosphatidic acid/cholesterol (23McCarthy M.P. Moore M.A. J. Biol. Chem. 1992; 267: 7655-7663Abstract Full Text PDF PubMed Google Scholar). This lipid composition supports both cation flux and agonist-induced conformational change. Affinity-purified membranes typically give a purity of 90–100% nAChR, as estimated by [125I]-bungarotoxin binding (24Méthot N. McCarthy M.P. Baenziger J.E. Biochemistry. 1994; 33: 7709-7717Crossref PubMed Scopus (46) Google Scholar) (see also theinset of Fig. 1). Proteinase K treatment was essentially as described previously for native nAChR membranes (18Gorne-Tschelnokow U. Strecker A. Kaduk C. Naumann D. Hucho F. EMBO J. 1994; 13: 338-341Crossref PubMed Scopus (100) Google Scholar), except that the affinity-purified nAChR membranes were dissolved in a solution containing 5 mg/ml proteinase K, 50 mm phosphate buffer, and 150 mm KCl at pH 8.0. Samples were incubated at for and of proteinase K were and with the nAChR membranes were with to to both of the nAChR The nAChR was at a of 5 mg/ml in mm phosphate buffer, pH 8.0. A containing mg/ml in was to give a of at the nAChR was for the resulting dissolved in a mg/ml Pronase solution containing and mm at pH and the nAChR membranes were incubated at for were with the of to a of 5 of was each was on for to and the membranes were to the integral membrane The were in mm phosphate and at of the nAChR was by on H. Biochem. PubMed Scopus Google Scholar) with the degraded nAChR was for with at for and on were to for with an FTIR spectra of the native and nAChR membranes were the on an with a A of nAChR containing the of of was on the of a and the was with a of The nAChR membranes were exposed to for spectra in the state. Each is a of and each was at of and for resolution were as described (24Méthot N. McCarthy M.P. Baenziger J.E. Biochemistry. 1994; 33: 7709-7717Crossref PubMed Scopus (46) Google Scholar). dichroism spectra were in a were with the infrared either or perpendicular to the of The dichroic is as the ratio of the of oriented perpendicular to the of with the H.H. Scholar, H.H. J. Full Text PDF PubMed Scopus Google Scholar). The was used to the data, as containing similar of nAChR are well the of the and the In an of is as and to the amide I and amide II and less respectively, a perpendicular to the bilayer surface. The of the transmembrane α-helices was to and H.H. J. Full Text PDF PubMed Scopus Google Scholar) from the amide II dichroism an between the amide II and of either or The amide II band in peptide hydrogens that are likely located the transmembrane domain, the amide I likely has from oriented extended or loop structures located at the membrane surface. Infrared spectra of affinity-purified nAChR exhibit two relatively protein located in the and The amide I in FTIR spectra is relatively and with centered near both 1655 and cm−1, of the and H. PubMed Scopus Google Scholar, M. H.H. Rev. Biochem. Mol. Biol. 1995; PubMed Scopus Google Scholar, Rev. PubMed Scopus Google Scholar). the amide I band and a analysis of the amide I a mixed α-helix/β-sheet protein with a of α-helical secondary structures (24Méthot N. McCarthy M.P. Baenziger J.E. Biochemistry. 1994; 33: 7709-7717Crossref PubMed Scopus (46) Google Scholar, F. M. Biochemistry. 1990; PubMed Scopus Google Scholar). The secondary structure estimated for affinity-purified nAChR is and similar to that been for the nAChR based on studies of native nAChR membranes D. C. Gorne-Tschelnokow U. Hucho F. Biochemistry. 1993; PubMed Scopus (60) Google Scholar). The in amide I band exposure suggests the of a of peptides forming (11Baenziger J.E. Méthot N. J. Biol. Chem. 1995; 270: 29129-29137Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). the structure of the pore-forming domain of the nAChR, the transmembrane structure was isolated by the extramembranous domains with one of two proteolytic proteinase K or of the transmembrane shows that both the and binding very near kDa for the proteinase and kDa in both the proteinase and were The is likely to of proteinase K that with the nAChR membranes (see the to Fig. the is likely to the proteolytically (see Fig. The the of for residues a to with hydrophobic transmembrane segments. This is consistent with the lack of observed for peptides in proteolytic studies of native nAChR membranes (18Gorne-Tschelnokow U. Strecker A. Kaduk C. Naumann D. Hucho F. EMBO J. 1994; 13: 338-341Crossref PubMed Scopus (100) Google Scholar). The transmembrane domain was by the segments with the hydrophobic M.P. Cohen J.B. Biochemistry. 