Altered kinetics and benzodiazepine sensitivity of a GABA _A receptor subunit mutation [γ ₂ (R43Q)] found in human epilepsy

Abstract

The ␥-aminobutyric acid type A (GABAA) receptor mediates fast inhibitory synaptic transmission in the CNS. Dysfunction of the GABA A receptor would be expected to cause neuronal hyperexcitability, a phenomenon linked with epileptogenesis. We have investigated the functional consequences of an arginine-toglutamine mutation at position 43 within the GABAA ␥2-subunit found in a family with childhood absence epilepsy and febrile seizures. Rapid-application experiments performed on receptors expressed in HEK-293 cells demonstrated that the mutation slows GABAA receptor deactivation and increases the rate of desensitization, resulting in an accumulation of desensitized receptors during repeated, short applications. In Xenopus laevis oocytes, two-electrode voltage-clamp analysis of steady-state currents obtained from ␣1␤2␥2 or ␣1␤2␥2(R43Q) receptors did not reveal any differences in GABA sensitivity. However, differences in the benzodiazepine pharmacology of mutant receptors were apparent. Mutant receptors expressed in oocytes displayed reduced sensitivity to diazepam and flunitrazepam but not the imidazopyridine zolpidem. These results provide evidence of impaired GABA A receptor function that could decrease the efficacy of transmission at inhibitory synapses, possibly generating a hyperexcitable neuronal state in thalamocortical networks of epileptic patients possessing the mutant subunit.rapid agonist application ͉ two-electrode voltage clamp ͉ Xenopus oocytes E pilepsy, one of the most common neurological disorders, is characterized by episodic seizures that are caused by paroxysmal, synchronized discharges of hyperexcitable neuronal populations. Many human epileptic syndromes have a genetic component, and the molecular basis of a few inherited epilepsies is now known (1). Most genes implicated in epilepsy to date code for ion channel subunits, both ligand-gated and voltage-gated (2). For example, mutations within the ␣-and ␤-subunits of the voltage-gated sodium channel family have been found in families with generalized epilepsy with febrile seizures plus (GEFSϩ; refs. 3 and 4). Mutations within the voltage-gated potassium channel subunits KCNQ2 and KCNQ3 cause the benign familial neonatal convulsions phenotype (5-9), whereas mutations in the acetylcholine ligand-gated receptor family are responsible for familial nocturnal frontal lobe epilepsy (10)(11)(12)(13)(14). In vitro investigations with the oocyte expression system and two-electrode voltage-clamp assay generally suggest that these mutations would result in neuronal hyperexcitability.The ␥-aminobutyric acid type A (GABA A ) receptor is the predominant ligand-gated Cl Ϫ ion channel conferring fast inhibitory synaptic transmission in the CNS and, therefore, is a prime candidate for involvement in epileptogenesis. The GABA A receptor is a heterooligomeric complex of five subunits. Each subunit has a large, N-terminal extracellular domain, four transmembrane domains, and a small, extracellular C-terminal tail. Multiple subunits have been cloned and divided into...

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

“…It is not likely that the ␤ 2 subunits detected on the cell surface are incorporated into anything other than pentamers, as previous studies have shown that partially assembled receptor intermediates containing ␣␥, ␣␤, or ␤␥ subunits are not expressed on the cell surface (19). In electrophysiological studies (14,15), GABA-evoked currents were measured from HEK 293 cells co-expressing wild-type ␣, ␤, and mutant ␥ 2 R43Q subunits, indicating expression of some ␤ 2 subunit containing receptors on the cell surface. Our data, however, suggest a decrease in surface GABAR protein in cells expressing the mutant ␥ 2 R43Q subunit compared with cells expressing WT subunits.…”

“…A recent study by Bowser et al (14), using rapid drug application and patch-clamp recordings of HEK 293 cells expressing mutant receptors found that ␣ 1 ␤ 2 ␥ 2 R43Q receptors displayed slowed deactivation, an increase in the fast component of desensitization, and an increase in paired-pulse desensitization, indicating that the mutation altered receptor kinetics. In contrast, a paper by Bianchi et al (15), using a similar recording technique and cell expression system, reported no differences in macroscopic GABAR kinetics or diazepam potentiation of GABA current in ␣ 1 ␤ 3 ␥ 2L R43Q receptors but observed reductions in peak GABA-induced currents instead.…”

“…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 receptors were observed (13). However, the ability of the BZDs flunitrazepam (FNZM) and diazepam to potentiate GABA-activated current was reduced in mutant receptors (13,14). Recombinant receptor expression in HEK 293 cells combined with patchclamping and rapid drug application techniques demonstrated that the ␥ 2 R43Q mutation slowed GABAR deactivation and speeded desensitization (14).…”

“…However, the ability of the BZDs flunitrazepam (FNZM) and diazepam to potentiate GABA-activated current was reduced in mutant receptors (13,14). Recombinant receptor expression in HEK 293 cells combined with patchclamping and rapid drug application techniques demonstrated that the ␥ 2 R43Q mutation slowed GABAR deactivation and speeded desensitization (14). In contrast, another study using expression in HEK 293 cells and similar recording techniques reported no differences in receptor kinetics or diazepam potentiation of GABA current between mutant and WT receptors but instead observed reductions in maximal GABA-induced current in cells expressing mutant receptors (15).…”

Mutational analysis of GABRG2 in a Japanese cohort with childhood epilepsies

“…Another study on this mutation was shown to accelerate deactivation of the receptor. 16 These lines of evidence support that the N40S mutation should attenuate the GABA A receptor functions whereby increasing the intracortical excitability of the brain. This notion accords with the recent findings with the GABRG2 mutations in the N-terminal domain.…”