The juvenile myoclonic epilepsy GABA_A receptor α1 subunit mutation A322D produces asymmetrical, subunit position-dependent reduction of heterozygous receptor currents and α1 subunit protein expression

Individuals with autosomal dominant juvenile myoclonic epilepsy are heterozygous for a GABA_A receptor α1 subunit mutation (α1A322D). GABA_A receptor αβγ subunits are arranged around the pore in a β-α-β-α-γ sequence (counterclockwise from the synaptic cleft). Therefore, each α1 subunit has different adjacent subunits, and heterozygous expression of α1(A322D), β, and γ subunits could produce receptors with four different subunit arrangements: β-α1-β-α1-γ (wild type); β-α1(A322D)-β-α1-γ (Het _βαβ); β-α1-β-α1(A322D)-γ (Het_βαγ); β-α1(A322D)-β- α1(A322D)-γ (homozygous). Expression of a 1:1 mixture of wild-type and α1(A322D) subunits with β2S and γ2S subunits (heterozygous transfection) produced smaller currents than wild type and much larger currents than homozygous mutant transfections. Western blot and biotinylation assays demonstrated that the amount of total and surface α1 subunit from heterozygous transfections was also intermediate between those of wild-type and homozygous mutant transfections. α1(A322D) mutations were then made in covalently tethered triplet (γ2S-β2S-α1) and tandem (β2S-α1) concatamers to target selectively α1(A322D) to each of the asymmetric α1 subunits. Coexpression of mutant and wild-type concatamers resulted in expression of either Het_βαβ or Het_βαγ receptors. Het _βαβ currents were smaller than wild type and much larger than He_βαγ and homozygous currents. Furthermore, Het_βαβ transfections contained less β-α concatamer than wild type but more than both Het _βαγ and homozygous mutant transfections. Thus, whole-cell currents and protein expression of heterozygous α1(A322D) β2Sγ2S receptors depended on the position of the mutant α1 subunit, and GABA_A receptor currents in heterozygous individuals likely result primarily from wild-type and Het_βαβ receptors with little contribution from Het_βαγ and homozygous receptors.