1994; 33: 2859-2872Crossref PubMed Scopus (211) Google Scholar, J. Annu. Rev. Biochem. 1993; PubMed Scopus Google Scholar) and the by H. Biochem. PubMed Scopus Google Scholar). is in the of for proteinase and the of the is in with in the 3–6 kDa with a in exposure is evidence of nAChR subunits and very evidence of with than analysis of the and of gel that than of the is to peptides in the results that the nAChR transmembrane domain is predominantly of kDa in consistent with the of containing the nAChR transmembrane segments (5Noda M. Takahashi H. Tanabe T. Toyosato M. Kikyotani S. Furutani Y. Hirose T. Takashima H. Inayama S. Miyata T. Numa S. Nature. 1983; 302: 528-532Crossref PubMed Scopus (533) Google Scholar, U. Strecker A. Kaduk C. Naumann D. Hucho F. EMBO J. 1994; 13: 338-341Crossref PubMed Scopus (100) Google Scholar, M. Takahashi H. Tanabe T. Toyosato M. Furutani Y. Hirose T. M. Inayama S. Miyata T. Numa S. Nature. PubMed Scopus Google Scholar). N-terminal of the analysis of the a peptide at consistent with the of a peptide the hydrophobic segments. The with the hydrophobic is also consistent with a transmembrane for the FTIR spectra of proteolytically nAChR exhibit several consistent with of the extramembranous domains two and The ratio of the to 1655 amide is to ratio Eur. J. Biochem. 1990; PubMed Scopus Google Scholar), proteolytic The of the in near suggests that of the nAChR protein has been consistent with the nAChR protein located the lipid bilayer (17Unwin N. J. Mol. Biol. 1993; 229: 1101-1124Crossref PubMed Scopus (716) Google Scholar, U. Strecker A. Kaduk C. Naumann D. Hucho F. EMBO J. 1994; 13: 338-341Crossref PubMed Scopus (100) Google Scholar). is also a in the of bands near cm−1, are to the of of and The of in suggests that proteolytic treatment the of located in extramembranous of the nAChR. The very remaining near in the proteinase spectra be to proteinase The proteolytically nAChR is relatively resistant to exchange. of the amide II band near observed and exposure to suggests that of the by proteinase K treatment in a form of exposure This the that exposure of proteins, and for the The of peptide hydrogens located in relatively segments to with supports a the relatively of the lipid nAChR is also resistant to peptide 1H/2H of the membranes with peptide 1H/2H rates to the proteinase In contrast to spectra of nAChR, spectra of the transmembrane domain exhibit relatively sharp symmetric amide I band contours centered near and 1655 and band and are of α-helical structures. The lack of in band exposure to suggests is little The near in to the observed in spectra of nAChR suggests is β-sheet and The α-helical character of the nAChR transmembrane domain is supported by a analysis of the amide I Curve-fitting suggests an α-helical of between and be as a very the with the amide I contours (see The remaining of the peptides adopt structures that in the and/or and of the spectra and the of of transmembrane bands in both are in the spectra of proteins such as myoglobin and bacteriorhodopsin, are of β-sheet (see Fig. and H. PubMed Scopus Google and Eur. J. Biochem. 1990; PubMed Scopus Google Scholar). The bands observed between and in the transmembrane domain be interpreted in terms of β-sheet in the transmembrane A of the and β-sheet character of the transmembrane segments was by the spectra with of proteins of secondary and structures The transmembrane domain spectra contrast from and proteins, such as the membrane protein exhibits predominantly β-sheet secondary structures H. PubMed Scopus Google Scholar, Eur. J. Biochem. 