Delta subunit susceptibility variants E177A and R220H associated with complex epilepsy alter channel gating and surface expression of alpha4beta2delta GABAA receptors

Abstract

Most human idiopathic generalized epilepsies (IGEs) are polygenic, but virtually nothing is known of the molecular basis for any of the complex epilepsies. Recently, two GABAA receptor delta subunit variants (E177A, R220H) were proposed as susceptibility alleles for generalized epilepsy with febrile seizures plus and juvenile myoclonic epilepsy. In human embryonic kidney 293T cells, recombinant halpha1beta2delta(E177A) and halpha1beta2delta(R220H) receptor currents were reduced, but the basis for the current reduction was not determined. We examined the mechanistic basis for the current reduction produced by these variants using the halpha4beta2delta receptor, an isoform more physiologically relevant and linked to epileptogenesis, by characterizing the effects of these variants on receptor cell surface expression and single-channel gating properties. Expression of variant alpha4beta2delta(R220H) receptors resulted in a decrease in surface receptor proteins, and a smaller, but significant, reduction was observed for variant alpha4beta2delta(E177A) receptors. For both variants, no significant alterations of surface expression were observed for mixed population of wild-type and variant receptors. The mean open durations of alpha4beta2delta(E177A) and alpha4beta2delta(R220H) receptor single-channel currents were both significantly decreased compared to wild-type receptors. These data suggest that both delta(E177A) and delta(R220H) variants may result in disinhibition in IGEs by similar cellular and molecular mechanisms, and in heterozygously affected individuals, a reduction in channel open duration of delta subunit-containing GABAA receptors may be the major contributor to the epilepsy phenotypes.

Figure 1.

GABA concentration responses were similar among α4β2δ GABA_A receptors containing wild-type or variant δ subunits. A, Examples of whole-cell current traces evoked by increasing GABA concentrations from wild-type α4β2δ and variant α4β2δ(E177A) or α4β2δ(R220H) receptors. B, The current responses were normalized to the maximal current of each cell. The squares denote the mean amplitudes of currents for wild-type α4β2δ receptors (n = 7), the triangles denote those for variant α4β2δ(E177A) receptors (n = 5), and the circles denote those for variant α4β2δ(R220H) receptors (n = 5). The solid bar above each current trace denotes the duration of GABA application (4 s). wt, Wild-type α4β2δ receptors. The error bars represent SEM. The holding potential was −50 mV for wild-type and variant receptors.

Figure 2.

The peak GABA current amplitudes were reduced, and the kinetic properties were altered for α4β2δ receptors containing mixed wild-type and variant or pure variant δ(E177A) receptors. A, Representative whole-cell current traces evoked by saturating GABA (1 mm) from wild-type α4β2δ, mixed wild-type and variant, and pure variant α4β2δ(E177A) receptors. B, Compared with wild-type receptors (n = 77), the mean current amplitude was smaller for mixed receptors (n = 26) as well as for pure variant receptors (n = 25). C, The mean extent of desensitization was decreased for mixed and pure variant receptors compared with wild-type receptors. D, Comparison of the mean rate of current deactivation among wild-type, mixed, and pure variant receptors. The solid bar above each current trace denotes the duration of GABA application (4 s). The open bars represent the mean current features for wild-type receptors, the hatched bars represent those for the mixed receptors, and the black bars represent those for the pure variant receptors. wt, Wild-type α4β2δ receptors; mix, presence of both receptors; var, pure variant receptors. The error bars denote SEM. The asterisks indicate significant difference from wild-type α4β2δ receptors (*p < 0.05, ** p < 0.01).

Figure 3.

The peak GABA current amplitudes were reduced, but the kinetic properties were not altered for α4β2δ receptors containing mixed wild-type and variant or pure variant δ(R220H) receptors. A, Representative whole-cell current traces evoked by saturating GABA (1 mm) from wild-type α4β2δ, mixed wild-type and variant, and pure variant α4β2δ(R220H) receptors. B, Compared with wild-type receptors (n = 77), the mean current amplitude was smaller for the mixed receptors (n = 20) as well as for pure variant receptors (n = 24). C, The mean extent of desensitization was not different among wild-type, mixed, and pure variant receptors. D, The mean rate of current deactivation of mixed, and pure variant receptors was not altered compared with wild-type receptors. The solid bar above each current trace denotes the duration of GABA application (4 s). The open bars represent the mean current features for wild-type receptors, the hatched bars represent those for the mixed receptors, and the black bars represent those for pure variant receptors. wt, Wild-type α4β2δ receptors; mix, presence of both receptors; var, pure variant receptors. The error bars denote SEM. The asterisks indicate significant difference from wild-type α4β2δ receptors (**p < 0.01).

