Background: A GABRB3 mutation has been associated with childhood absence epilepsy and ␤3 subunit hyperglycosylation. Results: The mutation altered subunit expression and reduced GABA A receptor function independent of N-glycosylation. Conclusion: The mutation introduced a charged residue predicted to alter subunit interactions. Significance: The distal N terminus of GABA A receptor subunits may play an unexpected role in receptor assembly and channel gating.

Despite its genetic heterogeneity, hereditary spastic paraplegia (HSP) is characterized by similar clinical phenotypes, suggesting that a common biochemical pathway underlies its pathogenesis. In support of this hypothesis, we used a combination of immunoprecipitation, confocal microscopy, and flow cytometry to demonstrate that two HSP-associated proteins, atlastin-1 and NIPA1, are direct binding partners, and interestingly, that the endogenous expression and trafficking of these proteins is highly dependant upon their coexpression. In addition, we demonstrated that the cellular distribution of atlastin-1:NIPA1 complexes was dramatically altered by HSP-causing mutations, as missense mutations in atlastin-1 (R239C and R495W) and NIPA1 (T45R and G106R) caused protein sequestration in the Golgi complex (GC) and endoplasmic reticulum (ER), respectively. Moreover, we demonstrated that HSP-causing mutations in both atlastin-1 and NIPA1 reduced axonal and dendritic sprouting in cultured rat cortical neurons. Together, these findings support the hypothesis that NIPA1 and atlastin-1 are members of a common biochemical pathway that supports axonal maintenance, which may explain in part the characteristic degeneration of long spinal pathways observed in patients with HSP.

Genetic epilepsies (GEs) account for approximately 50% of all seizure disorders, and familial forms include mutations in single GABAA receptor subunit genes (GABRs). In 144 sporadic GE cases (GECs), exome sequencing of 237 ion channel genes identified 520 GABR variants. Among these variants, 33 rare variants in 11 GABR genes were present in 24 GECs. To assess functional risk of variants in GECs, we selected 8 variants found in GABRA, 3 in GABRB, and 3 in GABRG and compared them to 18 variants found in the general population for GABRA1 (n = 9), GABRB3 (n = 7), and GABRG2 (n = 2). To identify deleterious variants and gain insight into structure-function relationships, we studied the gating properties, surface expression and structural perturbations of the 32 variants. Significant reduction of GABAA receptor function was strongly associated with variants scored as deleterious and mapped within the N-terminal and transmembrane domains. In addition, 12 out of 17 variants mapped along the β+/α- GABA binding interface, were associated with reduction in channel gating and were predicted to cause structural rearrangements of the receptor by in silico simulations. Missense or nonsense mutations of GABRA1, GABRB3 and GABRG2 primarily impair subunit biogenesis. In contrast, GABR variants affected receptor function by impairing gating, suggesting that different mechanisms are operating in GABR epilepsy susceptibility variants and disease-causing mutations. The functional impact of single GABR variants found in individuals with sporadic GEs warrants the use of molecular diagnosis and will ultimately improve the treatment of genetic epilepsies by using a personalized approach.

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.

The subunit stoichiometry and arrangement of synaptic αβγ GABAA receptors are generally accepted as 2α:2β:1γ with a β-α-γ-β-α counterclockwise configuration, respectively. Whether extrasynaptic αβδ receptors adopt the analogous β-α-δ-β-α subunit configuration remains controversial. Using flow cytometry, we evaluated expression levels of human recombinant γ2 and δ subunits when co-transfected with α1 and/or β2 subunits in HEK293T cells. Nearly identical patterns of γ2 and δ subunit expression were observed as follows: both required co-transfection with α1 and β2 subunits for maximal expression; both were incorporated into receptors primarily at the expense of β2 subunits; and both yielded similar FRET profiles when probed for subunit adjacency, suggesting similar underlying subunit arrangements. However, because of a slower rate of δ subunit degradation, 10-fold less δ subunit cDNA was required to recapitulate γ2 subunit expression patterns and to eliminate the functional signature of α1β2 receptors. Interestingly, titrating γ2 or δ subunit cDNA levels progressively altered GABA-evoked currents, revealing more than one kinetic profile for both αβγ and αβδ receptors. This raised the possibility of alternative receptor isoforms, a hypothesis confirmed using concatameric constructs for αβγ receptors. Taken together, our results suggest a limited cohort of alternative subunit arrangements in addition to canonical β-α-γ/δ-β-α receptors, including β-α-γ/δ-α-α receptors at lower levels of γ2/δ expression and β-α-γ/δ-α-γ/δ receptors at higher levels of expression. These findings provide important insight into the role of GABAA receptor subunit under- or overexpression in disease states such as genetic epilepsies.

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GABAA receptors, the major mediators of fast inhibitory neuronal transmission, are heteropentameric glycoproteins assembled from a panel of subunits, usually including α and β subunits with or without a γ2 subunit. The α1β2γ2 receptor is the most abundant GABAA receptor in brain. Co-expression of γ2 with α1 and β2 subunits causes conformational changes, increases GABAA receptor channel conductance, and prolongs channel open times. We reported previously that glycosylation of the three β2 subunit glycosylation sites, N32, N104 and N173, was important for α1β2 receptor channel gating. Here, we examined the hypothesis that steric effects or conformational changes caused by γ2 subunit co-expression alter the glycosylation of partnering β2 subunits. We found that co-expression of γ2 subunits hindered processing of β2 subunit N104 N-glycans in HEK293T cells. This γ2 subunit-dependent effect was strong enough that a decrease of γ2 subunit expression in heterozygous GABRG2 knockout (γ2+/−) mice led to appreciable changes in the endoglycosidase H (endo H) digestion pattern of neuronal β2 subunits. Interestingly, as measured by flow cytometry, γ2 subunit surface levels were decreased by mutating each of the β2 subunit glycosylation sites. The β2 subunit mutation N104Q also decreased GABA potency to evoke macroscopic currents and reduced conductance, mean open time and open probability of single channel currents. Collectively, our data suggested that γ2 subunits interacted with β2 subunit N-glycans and/or subdomains containing the glycosylation sites, and that γ2 subunit co-expression-dependent alterations in the processing of the β2 subunit N104 N-glycans were involved in altering the function of surface GABAA receptors.

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