1990; PubMed Scopus Google Scholar). that the β-sheet of that suggested for the nAChR transmembrane domain based on resolution electron microscopic The transmembrane domain spectra are with the 75% β-sheet suggested for the nAChR transmembrane domain (17Unwin N. J. Mol. Biol. 1993; 229: 1101-1124Crossref PubMed Scopus (716) Google Scholar). The transmembrane domain spectra exhibit much less in both the and β-sheet than is observed in spectra of and proteins with β-sheet H. PubMed Scopus Google Scholar). This suggests that much less than 20% of the nAChR transmembrane domain peptides adopt β-sheet secondary structures. A β-sheet of to that the of each hydrophobic the of at or (18Gorne-Tschelnokow U. Strecker A. Kaduk C. Naumann D. Hucho F. EMBO J. 1994; 13: 338-341Crossref PubMed Scopus (100) Google Scholar), the of bands to β-strands may be to in terms of transmembrane The bands observed between and likely to turn or extended structures located in the at the membrane as to transmembrane The α-helical of the transmembrane domain and the that the amide I from turn and extended are supported by a of the transmembrane domain spectra with of myoglobin exhibit relatively and symmetric amide I bands that little in exposure spectra of myoglobin are in Baenziger and J.E. J. Biochemistry. PubMed Scopus Google The amide I contours are similar in terms of the of α-helical and The bands also at similar In the between the spectra is that the α-helical structures in myoglobin at a to the α-helical structures of the transmembrane domain predominantly in the The is that α-helical including of nAChR, in by peptide 1H/2H (11Baenziger J.E. Méthot N. J. Biol. Chem. 1995; 270: 29129-29137Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 12Méthot N. Baenziger J.E. Biochemistry. 1998; 37: 14815-14822Crossref PubMed Scopus (31) Google Scholar, Rev. PubMed Scopus Google J.E. J. Biochemistry. PubMed Scopus Google Scholar). of the transmembrane domain peptide hydrogens to with is consistent with a hydrophobic transmembrane This shows that both myoglobin and the transmembrane domain preparations very similar secondary structural In the transmembrane domain amide I band is very similar in terms of the and of both α-helical and bands to that observed for the α-helical integral membrane bacteriorhodopsin, also exhibit β-sheet Eur. J. Biochem. 1990; PubMed Scopus Google Scholar). The spectra are consistent with a transmembrane domain composed predominantly of transmembrane the remaining peptides adopting turn or extended structures at the bilayer surface. The nAChR transmembrane exhibits dichroic of and for the amide I and amide II respectively, both a for α-helices perpendicular to the bilayer and contrast the dichroism for oriented transmembrane β-strands D. J. Full Text PDF PubMed Scopus Google Scholar). The of the transmembrane α-helical from the bilayer is both the of the transmembrane and the amide I and II band dichroism the from the bilayer (see the to Fig. The represents a The of the transmembrane α-helices is likely much less than The an α-helical structure for the transmembrane domain of the nAChR. This is based on the α-helical character of the infrared spectra from the nAChR transmembrane domain preparations as well as from dichroism α-helical structures perpendicular to the bilayer surface. In the amide I observed in spectra of the transmembrane domain exhibits much less β-sheet character than spectra of proteins such as and and β-sheet H. PubMed Scopus Google Scholar, Eur. J. Biochem. 1990; PubMed Scopus Google Scholar). The transmembrane domain that of the integral membrane Eur. J. Biochem. 1990; PubMed Scopus Google Scholar). the transmembrane domain amide I band is very similar to the band observed in spectra from myoglobin and the α-helical integral membrane protein Eur. J. Biochem. 