Figure 4.

Surface receptor expression was decreased for α4β2δ receptors containing pure variant δ subunits. A, Examples of Western blot bands for wild-type α4β2δ, mixed receptor population, and variant α4β2δ(E177A) receptors are presented. Compared with wild-type receptors, the surface receptor proteins were slightly decreased for pure variant receptors, but those for mixed receptors were not obviously altered. The total receptor proteins (surface plus intracellular compartments) were not different among wild-type and variant receptors. B, Examples of Western blot bands for wild-type α4β2δ, mixed, and pure variant α4β2δ(R220H) receptors. Compared with wild-type receptors, the surface receptor proteins for pure variant receptors were substantially reduced, but those for the mixed receptors were not different from wild-type receptors. The total receptor proteins were not altered for variant receptors compared with wild-type receptors. C, The surface receptor proteins of wild-type and δ(E177A) variant receptors were quantified by normalizing to β-actin and expressed as relative optical density. The relative optical density of pure variant receptors (n = 7) was reduced compared with wild-type receptors (n = 14). The relative optical density of mixed receptors (n = 7) was not different from that of wild-type and pure variant receptors. D, The relative optical density of pure variant α4β2δ(R220H) receptors (n = 7) was substantially reduced compared with wild-type receptors (n = 14). The relative optical density of mixed receptors (n = 7) was not different from that of wild-type receptors but was significantly different from that of pure variant receptors. A V5 epitope was tagged at the N terminus of wild-type or variant δ subunit. The total receptor proteins were analyzed by Western blot using antibody against the V5 epitope. The surface receptor proteins were captured by biotinylation and analyzed by Western blot. A protein band of ∼50 kDa was detected for the wild-type α4β2δ receptor or the α4β2δ receptor containing either of the variant subunits. wt, Wild-type α4β2δ receptors; mix, mixed population of receptors; var, pure variant receptors. The error bars denote SEM. In both A and B, β-actin was applied as internal loading control. The asterisks indicate significant difference from wild-type α4β2δ receptors (*p < 0.05, ***p < 0.001). ^###p < 0.001, significant difference from mixed wild-type and variant α4β2δ(R220H) receptors.

Figure 5.

Mean open duration of single-channel currents was reduced for α4β2δ receptors containing pure variant δ subunits. A1–C1, Examples of single-channel currents evoked by 1 mm GABA from wild-type α4β2δ, and pure variant α4β2δ(E177A) and α4β2δ(R220H) receptors are presented. The single-channel current open duration was longer for the wild-type α4β2δ receptor than for variant α4β2δ(E177A) or α4β2δ(R220H) receptor. A2–C2, The distributions of open states of the wild-type α4β2δ, and variant α4β2δ(E177A) and α4β2δ(R220H) receptors were plotted. There were 4387 open events used for A2, 2331 open events for B2, and 2931 open events for C2. Each histogram contained data from a single patch. D, The single-channel current mean open duration was decreased for variant α4β2δ(E177A) (n = 7) and α4β2δ(R220H) receptors (n = 8) compared with wild-type α4β2δ receptors (n = 14). E, Comparison of the mean value of τ₁, τ₂, and τ₃ among wild-type α4β2δ and variant α4β2δ(E177A) and α4β2δ(R220H) receptors. F, Comparison of the mean A₁, A₂, and A₃ among wild-type α4β2δ and variant α4β2δ(E177A) and α4β2δ(R220H) receptors. The average open events used for each patch were 2559, 2414, and 2921 for wild-type α4β2δ, variant α4β2δ(E177A), and α4β2δ(R220H) receptors, respectively. wt, Wild-type α4β2δ receptors. The error bars represent SEM. Asterisks indicate significant difference from wild-type α4β2δ receptor (*p < 0.05, **p < 0.01, and ***p < 0.001).