title: Functional effects of periodic tryptophan substitutions in the alpha M4 transmembrane domain of the Torpedo californica nicotinic acetylcholine receptor.
abstract: Previous amino acid substitutions at the M4 domain of the Torpedo californica and mouse acetylcholine receptor suggested that the location of the substitution relative to the membrane-lipid interface and perhaps to the ion pore can be critical to the channel gating mechanism [Lasalde, J. A., Tamamizu, S., Butler, D. H., Vibat, C. R. T., Hung, B., and McNamee, M. G. (1996) Biochemistry 35, 14139-14148; Ortiz-Miranda, S. I., Lasalde, J. A., Pappone, P. A., and McNamee, M. G. (1997) J. Membr. Biol. 158, 17-30; Tamamizu, S., Lee, Y. H., Hung, B., McNamee, M. G., and Lasalde-Dominicci, J. A. (1999) J. Membr. Biol. 170, 157-164]. In this study, we introduce tryptophan substitutions at 12 positions (C412W, M415W, L416W, I417W, C418W, I419W, I420W, G421W, T422W, V423W, S424W, and V425W) along this postulated lipid-exposed segment M4 so that we can examine functional consequences on channel gating. The expression levels of mutants C412W, G421W, S424W, and V425W were almost the same as that of the wild type, whereas other mutants (M415W, L416W, C418W, I419W, I420W, T422W, and V423W) had relatively lower expression levels compared to that of the wild type as measured by iodinated alpha-bungarotoxin binding ([(125)I]-alpha-BgTx). Two positions (L416W and I419W) had less than 20% of the wild type expression level. I417W gave no detectable [(125)I]BgTx binding on the surface of oocyte, suggesting that this position might be involved in the AChR assembly, oligomerization, or transport to the cell membrane. The alphaV425W mutant exhibited a significant increase in the open channel probability with a moderate increase in the macroscopic response at higher ACh concentrations very likely due to channel block. The periodicity for the alteration of receptor assembly and ion channel function seems to favor a potential alpha-helical structure. Mutants that have lower levels of expression are clustered on one side of the postulated alpha-helical structure. Mutations that display normal expression and functional activity have been shown previously to face the membrane lipids by independent labeling studies. The functional analysis of these mutations will be presented and discussed in terms of possible structural models.
authors.affiliation: Section of Molecular and Cellular Biology, University of California, Davis, California 95616, USA.
authors.forename: S
authors.initials: S
authors.lastname: Tamamizu
authors.forename: G R
authors.initials: GR
authors.lastname: Guzmán
authors.forename: J
authors.initials: J
authors.lastname: Santiago
authors.forename: L V
authors.initials: LV
authors.lastname: Rojas
authors.forename: M G
authors.initials: MG
authors.lastname: McNamee
authors.forename: J A
authors.initials: JA
authors.lastname: Lasalde-Dominicci
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology; D000495:Allosteric Site; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D002038:Bungarotoxins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004553:Electric Conductivity; D015870:Gene Expression; D015640:Ion Channel Gating / Q000187:drug effects; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009865:Oocytes; D018408:Patch-Clamp Techniques; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014101:Torpedo / Q000235:genetics; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D002038:Bungarotoxins; D011978:Receptors, Nicotinic; D014364:Tryptophan; D000109:Acetylcholine