A mutation in the GABAA receptor α1 subunit linked to human epilepsy affects channel gating properties
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 GABAA 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.
Section snippets
Transfection of mammalian cells
Full-length cDNAs in the pCMVNeo (Dr. Robert Macdonald, Vanderbilt University) or pcDNA1.1/Amp (Invitrogen, Carlsbad, CA) expression vectors were transfected into the human embryonic kidney cell line HEK-293T (GenHunter, Nashville, TN). For selection of transfected cells, the plasmid pHook™-1 (Invitrogen) containing cDNA encoding the surface antibody sFv was also transfected into the cells (Chesnut et al., 1996). Cells were maintained in Dulbecco’s modified Eagle medium (DMEM) plus 10% fetal
Mutation of alanine294 in the α1 subunit affects GABA sensitivity
As the α1(A294D) mutation is associated with the development of epilepsy, we examined the effects of this mutation as well as changes to other amino acid residues on the sensitivity of the receptors to GABA. All mutant constructs produced GABA-sensitive currents when combined with wild-type β3 and γ2L subunits. The mutations of A294 had variable effects on GABA sensitivity (Fig. 2). Cells were voltage-clamped at −50 mV, and whole-cell currents were recorded in response to 5 s applications of
Discussion
These results demonstrate that alanine294 in the TM3 of the α1 GABAR subunit plays an important role in regulating channel gating kinetics. Mutation of this residue to aspartate substantially reduced the sensitivity of the receptor to GABA, increased the deactivation rate, and decreased channel mean open time. These alterations are consistent with a role for this mutation in the development of human epilepsy (Cossette et al., 2002), as they would be expected to reduce the amplitude and duration
Acknowledgements
This work was supported by the University of South Carolina School of Medicine Research Development Fund and the PhRMA Foundation. Thanks to Kathryn Long and Brandon Drafts for technical assistance.
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