title: Spontaneous cross-link of mutated alpha1 subunits during GABA(A) receptor assembly.
abstract: gamma-Aminobutyric acid, type A (GABA(A)) receptor alpha1 subunits containing a cysteine mutation at a position in the channel mouth (H109C) surprisingly formed a spontaneous cross-link with each other in receptors composed of alpha1H109C, beta3, and gamma2 subunits. Cross-linking of two alpha1H109C subunits did not significantly change the affinity of [(3)H]muscimol or [(3)H]Ro15-1788 binding in alpha1H109Cbeta3gamma2 receptors, but GABA displayed a reduced potency for activating chloride currents. On reduction of the disulfide bond, however, GABA activation as well as diazepam modulation was similar in mutated and wild-type receptors, suggesting that these receptors exhibited the same subunit stoichiometry and arrangement. Disulfide bonds could not be reoxidized by copper phenanthroline after having been reduced in completely assembled receptors, suggesting that cross-linking can only occur at an early stage of assembly. The cross-link of alpha1H109C subunits and the subsequent transport of the resulting homodimers to the cell surface caused a reduction of the intracellular pool of alpha1H109C subunits and a reduced formation of completely assembled receptors. The formation of alpha1H109C homodimers as well as of correctly assembled GABA(A) receptors containing cross-linked alpha1H109C subunits could indicate that homodimerization of alpha1 subunits via contacts located in the channel mouth might be one starting point of GABA(A) receptor assembly. Alternatively the assembly mechanism might have started with the formation of heterodimers followed by a cross-link of mutated alpha1 subunits at the heterotrimeric stage. The formation of cross-linked alpha1H109C homodimers would then have occurred independently in a separate pathway.
authors.affiliation: Division of Biochemistry and Molecular Biology, Center for Brain Research, Medical University of Vienna and A-1090 Vienna, Austria.
authors.forename: Isabella
authors.initials: I
authors.lastname: Sarto-Jackson
authors.affiliation: Division of Biochemistry and Molecular Biology, Center for Brain Research, Medical University of Vienna and A-1090 Vienna, Austria.
authors.forename: Roman
authors.initials: R
authors.lastname: Furtmueller
authors.affiliation: Division of Biochemistry and Molecular Biology, Center for Brain Research, Medical University of Vienna and A-1090 Vienna, Austria.
authors.forename: Margot
authors.initials: M
authors.lastname: Ernst
authors.affiliation: Division of Biochemistry and Molecular Biology, Center for Brain Research, Medical University of Vienna and A-1090 Vienna, Austria.
authors.forename: Sigismund
authors.initials: S
authors.lastname: Huck
authors.affiliation: Division of Biochemistry and Molecular Biology, Center for Brain Research, Medical University of Vienna and A-1090 Vienna, Austria; Section of Biochemical Psychiatry, University Clinic for Psychiatry, A-1090 Vienna, Austria. Electronic address: werner.sieghart@meduniwien.ac.at.
authors.forename: Werner
authors.initials: W
authors.lastname: Sieghart
mesh_terms: D000818:Animals; D018118:Chloride Channels / Q000737:chemistry / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D009118:Muscimol / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010618:Phenanthrolines / Q000737:chemistry; D017354:Point Mutation; D020836:Protein Structure, Quaternary / Q000235:genetics; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics; D051381:Rats; D011963:Receptors, GABA-A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013440:Sulfides / Q000737:chemistry / Q000378:metabolism
chemical_list: D018118:Chloride Channels; D010618:Phenanthrolines; D021122:Protein Subunits; D011963:Receptors, GABA-A; D013440:Sulfides; D009118:Muscimol; D003545:Cysteine