title: Tryptophan scanning mutagenesis of the gammaM4 transmembrane domain of the acetylcholine receptor from Torpedo californica.
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 gammaM4 transmembrane domain (gammaTM4) by introducing tryptophan, at residues 451-462, along the gammaTM4. Wild type (WT) and mutant AChR were expressed in Xenopus laevis oocytes. Using [(125)I]alpha-bungarotoxin binding assays and voltage clamp, we determined that the nAChR expression, EC(50), and Hill coefficient values for WT are 1.8 +/- 0.4 fmol, 30.3 +/- 1.1 microM, 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 EC(50) (19.5 +/- 1.7, 11.4 +/- 0.7, 16.4 +/- 3.8, and 19.1 +/- 2.6 microM, respectively), thus suggesting a gain in function when compared with WT. In contrast, mutation L458W induced an increase in EC(50) (42.8 +/- 6.8 microM) 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 (EC(50) 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 gammaTM4.
authors.affiliation: University of Puerto Rico, Department of Biology, San Juan, Puerto Rico 00931-3360.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Ortiz-Acevedo
authors.forename: Mariel
authors.initials: M
authors.lastname: Melendez
authors.forename: Aloysha M
authors.initials: AM
authors.lastname: Asseo
authors.forename: Nilza
authors.initials: N
authors.lastname: Biaggi
authors.forename: Legier V
authors.initials: LV
authors.lastname: Rojas
authors.forename: José A
authors.initials: JA
authors.lastname: Lasalde-Dominicci
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D008845:Microinjections; D009865:Oocytes; D018408:Patch-Clamp Techniques; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D011950:Receptors, Cholinergic / Q000737:chemistry* / Q000235:genetics; D014101:Torpedo; D014364:Tryptophan; D014981:Xenopus
chemical_list: D021122:Protein Subunits; D011950:Receptors, Cholinergic; D014364:Tryptophan