Identification of amino acid residues important for assembly of GABAA receptor a1 and c2 subunits

Comparative models of GABAA receptors composed of a1b3c2 subunits were generated using the acetylcholinebinding protein (AChBP) as a template and were used for predicting putative engineered cross-link sites between the a1 and the c2 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 GABAA receptors. Whereas residue a1A108 was important for the formation of assembly intermediates with b3 and c2 subunits consistent with its proposed location at the a1(+) side of GABAA receptors, residues c2T125 and c2P127 were important for assembly with b3 subunits. Mutation of each of these residues also caused an impaired expression of receptors at the cell surface. In contrast, mutated residues a1F99C, a1S106C or c2T126C 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.