The juvenile myoclonic epilepsy GABA(A) receptor alpha1 subunit mutation A322D produces asymmetrical, subunit position-dependent reduction of heterozygous receptor currents and alpha1 subunit protein expression

Abstract

Individuals with autosomal dominant juvenile myoclonic epilepsy are heterozygous for a GABA(A) receptor alpha1 subunit mutation (alpha1A322D). GABA(A) receptor alphabetagamma subunits are arranged around the pore in a beta-alpha-beta-alpha-gamma sequence (counterclockwise from the synaptic cleft). Therefore, each alpha1 subunit has different adjacent subunits, and heterozygous expression of alpha1(A322D), beta, and gamma subunits could produce receptors with four different subunit arrangements: beta-alpha1-beta-alpha1-gamma (wild type); beta-alpha1(A322D)-beta-alpha1-gamma (Het(betaalphabeta)); beta-alpha1-beta-alpha1(A322D)-gamma (Het(betaalphagamma));beta-alpha1(A322D)-beta-alpha1(A322D)-gamma (homozygous). Expression of a 1:1 mixture of wild-type andalpha1(A322D) subunits with beta2S and gamma2S 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 alpha1 subunit from heterozygous transfections was also intermediate between those of wild-type and homozygous mutant transfections. alpha1(A322D) mutations were then made in covalently tethered triplet (gamma2S-beta2S-alpha1) and tandem (beta2S-alpha1) concatamers to target selectively alpha1(A322D) to each of the asymmetric alpha1 subunits. Coexpression of mutant and wild-type concatamers resulted in expression of either Het(betaalphabeta) or Het(betaalphagamma) receptors. Het(betaalphabeta) currents were smaller than wild type and much larger than Het(betaalphagamma) and homozygous currents. Furthermore, Het(betaalphabeta) transfections contained less beta-alpha concatamer than wild type but more than both Het(betaalphagamma) and homozygous mutant transfections. Thus, whole-cell currents and protein expression of heterozygous alpha1(A322D)beta2Sgamma2S receptors depended on the position of the mutant alpha1 subunit, and GABA(A) receptor currents in heterozygous individuals likely result primarily from wild-type and Het(betaalphabeta) receptors with little contribution from Het(betaalphagamma) and homozygous receptors.

Figure 1.

Whole-cell currents of untethered GABA_A receptors. Representative current traces obtained from wild-type (A), heterozygous (B), or homozygous (C) mutant transfections after a 400 msec step into 1 mm GABA. The black trace in C is depicted on the same voltage scale (top scale bar) as the traces in A and B, but the gray trace is plotted in an expanded scale (bottom scale bar) to display the time course of the current kinetics. D, The mean peak currents are depicted ±SEM. Peak currents from heterozygous transfections (n = 16) were significantly larger than homozygous mutant peak currents (n = 5; p < 0.001) and significantly smaller than wild-type peak currents (n = 17; p < 0.05). E, The GABA EC₅₀ values for wild-type (•), heterozygous (▴), and homozygous (▪) mutant transfections were determined by 6 sec, rapid-perfusion, whole-cell response in increasing GABA concentrations. Cells were voltage clamped at -20 mV. Currents were normalized to the maximal peak current for each cell, and the EC₅₀ values were calculated and fit as described in Materials and Methods.

Figure 2.

Western blot analysis of total and surface α1 receptor protein. HEK293T cells were transfected with wild-type β2 and γ2 subunits and either wild-type α1 subunit (WT), a 1:1 mixture of wild-type andα1(A322D) subunit (Het),α1(A322D) subunit (Hom), or onlyβ- andγ - subunits but no α1 subunit (βγ). A, Western blots of whole-cell lysates (20 μg of protein) demonstrated a specific immunoreactive band at 50 kDa. B, Transfected HEK293T cells were incubated with sulfo-NHS-SS-biotin, and the biotinylated proteins were analyzed by Western blot.

Figure 3.

Whole-cell currents of tethered GABA_A receptors. A-C, Representative current traces obtained from wild type (A; WT), Het_βαβ (B) or Het_βαγ (C) receptors after a 400msec step into 1 mm GABA. The black trace in C is depicted on the same voltage scale (top scale bar) as the traces in A and B, but the gray trace is plotted in an expanded scale (bottom scale bar) to display the time course of the current kinetics. D, The mean peak currents are depicted ±SEM. Het_βαγ peak currents (n = 8) were significantly smaller than both WT (n = 20) and Het_βαβ (n = 17) currents (p < 0.01). Mean Het_βαβ currents were smaller than WT currents (p < 0.05). Het_βαβ currents had a greater component of fast desensitization and total desensitization than wild-type tethered GABA_A receptor currents. There was no significant change in the component of intermediate desensitization or in the desensitization time constants (Table 2). E, The GABA EC₅₀ values for tethered wild-type (•) and Het_βαβ (▴) receptors were determined by 1 sec, rapid-perfusion, whole-cell response in increasing GABA concentrations. Cells were voltage clamped at -20mV. Currents were normalized to the maximal peak current for each cell, and the EC₅₀ values were calculated and fit as described in Materials and Methods. There was no significant difference between the EC₅₀ value of wild-type (42 ± 0.4 μm) and Het_βαβ receptors (32 ± 0.4 μm; p > 0.05).

Figure 4.

Western blot analysis of tethered α1(A322D) GABA_A receptors. Lysates (30 μg of protein) from untransfected HEK293T cells (UT) or HEK293T cells transfected with tethered wild-type (WT), Het_βαβ (βαβ), Het_βαγ (βαγ), or homozygous mutant (Hom) GABA_A receptors were analyzed by Western blots and probed with a monoclonal antibody targeted against the β2 subunit.

Figure 5.

Subunit combinations in a heterozygous expression. There are four receptor phenotypes formed with heterozygous expression of wild-type (white) and mutant α1(A322D) (shaded) subunits. The Het_βαγ and homozygous mutant receptors produced <7% of the mean current of wild-type receptors. Therefore, the currents of the heterozygous receptors were determined by the wild-type (WT) and Het_βαβ receptors.