title: 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.
abstract: Our previous amino-acid substitutions at the postulated lipid-exposed transmembrane segment M4 of the Torpedo californica acetylcholine receptor (AChR) focused on the alpha subunit. In this study we have extended the mutagenesis analysis using single tryptophan replacements in seven positions (I288, M291, F292, S294, L296, M299 and N300) near the center of the third transmembrane domain of the gamma subunit (gamma M3). All the tryptophan substitution mutants were expressed in Xenopus laevis oocytes following mRNA injections at levels close to wild type. The functional response of these mutants was evaluated using macroscopic current analysis in voltage-clamped oocytes. For all the substitutions the concentration for half-maximal activation, EC(50), is similar to wild type using acetylcholine. For F292W, L296W and M299W the normalized macroscopic responses are 2- to 3-fold higher than for wild type. Previous photolabeling studies demonstrated that these three positions were in contact with membrane lipids. Each of these M3 mutations was co-injected with the previously characterized alpha C418W mutant to examine possible synergistic effects of single lipid-exposed mutations on two different subunits. For the gamma M3/alpha M4 double mutants, the EC(50)s were similar to those measured for the alpha C418W mutant alone. Tryptophan substitutions at positions that presumably face the interior of the protein (S294 and M291) or neighboring helices (I288) did not cause significant inhibition of channel function or surface expression of AChRs.
authors.affiliation: Department of Biology, University of Puerto Rico, P.O. Box 23360, San Juan, Puerto Rico 00931-3360, USA.
authors.forename: A
authors.initials: A
authors.lastname: Cruz-Martín
authors.forename: J L
authors.initials: JL
authors.lastname: Mercado
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 / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D015870:Gene Expression; D007473:Ion Channels / Q000502:physiology*; D050356:Lipid Metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014101:Torpedo; D014364:Tryptophan / Q000235:genetics / Q000502:physiology*; D014982:Xenopus laevis
chemical_list: D007473:Ion Channels; D011978:Receptors, Nicotinic; D014364:Tryptophan; D000109:Acetylcholine