Co-expression of γ2 Subunits Hinders Processing of N-Linked Glycans Attached to the N104 Glycosylation Sites of GABAA Receptor β2 Subunits

Abstract

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.

Deleterious Rare Variants Reveal Risk for Loss of GABAA Receptor Function in Patients with Genetic Epilepsy and in the General Population

“…Conversely, (-)β3R194Q, (-)β3D197N and (+)α1T441M variants propagated rearrangements (RMS > 0.5–3 Å) from the binding site (loops G and F, β8-strand) to the coupling interface (Cys-loop) at the γ+/β-, α+/β-, and α+/γ- interfaces. Despite being opposite to the main interface, the interface convergence form homologous functional domains can impair receptor gating as reported previously [ 34 – 37 ]. In contrast, ESP (-)β3V200I, (-)β3I448V, (+)α1H129Y, (-)α1P29S and (-)γ2L57F variants that had no effects on channel gating produced perturbations that were mainly located on complementary GABA A receptor subunit interfaces (α+/β-, α+/γ-, and γ+/β-) ( S2 Fig ).…”

Epileptic encephalopathy de novo GABRB mutations impair γ‐aminobutyric acid type A receptor function

“…The β3(N110D) subunit mutation caused structural changes similar to those caused by the β3(D120N) subunit mutation, but in the principal (+) side of both α and γ subunits. Interestingly only at the γ+/β− interface, the β3(N110D) subunit mutation predicted changes that were extended across the α−β1 loop of the γ2 subunit, a motif previously established to impair receptor gating and βγ subunit interaction 15, 16, 25 . The β3(N110D) subunit mutation lies in the inner β3-sheet and it seems unlikely that being on the opposite side of the ligand binding-channel gating coupling interface would reduce the gating of the channel, but similar results were found after glycine insertions in the inner β4-β5 sheets at the β3− interface with decreased GABA A receptor activation 26 .…”

“…Interestingly only at the c1/b2 interface, the b3(N110D) subunit mutation predicted changes that were extended across the a-b1 loop of the c2 subunit, a motif previously established to impair receptor gating and bc subunit interaction. 15,16,25 The b3(N110D) subunit mutation lies in the inner b3 sheet, and it seems unlikely that being on the opposite side of the ligand binding channel gating coupling interface would reduce the gating of the channel, but similar results were found after glycine insertions in the inner b4-b5 sheets at the b32 interface with decreased GABA A receptor activation. 26 In line with this, IS-associated mutations mainly affected the kinetic properties of the channel and altered similar domains at the homologous c1/b2 and a1/b2 interfaces required for receptor expression and function.…”

De novo GABRG2mutations associated with epileptic encephalopathies

“…De novo g2 subunit mutations decreased GABA potency by disrupting structural domains important for GABA A receptor function Changes in GABA A receptor potency appeared to be correlated with well-defined structural domains, which have been described as essential to receptor function (Klausberger et al, 2000;Bianchi et al, 2001;Bianchi and Macdonald, 2002;Sarto et al, 2002;Venkatachalan and Czajkowski, 2012;Althoff et al, 2014;Lo et al, 2014). To determine whether predicted changes in channel structure caused by the g2 subunit mutations are related to changes in the GABA A receptor potency, we first generated wild-type and mutant pentameric abg GABA A receptor simulations (Fig.…”