Identification of amino acid residues important for assembly of GABA receptor alpha1 and gamma2 subunits

Comparative models of GABA(A) receptors composed of alpha1 beta3 gamma2 subunits were generated using the acetylcholine-binding protein (AChBP) as a template and were used for predicting putative engineered cross-link sites between the alpha1 and the gamma2 subunit. The respective amino acid residues were substituted by cysteines and disulfide bond formation between subunits was investigated on co-transfection into human embryonic kidney (HEK) cells. Although disulfide bond formation between subunits could not be observed, results indicated that mutations studied influenced assembly of GABA(A) receptors. Whereas residue alpha1A108 was important for the formation of assembly intermediates with beta3 and gamma2 subunits consistent with its proposed location at the alpha1(+) side of GABA(A) receptors, residues gamma2T125 and gamma2P127 were important for assembly with beta3 subunits. Mutation of each of these residues also caused an impaired expression of receptors at the cell surface. In contrast, mutated residues alpha1F99C, alpha1S106C or gamma2T126C only impaired the formation of receptors at the cell surface when co-expressed with subunits in which their predicted interaction partner was also mutated. These data are consistent with the prediction that the mutated residue pairs are located close to each other.