Spontaneous cross-link of mutated α1 subunits during GABAA receptor assembly

gamma-Aminobutyric acid, type A (GABA(A)) receptor alpha 1 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 alpha 1H109C, beta 3, and gamma 2 subunits. Cross-linking of two alpha 1H109C subunits did not significantly change the affinity of [H-3]muscimol or [H-3]Ro15-1788 binding in alpha 1H109C beta 3 gamma 2 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 alpha 1H109C subunits and the subsequent transport of the resulting homodimers to the cell surface caused a reduction of the intracellular pool of alpha 1H109C subunits and a reduced formation of completely assembled receptors. The formation of alpha 1H109C homodimers as well as of correctly assembled GABAA receptors containing cross-linked alpha 1H109C subunits could indicate that homodimerization of alpha 1 subunits via contacts located in the channel mouth might be one starting point of GABA, receptor assembly. Alternatively the assembly mechanism might have started with the formation of heterodimers followed by a cross-link of mutated alpha 1 subunits at the hetero trimeric stage. The formation of cross-linked alpha 1H109C homodimers would then have occurred independently in a separate pathway.