A mutation in the GABA_A receptor α1 subunit linked to human epilepsy affects channel gating properties

Abstract

A genetic component is associated with the development of many forms of epilepsy. Recently, mutations in the GABA_A receptor have been linked to several inherited epilepsies. One of these mutations is a non-conservative change of alanine to aspartate in the third transmembrane domain of the α1 subunit. To determine the functional consequences of this alteration, mutated α subunits were transiently transfected along with wild-type β3 and γ2L subunits into HEK-293T cells. The mutated α1_(A294D) subunit reduced GABA sensitivity of the receptor, increased the deactivation rate and slowed desensitization. The mutation caused a reduction in channel open time but no change in single channel conductance. Studies with additional mutants, altering the charge and/or size of the side-chain, indicated that both size and hydrophobicity of the residue at this location influence channel gating. The effects on GABA sensitivity, deactivation rate and channel open time are consistent with a reduced efficacy of channel gating, and would be expected to decrease GABAergic neurotransmission. The α1 subtype is the most widely expressed of the α subunits, with expression increasing throughout development. Therefore, production of the mutated subunit could cause global hyperexcitability throughout the brain, leading to generalized seizures with juvenile onset.

Introduction

Epilepsy affects nearly 1% of the US population and a genetic component often underlies its development (Kaneko et al., 2002). In most cases, the cause of initiation and maintenance of seizure activity are unknown. Many drugs used clinically as anti-convulsants act by increasing the activity of the GABA_A receptor (GABAR) (Korpi et al., 2002). The GABAR is a ligand-gated ion channel permeable to chloride and is responsible for most fast inhibitory neurotransmission in the mammalian central nervous system. The structure of the GABAR is complex, with 16 different subunit subtypes from seven different families (α, β, γ, δ, ε, π and θ). These different subtypes show distinct expression patterns throughout the brain and their levels change with development (Laurie et al., 1992a, Laurie et al., 1992b, Wisden et al., 1992). The subunit subtype composition of the receptor plays a significant role in determining the functional and pharmacological properties of the receptor (Korpi et al., 2002).

Recently, several mutations in two GABAR subunits have been linked to inherited forms of epilepsy (Baulac et al., 2001, Wallace et al., 2001, Cossette et al., 2002, Kananura et al., 2002, Harkin et al., 2002). Some of these mutations lead to the formation of non-functional subunits (Kananura et al., 2002, Harkin et al., 2002) while others alter the behavior of receptors that incorporate the mutated subunits (Baulac et al., 2001, Wallace et al., 2001, Cossette et al., 2002). The functional effects of these mutations can provide insight into the structures that regulate GABAR channel properties and may suggest appropriate treatments for these disorders. Two mutations in different regions of the γ2 subunit were found to alter the pharmacological and/or kinetic properties of the receptors (Baulac et al., 2001, Wallace et al., 2001, Bianchi et al., 2002, Bowser et al., 2002). Recently, a mutation in the α1 subunit (Fig. 1) was identified in a family with juvenile idiopathic generalized epilepsy (Cossette et al., 2002). The α1 subtype is the most highly and widely expressed of the α family subunits in the adult brain and may be incorporated into over half of the GABARs in adult neurons (McKernan and Whiting, 1996). The mutation causes a non-conservative missense coding change from an alanine to an aspartate residue within the third transmembrane domain (TM3). This alanine residue is conserved among all α subunit subtypes, and therefore may play an important role in regulating channel function.

Effects of mutations at alanine294 of the α1 subunit on the pharmacological and kinetic properties of recombinant GABARs were examined at the macroscopic and single channel levels. The mutation altered the receptor’s kinetic properties in a manner consistent with reduced gating efficacy. We also examined the structural dependence for functional effects at this site. Because of its widespread expression pattern, decreasing function of the α1 subunit would be expected to lead to generalized epilepsy, as observed in the patient population carrying the α1_(A294D) mutation. Understanding the mechanisms underlying these genetic disorders may lead to more effectively targeted treatments for inherited forms of epilepsy.