Tryptophan Scanning Mutagenesis of the γM4 Transmembrane Domain of the Acetylcholine Receptor from Torpedo californica
dc.contributor.author | Ortiz-Acevedo, Alejandro | |
dc.contributor.author | Meléndez, Mariel | |
dc.contributor.author | Asseo-García, Aloysha M. | |
dc.contributor.author | Biaggi, Nilza | |
dc.contributor.author | Rojas, Legier V. | |
dc.contributor.author | Lasalde-Dominicci, José A. | |
dc.date.accessioned | 2017-03-23T22:11:30Z | |
dc.date.available | 2017-03-23T22:11:30Z | |
dc.date.copyright | The NIH requires authors of all publications resulting from any NIH-supported research deposit the final accepted version of their manuscripts in PubMed Central. All manuscripts accepted by ASBMB publications appear as Papers in Press immediately upon acceptance, and these remain freely accessible on the journal site at all times after the initial posting. | en_USA |
dc.date.issued | 2004-10 | |
dc.identifier.citation | Ortiz-Acevedo, A., et al. “Tryptophan Scanning Mutagenesis of the γM4 Transmembrane Domain of the Acetylcholine Receptor from Torpedo Californica.” Journal of Biological Chemistry, vol. 279, no. 40, Sept. 2004, pp. 42250–42257., doi:10.1074/jbc.m405132200. | en_US |
dc.identifier.issn | 1083-351X | |
dc.identifier.uri | http://hdl.handle.net/11721/1570 | |
dc.description.abstract | The periodicity of structural and functional effects induced by tryptophan scanning mutagenesis has been successfully used to define function and secondary structure of various transmembrane domains of the acetylcholine receptor of Torpedo californica. We expand the tryptophan scanning of the AchR of T. californica to the γM4 transmembrane domain (γTM4) by introducing tryptophan, at residues 451–462, along the γTM4. Wild type (WT) and mutant AChR were expressed in Xenopus laevis oocytes. Using [125I]α-bungarotoxin binding assays and voltage clamp, we determined that the nAChR expression, EC50, and Hill coefficient values for WT are 1.8 ± 0.4 fmol, 30.3 ± 1.1 μM, and 1.8 ± 0.3, respectively. Mutations L456W, F459W, and G462W induce a significant increase in nAChR expression (2.8 ± 0.5, 3.6 ± 0.6, and 3.0 ± 0.5 fmol, respectively) when compared with WT. These data suggest that these residues are important for AChR oligomerization. Mutations A455W, L456W, F459W, and G462W result in a significant decrease in EC50 (19.5 ± 1.7, 11.4 ± 0.7, 16.4 ± 3.8, and 19.1 ± 2.6 μM, respectively), thus suggesting a gain in function when compared with WT. In contrast, mutation L458W induced an increase in EC50 (42.8 ± 6.8 μM) or loss in function when compared with WT. The Hill coefficient values were the same for WT and all of the mutations studied. The periodicity in function (EC50 and macroscopic peak current) and nAChR expression reveals an average of 3.3 and 3.0 amino acids respectively, thus suggesting a helical secondary structure for the γTM4. | en_US |
dc.description.sponsorship | This work was supported by NIGMS, National Institutes of Health Grants GM56371-05 and GM08102-27. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Supported by a Postdoctoral Supplement to Grant GMRO156371. Supported by Minority Access to Research Careers-National Institutes of Health Grant 5T34GM07821 and the SCORE Program. | en_USA |
dc.Format.extent | 1.13 MB | |
dc.language.iso | en_US | en_US |
dc.publisher | American Society for Biochemistry and Molecular Biology | en_US |
dc.subject.mesh | Amino Acid Substitution | en_USA |
dc.subject.mesh | Receptors, Cholinergic/chemistry | en_USA |
dc.subject.mesh | Torpedo | en_USA |
dc.subject.mesh | Tryptophan | en_USA |
dc.title | Tryptophan Scanning Mutagenesis of the γM4 Transmembrane Domain of the Acetylcholine Receptor from Torpedo californica | en_US |
dc.type | Article | en_US |
dc.rights.licence | The NIH requires authors of all publications resulting from any NIH-supported research deposit the final accepted version of their manuscripts in PubMed Central. All manuscripts accepted by ASBMB publications appear as Papers in Press immediately upon acceptance, and these remain freely accessible on the journal site at all times after the initial posting. | en_USA |
dcterms.rights | The NIH requires authors of all publications resulting from any NIH-supported research deposit the final accepted version of their manuscripts in PubMed Central. The JBC automatically deposits all papers into PubMed Central. | en_USA |
dcterms.rightsHolder | The NIH requires authors of all publications resulting from any NIH-supported research deposit the final accepted version of their manuscripts in PubMed Central.The JBC automatically deposits all papers into PubMed Central. | en_USA |
dc.identifier.doi | 10.1074/jbc.M405132200 | |
dc.local.Department | Department of Biology | en_USA |
dc.local.Faculty | College of Natural Sciences | en_USA |
dc.contributor.campus | University of Puerto Rico, Río Piedras Campus |
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Except where otherwise noted, this item's license is described as The NIH requires authors of all publications resulting from any NIH-supported research deposit the final accepted version of their manuscripts in PubMed Central. All manuscripts accepted by ASBMB publications appear as Papers in Press immediately upon acceptance, and these remain freely accessible on the journal site at all times after the initial posting.