Functional genomics of epilepsy-associated mutations in the GABAA receptor subunits reveal that one mutation impairs function and two are catastrophic

A number of epilepsy-causing mutations have recently been identified in the genes of the 1, 3, and 2 subunits comprising the -aminobutyric acid type A (GABA(A)) receptor. These mutations are typically dominant, and in certain cases, such as the 1 and 3 subunits, they may lead to a mix of receptors at the cell surface that contain no mutant subunits, a single mutated subunit, or two mutated subunits. To determine the effects of mutations in a single subunit or in two subunits on receptor activation, we created a concatenated protein assembly that links all five subunits of the 132 receptor and expresses them in the correct orientation. We created nine separate receptor variants with a single-mutant subunit and four receptors containing two subunits of the 2(R323Q), 3(D120N), 3(T157M), 3(Y302C), and 3(S254F) epilepsy-causing mutations. We found that the singly mutated 2(R323Q) subunit impairs GABA activation of the receptor by reducing GABA potency. A single 3(D120N), 3(T157M), or 3(Y302C) mutation also substantially impaired receptor activation, and two copies of these mutants within a receptor were catastrophic. Of note, an effect of the 3(S254F) mutation on GABA potency depended on the location of this mutant subunit within the receptor, possibly because of the membrane environment surrounding the transmembrane region of the receptor. Our results highlight that precise functional genomic analyses of GABA(A) receptor mutations using concatenated constructs can identify receptors with an intermediate phenotype that contribute to epileptic phenotypes and that are potential drug targets for precision medicine approaches.