A comparison between the Torpedo and muscle-type acetylcholine receptors (AChRs) reveals differences in several lipid-exposed amino acids, particularly in the polarity of those residues. The goal of this study was to characterize the role of eight lipid-exposed residues in the functional differences between the Torpedo and muscle-type AChRs. To this end, residues αS287, αC412, βY441, γM299, γS460, δM293, δS297 and δN305 in the Torpedo AChR were replaced with those found in the muscle-type receptor. Mutant receptor expression was measured in Xenopus oocytes using [125I]-α-bungarotoxin, and AChR ion channel function was evaluated using the two-electrode voltage clamp. Eight mutant combinations resulted in an increase (1.5- to 5.2-fold) in AChR expression. Four mutant combinations produced a significant 46% decrease in the ACh 50% inhibitory concentration (EC50), while three mutant combinations resulted in 1.7- to 2-fold increases in ACh EC50. Finally, seven mutant combinations resulted in a decrease in normalized, ACh-induced currents. Our results suggest that these residues, although remote from the ion channel pore, (1) contribute to ion channel gating, (2) may affect trafficking of AChR into specialized membrane domains and (3) account for the functional differences between Torpedo and muscle-type AChR. These findings emphasize the importance of the lipid-protein interface in the functional differences between the Torpedo and muscle-type AChRs.
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
Akabas M.H., Karlin A. 1995. Identification of acetylcholine receptor channel-lining residues in the M1 segment of the alpha-subunit. Biochemistry 34:12496–12500
Blanton M.P., Cohen J.B. 1992. Mapping the lipid-exposed regions in the Torpedo californica nicotinic acetylcholine receptor. Biochemistry 31:3738–3750. Erratum in Biochemistry 31:5951
Blanton M.P., Cohen J.B. 1994. Identifying the lipid-protein interface of the Torpedo nicotinic acetylcholine receptor: Secondary structure implications. Biochemistry 33:2859–2872
Blanton M.P., Dangott L.J., Raja S.K., Lala A.K., Cohen J.B. 1998. Probing the structure of the nicotinic acetylcholine receptor ion channel with the uncharged photoactivable compound 3H-diazofluorene. J. Biol. Chem. 273:8659–8668
Bouzat C., Bren N., Sine S.M. 1994. Structural basis of the different gating kinetics of fetal and adult acetylcholine receptors. Neuron 13:1395–1402
Bouzat C., Roccamo A.M., Garbus. I., Barrantes F.J. 1998. Mutations at lipid-exposed residues of the acetylcholine receptor affect its gating kinetics. Mol. Pharmacol. 54:146–153
Butler, D.H., Lasalde, J.A., Butler, J.K., Tamamizu, S., Zimmerman, G., McNamee, M.G. 1997. Mouse-Torpedo chimeric alpha-subunit used to probe channel-gating determinants on the nicotinic acetylcholine receptor primary sequence. Cell. Mol. Neurobiol. 17:13–33
Chothia C. 1975. Structural invariants in protein folding. Nature 254:304–308
Couet J., Li S., Okamoto T., Ikezu T., Lisanti M.P. 1997. Identification of peptide and protein ligands for the caveolin-scaffolding domain. Implications for the interaction of caveolin with caveolae-associated proteins. J. Biol. Chem. 272:6525–6533
Cruz-Martin A., Mercado J.L., Rojas L.V., McNamee M.G., Lasalde-Dominicci J.A. 2001. Tryptophan substitutions at lipid-exposed positions of the gamma M3 transmembrane domain increase the macroscopic ionic current response of the Torpedo californica nicotinic acetylcholine receptor. J. Membr. Biol. 183:61–70
Guzman G.R., Santiago J., Ricardo A., Marti-Arbona R., Rojas L.V., Lasalde-Dominicci J.A. 2003. Tryptophan scanning mutagenesis in the alpha M3 transmembrane domain of the Torpedo californica acetylcholine receptor: Functional and structural implications. Biochemistry 42:12243–12250
Lasalde, J.A., Tamamizu, S., Butler, D.H., Vibat, C.R., Hung, B., McNamee, M.G. 1996. Tryptophan substitutions at the lipid-exposed transmembrane segment M4 of Torpedo californica acetylcholine receptor govern channel gating. Biochemistry :14139–14148
