A γ2(R43Q) Mutation, Linked to Epilepsy in Humans, Alters GABAA Receptor Assembly and Modifies Subunit Composition on the Cell Surface

Abstract

Genetic defects leading to epilepsy have been identified in ␥2 GABA A receptor subunit. A ␥2(R43Q) substitution is linked to childhood absence epilepsy and febrile seizure, and a ␥2(K289M) mutation is associated with generalized epilepsy with febrile seizures plus. To understand the effect of these mutations, surface targeting of GABA A receptors was analyzed by subunit-specific immunofluorescent labeling of living cells. We first transfected hippocampal neurons in culture with recombinant ␥2 constructs and showed that the ␥2(R43Q) mutation prevented surface expression of the subunit, unlike ␥2(K289M) substitution. Several ␥2-subunit constructs, bearing point mutations within the Arg-43 domain, were expressed in COS-7 cells with ␣3-and ␤3-subunits. R43Q and R43A substitutions dramatically reduced surface expression of the ␥2-subunit, whereas R43K, P44A, and D39A substitutions had a lesser, but still significant, impact and K289M substitution had no effect. Whereas the mutant ␥2(R43Q) was retained within intracellular compartments, ␣␤ complexes were still targeted at the cell membrane. Coimmunoprecipitation experiments showed that ␥2(R43Q) was able to associate with ␣3-or ␤3-subunits, although the stoichiometry of the complex with ␣3 was altered. Our data show that ␥2(R43Q) is not a dominant negative and that the mutation leads to a modification of GABA A receptor subunit composition on the cell surface that impairs the synaptic targeting in neurons. This study reveals an involvement of the ␥2-Arg-43 domain in the control of receptor assembly that may be relevant to the effect of the heterozygous ␥2(R43Q) mutation leading to childhood absence epilepsy and febrile seizure.

An Epilepsy-Related Region in the GABAA Receptor Mediates Long-Distance Effects on GABA and Benzodiazepine Binding Sites

“…Immunofluorescence studies have shown that mutation of ␥ 2 Arg43 to glutamine, lysine, or alanine impairs surface expression of the ␥ 2 subunit, probably by trapping it in the endoplasmic reticulum (Kang and Macdonald, 2004;Sancar and Czajkowski, 2004;Eugène et al, 2007;Frugier et al, 2007;Tan et al, 2007). It remains unclear whether the reduced ␥ 2 subunit expression reflects the loss of all the constituent subunits in those receptors, such as would occur if the trapped ␥ 2 subunits were part of fully assembled channels (Kang and Macdonald, 2004;Hales et al, 2005), or whether the removal of ␥ 2 leaves behind ␣ 1 ␤ 2 receptors (Sancar and Czajkowski, 2004;Frugier et al, 2007;Tan et al, 2007). However, methods such as immunofluorescence labeling or radioligand binding cannot distinguish between a reduction in functional versus nonfunctional receptors.…”

“…These mice have reduced cell-surface expression of the ␥ 2 subunit and reduced miniature inhibitory postsynaptic currents. In heterologous expression systems, the mutation alters receptor kinetics (Bowser et al, 2002; but see Bianchi et al, 2002) and impairs receptor assembly or trafficking (Kang and Macdonald, 2004;Sancar and Czajkowski, 2004;Hales et al, 2005;Eugène et al, 2007;Frugier et al, 2007). Either kinetic or trafficking effects could result in hyperexcitability and seizures: the former by accumulation of desensitized receptors during high frequency transmission and the latter by decreasing the number of functional receptors.…”

GABAAReceptor γ2 Subunit Mutations Linked to Human Epileptic Syndromes Differentially Affect Phasic and Tonic Inhibition

“…In contrast, ␥2 bearing the R43Q mutation was not expressed at the membrane of 21 of 30 transfected neurons. In these cells, recombinant ␥2 was retained in the cytoplasm as in heterologous systems (Kang and Macdonald, 2004;Hales et al, 2005;Frugier et al, 2006). In the other 9 neurons, recombinant ␥2 formed surface clusters of lower density (0.64 Ϯ 0.08 m Ϫ2 , p Ͻ 0.03) and smaller size (0.06 Ϯ 0.01 m 2 , p Ͻ 0.001) than wildtype ␥2.…”

GABRB3 Mutation, G32R, Associated with Childhood Absence Epilepsy Alters α1β3γ2L γ-Aminobutyric Acid Type A (GABAA) Receptor Expression and Channel Gating

“…Interestingly, the side chain of the ␤3 subunit residue Lys-32 angled toward the cell membrane and formed a salt bridge with the ␥2 subunit residue Glu-217, which participates in a salt bridge network disrupted by the epilepsy-associated ␥2(R82Q) mutation (Fig. 9C) (36). The side chain of the ␤3 subunit residue Glu-32, conversely, extended toward the ␥2 subunit but did not come within 4 Å of any ␥2 subunit atoms (Fig.…”

“…It is somewhat less clear how salt bridge formation between Arg-32 and ␥2(Asp-123) could discourage incorporation of ␥2 subunits; however, it is important to note that salt bridges can be destabilizing (43) and that ␥2 subunit (122-129) integrity was essential for ␥2-␤3 subunit interaction (35). It is also worth mentioning that the epilepsy- associated mutation ␥2(R82Q), which has been shown to disrupt receptor assembly (36,44), is located in the ␥2 subunit loop closest to the N-terminal domain of the ␤3 subunit ␣-helix. Indeed, point mutations throughout this loop impaired ␥2 subunit incorporation.…”