A major challenge in understanding complex idiopathic generalized epilepsies has been the characterization of their underlying molecular genetic basis. Here, we report that genetic variation within the GABRD gene, which encodes the GABA_A receptor δ subunit, affects GABA current amplitude consistent with a model of polygenic susceptibility to epilepsy in humans. We have found a GABRD Glu177Ala variant which is heterozygously associated with generalized epilepsy with febrile seizures plus. We also report an Arg220His allele in GABRD which is present in the general population. Compared with wild-type receptors, α1β2Sδ GABA_A receptors containing δ Glu177Ala or Arg220His have decreased GABA_A receptor current amplitudes. As GABA_A receptors mediate neuronal inhibition, the reduced receptor current associated with both variants is likely to be associated with increased neuronal excitability. Since δ subunit-containing receptors localize to extra- or peri-synaptic membranes and are thought to be involved in tonic inhibition, our results suggest that alteration of this process may contribute to the common generalized epilepsies.

GABA_A receptors are modulated by a variety of compounds, including the neurosteroids and barbiturates. Although the effects of barbiturates on αβγ isoforms, thought to dominate phasic (synaptic) GABAergic inhibition, have been extensively studied, the effects of pentobarbital on kinetic properties of αβδ GABA_A receptors, thought to mediate tonic (extra- or perisynaptic) inhibition, are unknown. Using ultrafast drug delivery and single channel recording techniques, we demonstrate isoform-specific pentobarbital modulation of low-efficacy, minimally desensitizing α1β3 currents and high-efficacy, rapidly desensitizing α1β3γ2L currents. Specifically, with saturating concentrations of GABA, pentobarbital substantially potentiated peak α1β3δ receptor currents but failed to potentiate peak α1β3γ2L receptor currents. Also, pentobarbital had opposite effects on the desensitization of α1β3δ (increased) and α1β3γ2L (decreased) receptor currents evoked by saturating GABA. Pentobarbital increased steady-state α1β3δ receptor single channel open duration primarily by introducing a longer duration open state, whereas for α1β3γ2L receptor channels, pentobarbital increased mean open duration by increasing the proportion and duration of the longest open state. The data support previous suggestions that GABA may be a partial agonist at αβδ isoforms, which may render them particularly sensitive to allosteric modulation. The remarkable increase in gating efficacy of α1β3δ receptors suggests that αβδ isoforms, and by inference tonic forms of inhibition, may be important targets for barbiturates.

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 GABA_Areceptor δ 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 hα1β2δ(E177A) and hα1β2δ(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 hα4β2δ 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 α4β2δ(R220H) receptors resulted in a decrease in surface receptor proteins, and a smaller, but significant, reduction was observed for variant α4β2δ(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 α4β2δ(E177A) and α4β2δ(R220H) receptor single-channel currents were both significantly decreased compared to wild-type receptors. These data suggest that both δ(E177A) and δ(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 δ subunit-containing GABA_Areceptors may be the major contributor to the epilepsy phenotypes.