The GABRA6 mutation, R46W, associated with childhood absence epilepsy, alters α6β2γ2 and α6β2δ GABA_A receptor channel gating and expression

Abstract

Non-technical summary Childhood absence epilepsy (CAE) is a genetic form of epilepsy that typically develops at 4-8 years of age with brief losses of consciousness and frequent staring spells. Genetic defects or mutations associated with this disorder have been found in specialized membrane proteins called GABA A receptor channels. GABA A receptors are ligand-gated chloride channels, and the majority are thought to be composed of α, β and γ or α, β and δ subunit proteins that mediate both rapid, phasic inhibitory synaptic transmission and steady-state, tonic perisynaptic inhibition in the nervous system. Here we showed that a novel GABA A receptor α6 subunit mutation linked with CAE, R46W, impaired gating and assembly of both αβγ and αβδ GABA A receptors. These findings suggested that the CAE-associated α6(R46W) subunit mutation could cause neuronal disinhibition and thus increase susceptibility to generalized seizures through a reduction of αβγ and αβδ receptor function and expression.Abstract A GABA A receptor α6 subunit mutation, R46W, was identified as a susceptibility gene that may contribute to the pathogenesis of childhood absence epilepsy (CAE), but the molecular basis for alteration of GABA A receptor function is unclear. The R46W mutation is located in a region homologous to a GABA A receptor γ2 subunit missense mutation, R82Q, that is associated with CAE and febrile seizures in humans. To determine how this mutation reduces GABAergic inhibition, we expressed wild-type (α6β2γ2L and α6β2δ) and mutant (α6(R46W)β2γ2L and α6(R46W)β2δ) receptors in HEK 293T cells and characterize their whole-cell and single-channel currents, and surface and total levels. We demonstrated that gating and assembly of both α6(R46W)β2γ2L and α6(R46W)β2δ receptors were impaired. Compared to wild-type currents, α6(R46W)β2γ2L and α6(R46W)β2δ receptors had a reduced current density, α6(R46W)β2γ2L currents desensitized to a greater extent and deactivated at a slower rate, α6(R46W)β2δ receptors did not desensitize but deactivated faster and both α6(R46W)β2γ2L and α6(R46W)β2δ single-channel current mean open times and burst durations were reduced. Surface levels of coexpressed α6(R46W), β2 and δ, but not γ2L, subunits were decreased. 'Heterozygous' coexpression of α6(R46W) and α6 subunits with β2 and γ2L subunits produced intermediate macroscopic current amplitudes by increasing incorporation of wild-type and decreasing incorporation of mutant subunits into receptors trafficked to the surface. Finally, these findings suggest that similar to the γ2(R82Q) mutation, the CAE-associated α6(R46W) mutation could cause neuronal disinhibition and thus increase susceptibility to generalized seizures through a reduction of αβγ and αβδ receptor function and expression. Abbreviations AChR, acetylcholine receptor; CAE, childhood absence epilepsy; HEK 293T, human embryonic kidney cells; nAChR, nicotinic acetylcholine receptor; wt, wild-type.

Three epilepsy-associated GABRG2 missense mutations at the γ+/β− interface disrupt GABAA receptor assembly and trafficking by similar mechanisms but to different extents

“…Whole cell voltage-clamp recordings were performed at room temperature on lifted HEK293T cells 36-48 hrs after transfection with GABA A receptor subunits as described previously (Hernandez et al, 2011). Cells were bathed in an external solution containing 142 mM NaCl, 1 mM CaCl 2 , 8 mM KCl, 6 mM MgCl 2 , 10 mM glucose, and 10 mM HEPES (pH 7.4, ~325 mOsM).…”

The GABRG2 nonsense mutation, Q40X, associated with Dravet syndrome activated NMD and generated a truncated subunit that was partially rescued by aminoglycoside-induced stop codon read-through

“…Whole cell voltage-clamp recordings were performed at room temperature on lifted HEK293T cells 24-72 hrs after transfection with GABA A receptor subunits as described previously (Hernandez et al, 2011). Successfully transfected cells were identified by the presence of GFP fluorescence (see Cell culture and transfection, above).…”

Genetic and epigenetic mechanisms of epilepsy: a review

“…Mutations in GABRA1 and GABRG2 have been recommended by the ILAE for genetic testing in epilepsy. Recently, mutation R46W in the GABAR GABRA6 was detected in patients with CAE, and it was located in a region homologous to a GABRG2 missense mutation, R82Q, that was associated with both CAE and febrile seizures in human beings 122. Mutations (P11S, S15F, and G32R) in the GABAR β 3 subunit gene ( GABRB3 ) are found in multiple families with CAE 123…”