1990; PubMed Scopus Google Scholar), both lack an α-helical character for the transmembrane A analysis of the transmembrane domain spectra supports our and suggests an α-helical of is consistent with the amide I band be that of amide I contours composed of bands is band and as well as a of the of bands and are a analysis of secondary structural based on amide I band as the analysis is by the with and an of the of the and β-sheet The that less than 20% of the nAChR transmembrane peptides adopt a β-sheet that all bands in of the spectra are to β structures. that β-sheet is to for the transmembrane β-strands to for one transmembrane of in each the between the amide I band in spectra of the transmembrane domain and myoglobin suggests similar secondary structures for two proteins and consistent with a transmembrane domain composed of at with the remaining peptides adopting extended or turn structures. that the studied transmembrane are 3–6 kDa in the most of our is that the transmembrane domain is composed of α-helical transmembrane structures with extended or turn structures at the bilayer surface. This a recent of the transmembrane domain of the nAChR proteolytic treatment of native nAChR membranes (18Gorne-Tschelnokow U. Strecker A. Kaduk C. Naumann D. Hucho F. EMBO J. 1994; 13: 338-341Crossref PubMed Scopus (100) Google Scholar). The a secondary structural of and turn and and a mixed α-helix/β-sheet transmembrane The between the two studies can be to a of the native membrane preparations used in the may such as a of the transmembrane channel M. A. J. in Scholar). This protein with the and in the native transmembrane preparations to the β-sheet character of the the secondary structural analysis of Gorne-Tschelnokow (18Gorne-Tschelnokow U. Strecker A. Kaduk C. Naumann D. Hucho F. EMBO J. 1994; 13: 338-341Crossref PubMed Scopus (100) Google Scholar) was on spectra the amide I band in a to from the spectra to the β-sheet of the transmembrane the analysis of Gorne-Tschelnokow U. Strecker A. Kaduk C. Naumann D. Hucho F. EMBO J. 1994; 13: 338-341Crossref PubMed Scopus (100) Google Scholar) into the that can in of FTIR spectra to as has been observed in spectra of α-helical proteins H. PubMed Scopus Google and Eur. J. Biochem. 1990; PubMed Scopus Google Scholar). the of such bands to β-sheet turn and/or extended structures is is to that the β-sheet suggested by Gorne-Tschelnokow for the transmembrane domain is similar to that has been for nAChR amide I band for the and the amide I band observed for the transmembrane domain both and in the of Gorne-Tschelnokow (18Gorne-Tschelnokow U. Strecker A. Kaduk C. Naumann D. Hucho F. EMBO J. 1994; 13: 338-341Crossref PubMed Scopus (100) Google Scholar) exhibit to the of β-sheet character that is observed in spectra of proteins that are at the of all FTIR of protein secondary structure supported by that the nAChR transmembrane domain a large of transmembrane In we the is evidence in our spectra for the of β-sheet in the transmembrane domain preparations of the nAChR. The α-helical character of the nAChR transmembrane segments is consistent with biophysical studies of transmembrane segments (13Corbin J. Méthot N. Wang H.H. Baenziger J.E. Blanton M.P. J. Biol. Chem. 1998; 273: 771-777Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 14Opella S.J. Marassi F.M. Gesell J.J. Valente A.P. Kim Y. Oblatt-Montal M. Montal M. Nat. Struct. Biol. 1999; 6: 374-379Crossref PubMed Scopus (299) Google Scholar, 15Lugovskoy A.A. Maslennikov I.V. Utkin Y.N. Tsetlin V.I. Cohen J.B. Arseniev A.S. Eur. J. Biochem. 1998; 255: 455-461Crossref PubMed Scopus (45) Google Scholar, 16Pashkov V.S. Maslennikov I.V. Tchikin L.D. Efremov R.G. Ivanov V.T. Arseniev A.S. FEBS Lett. 1999; 457: 117-121Crossref PubMed Scopus (29) Google Scholar) and both and studies of nAChR M.P. Cohen J.B. Biochemistry. 1994; 33: 2859-2872Crossref PubMed Scopus (211) Google Scholar, 9Blanton M.P. Cohen J.B. Biochemistry. 1992; 31: 3738-3750Crossref PubMed Scopus (137) Google Scholar, 10Tamamizu S. Guzman G.R. Santiago J. Rojas L.V. McNamee M.G. Lasalde-Dominicci J.A. Biochemistry. 2000; 39: 4666-4673Crossref PubMed Scopus (60) Google Scholar, J.E. Méthot N. J. Biol. Chem. 1995; 270: 29129-29137Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 12Méthot N. Baenziger J.E. Biochemistry. 1998; 37: 14815-14822Crossref PubMed Scopus (31) Google Scholar), M1 may form a A. Biochemistry. 1995; PubMed Scopus Google Scholar, F.J. Blanton M.P. M. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). Our results strongly that the transmembrane domain of the nAChR and likely of the ligand-gated ion channel is from five bundles of four transmembrane α-helices. for the

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