Lee Y.H., Li L., Lasalde J., Rojas L., McNamee M., Ortiz-Miranda S.I., Pappone P. 1994. Mutations in the M4 domain of Torpedo californica acetylcholine receptor dramatically alter ion channel function. Biophys. J. 66:646–653
Miyazawa A., Fujiyoshi Y., Unwin N. 2003. Structure and gating mechanism of the acetylcholine receptor pore. Nature 423:949–955
Naranjo D., Brehm P. 1993. Modal shifts in acetylcholine receptor channel gating confer subunit-dependent desensitization. Science 260:1811–1814
Navedo M., Nieves M., Rojas L., Lasalde-Dominicci J.A. 2004. Tryptophan substitutions reveal the role of nicotinic acetylcholine receptor alpha-TM3 domain in channel gating: differences between Torpedo and muscle-type AChR. Biochemistry 43:78–84
Noda M., Takahashi H., Tanabe T., Toyosato M., Kikyotani S., Furutani Y., Hirose T., Takashima H., Inayama S., Miyata T., Numa S. 1983. Structural homology of Torpedo californica acetylcholine receptor subunits. Nature 302:528–532
Mitra A., Bailey T.D., Auerbach A.L. 2004. Structural dynamics of the M4 transmembrane segment during acetylcholine receptor gating. Structure 12:1909–1918
Ortiz-Miranda S.I., Lasalde J.A., Pappone P.A., McNamee M.G. 1997. Mutations in the M4 domain of the Torpedo californica nicotinic acetylcholine receptor alter channel opening and closing. J. Membr. Biol. 158:17–30
Otero-Cruz J.D., Báez-Pagán C.A., Caraballo-González I.M., Lasalde-Dominicci J.A. 2006. A lipid-exposed transmembrane domain of ligand-gated ion channel receptor displays a tilted spring motion during channel activation, comparing Torpedo versus muscle-type acetylcholine Receptors. A SPRING MODEL REVEALED. J. Biol. Chem. 282:9162–9171
Santiago J., Guzman G.R., Rojas L.V., Marti R., Asmar-Rovira G.A., Santana L.F., McNamee M., Lasalde-Dominicci J.A. 2001. Probing the effects of membrane cholesterol in the Torpedo californica acetylcholine receptor and the novel lipid-exposed mutation alpha C418W in Xenopus oocytes. J. Biol. Chem. 276:46523–46532
Tamamizu S., Lee Y., Hung B., McNamee M.G., Lasalde-Dominicci J.A. 1999. Alteration in ion channel function of mouse nicotinic acetylcholine receptor by mutations in the M4 transmembrane domain. J. Membr. Biol. 170:157–164. Erratum in J. Membr. Biol. 1999;172:89
Tamamizu S., Guzman G.R., Santiago J., Rojas L.V., McNamee M.G., Lasalde-Dominicci J.A. 2000. Functional effects of periodic tryptophan substitutions in the alpha M4 transmembrane domain of the Torpedo californica nicotinic acetylcholine receptor. Biochemistry 39:4666–4673
Unwin N. 2005. Refined structure of the nicotinic acetylcholine receptor at 4Å resolution. J. Mol. Biol. 346:967–989
Wang H.L., Milone M., Ohno K., Shen X.M., Tsujino A., Batocchi A.P., Tonali P., Brengman J., Engel A.G., Sine S.M. 1999. Acetylcholine receptor M3 domain: Stereochemical and volume contributions to channel gating. Nat. Neurosci. 2:226–233. Erratum in Nat. Neurosci. 1999;2:485
Yu, L., Leonard, R.J., Davidson, N., Lester, H.A. 1991. Single-channel properties of mouse-Torpedo acetylcholine receptor hybrids expressed in Xenopus oocytes. Brain Res. Mol. Brain Res. :203–211
This work was supported in part by grants from the National Institutes of Health: 2RO1GM5637-10 and GM08102-27, as well as UPR Institutional Funds for Research awarded to J.A. Lasalde-Dominicci. G. Guzman was supported by NSF-AGEP grant HRD-9817642 and A. Ricardo by NIH-MARC grant 5T34GM07821.
About this article
Cite this article
Guzmán, G.R., Ortiz-Acevedo, A., Ricardo, A. et al. The Polarity of Lipid-Exposed Residues Contributes to the Functional Differences between Torpedo and Muscle-Type Nicotinic Receptors. J Membrane Biol 214, 131–138 (2006). https://doi.org/10.1007/s00232-006-0051-0
- Acetylcholine receptor
- Site-directed mutagenesis
- Xenopus oocyte
- Lipid-exposed residue
- Torpedo californica
- Muscle-type receptor