A GABAA Receptor Mutation Linked to Human Epilepsy (γ2R43Q) Impairs Cell Surface Expression of αβγ Receptors

Abstract

A mutation in the ␥ 2 subunit of the ␥-aminobutyric acid (GABA) type A receptor (GABAR), which changes an arginine to a glutamine at position 43 (R43Q), is linked to familial idiopathic epilepsies. We used radioligand binding, immunoblotting, and immunofluorescence techniques to examine the properties of wild-type ␣ 1 ␤ 2 ␥ 2 and mutant ␣ 1 ␤ 2 ␥ 2 R43Q GABARs expressed in HEK 293 cells. The ␥ 2 R43Q mutation had no affect on the binding affinity of the benzodiazepine flunitrazepam. However, in cells expressing ␣ 1 ␤ 2 ␥ 2 R43Q GABARs, the number of binding sites for [ 3 H]flunitrazepam relative to wild-type receptors was decreased 75%. Using surface protein biotinylation, affinity purification, and immunoblotting, we demonstrated that expression of cell surface ␣ 1 ␤ 2 ␥ 2 R43Q GABARs was decreased. Surface immunostaining of HEK 293 cells expressing ␣ 1 ␤ 2 ␥ 2 R43Q GABARs confirmed that surface expression of the ␥ 2 R43Q subunit was reduced. These data demonstrate that the ␥ 2 R43Q mutation impairs expression of cell surface GABARs. A deficit in surface GABAR expression would reduce synaptic inhibition and result in neuronal hyperexcitability, which could explain why families possessing the ␥ 2 R43Q subunit have epilepsy.Idiopathic partial and generalized epilepsies are the most common forms of heritable seizure disorders, accounting for 40% of all epilepsies (1). Idiopathic epilepsies are those seizure disorders that are not preceded by or concomitant with trauma or other disorders but are due mainly to genetic factors. Because of the ubiquitous role of the neurotransmitter GABA 1 in mediating cortical inhibition, deficits in GABAergic transmission have long been surmised to play a role in the pathogenesis of epilepsy. Recently, several mutations in the GABA A receptor (GABAR) have been identified through the use of genetic linkage analysis in families with idiopathic generalized epilepsies.GABARs are heteropentameric ligand gated chloride channels that mediate fast synaptic inhibition in the brain. The receptors are assembled from a number of different subunits and subunit isoforms, including ␣ 1-6 , ␤ 1-3 , ␥ 1-3 , ␦, ⑀, , and (2, 3). The majority of GABARs in the brain are believed to consist of 2 ␣, 2 ␤, and 1 ␥ subunits (4, 5). The inclusion of a ␥ subunit confers benzodiazepine (BZD) sensitivity to GABARs (6) and also influences receptor expression, synaptic targeting, and trafficking (7,8). To date, four mutations in the ␥ 2 subunit of the GABAR (missense mutations R43Q and K289M, the nonsense mutation Q351X, and the anomalous splice-donor site mutation IVS6 ϩ 2T 3 G) and one mutation in the ␣ 1 subunit of the receptor (missense mutation A322D) have been associated with idiopathic epileptic syndromes (9 -13).Several groups have explored the effects of the ␥ 2 R43Q mutation on GABAR function. Using recombinant GABAR expression in Xenopus laevis oocytes and two-electrode voltage clamp recording techniques, no differences in GABA EC 50 values between wild-type (WT)␣ 1 ␤ 2 ␥ 2 and mutant ␣ 1 ␤ 2 ␥ 2 R43Q r...

Reduced cortical inhibition in a mouse model of familial childhood absence epilepsy

“…Furthermore, loss of ␥2(R43Q) could alter trafficking (39) such that density of receptors at synapses is reduced, causing the observed reduction in mIPSCs. In partial contrast to our in vivo finding, heterologous expression studies performed in HEK cells demonstrate both a reduction in ␥2 subunit expression and in fully formed heteromeric GABA A receptors at the cell surface, suggesting a dominant-negative effect of the R43Q mutation (16,(18)(19)(20). These studies highlight a need for caution when using the results from cell-based experiments to explain the genesis of complex diseases.…”

“…The reduced cortical mIPSCs described above could be explained by intracellular retention of receptors comprising the mutant ␥2(R43Q) subunit. This possibility has been raised in studies that used cell culture systems to heterologously express mutant subunits and assess function and membrane trafficking (16,19,20). To determine whether incorporation of the ␥2(R43Q) subunit causes retention of GABA A receptor complexes by a dominant-negative effect, we cultured primary neurons from C57BL/6 embryonic cortices (E15-E16) and biotin-labeled cell surface receptors.…”

“…Epilepsy itself is a complex phenomenon involving the interaction of multiple cell types in networks within and between different brain regions that are likely to be influenced by GABA A receptor dysfunction caused by the R43Q mutation. Furthermore, in vitro analyses of the consequences of this mutation have shown inconsistent findings with a range of deficits in receptor pharmacology, trafficking, kinetics, or assembly (16)(17)(18)(19)(20)(21)(22)(23) potentially implicated in disease pathogenesis.…”

δ Subunit Susceptibility Variants E177A and R220H Associated with Complex Epilepsy Alter Channel Gating and Surface Expression of α4β2δ GABAAReceptors

“…Another ␥2 subunit mutation, ␥2(R43Q), reduced surface expression of GABA A receptors Sancar and Czajkowski, 2004;Hales et al, 2005), and like the ␥2(R43Q) mutation, both ␦(E177A) and ␦(R220H) variants are located in the subunit N terminus. The ␥2 subunit N terminus interacts with ␣ and ␤ subunits during receptor assembly (Klausberger et al, 2000;Jin et al, 2004), and the ␥2(R43Q) subunit mutation resulted in abnormal receptor assembly and trafficking Hales et al, 2005).…”

The GABA A receptor α1 subunit epilepsy mutation A322D inhibits transmembrane helix formation and causes proteasomal degradation

“…Three epilepsy genes are GABA A receptor subunits: ␣1, ␥2, and ␦ subunits. The ␥2(K289M), ␥2(R139G), ␦(E177A), and ␦(R220H) epilepsy mutations/polymorphisms alter receptor function (28)(29)(30), whereas the ␥2(R43Q) and ␥2(Q351X) epilepsy mutations reduce cell surface expression without altering total cellular subunit expression, likely through altering oligomer assembly and cell surface trafficking (31)(32)(33). Recently, Maljevic et al (34) identified a 2-bp deletion in the GABRA1 gene, ␣1(S326fs328X), in a patient with childhood absence epilepsy.…”