An Asymmetric Contribution to γ-Aminobutyric Type A Receptor Function of a Conserved Lysine within TM2–3 of α1, β2, and γ2 Subunits

Abstract

Mutations that impair the expression and/or function of ␥-aminobutyric acid type A (GABA A ) receptors can lead to epilepsy. The familial epilepsy ␥2(K289M) mutation affects a basic residue conserved in the TM2-3 linker of most GABA A subunits. We investigated the effect on expression and function of the Lys 3 Met mutation in mouse ␣1(K278M), ␤2(K274M), and ␥2(K289M) subunits. Compared with cells expressing wild-type and ␣1␤2␥2(K289M) receptors, cells expressing ␣1(K278M)␤2␥2 and ␣1␤2(K274M)␥2 receptors exhibited reduced agonist-evoked current density and reduced GABA potency, with no change in single channel conductance. The low current density of ␣1␤2(K274M)␥2 receptors coincided with reduced surface expression. By contrast the surface expression of ␣1(K278M)␤2␥2 receptors was similar to wild-type and ␣1␤2␥2(K289M) receptors suggesting that the ␣1(K278M) impairs function. In keeping with this interpretation GABA-activated channels mediated by ␣1(K278M)␤2␥2 receptors had brief open times. To a lesser extent ␥2(K289M) also reduced mean open time, whereas ␤2(K274M) had no effect. We used propofol as an alternative GABA A receptor agonist to test whether the functional deficits of mutant subunits were specific to GABA activation. Propofol was less potent as an activator of ␣1(K278M)␤2␥2 receptors. By contrast, neither ␤2(K274M) nor ␥2(K289M) affected the potency of propofol. The ␤2(K274M) construct was unique in that it reduced the efficacy of propofol activation relative to GABA. These data suggest that the ␣1 subunit Lys-278 residue plays a pivotal role in channel gating that is not dependent on occupancy of the GABA binding site. Moreover, the conserved TM2-3 loop lysine has an asymmetric function in different GABA A subunits. ␥-Aminobutyric acid type A (GABA A )3 receptors belong to the homologous Cys-loop superfamily of ion channels that includes the nicotinic acetylcholine, 5-hydroxytryptamine type-3 and glycine receptors, and the Zn 2ϩ -activated ion channel (1, 2). GABA A receptors are pentameric, composed from distinct subunit classes, including ␣ (1-6), ␤ (1-3), ␥ (1-3), ␦, ⑀, , and . GABA A receptors mediate rapid synaptic inhibition. Impairment of GABAergic inhibition is associated with anxiety (3) and is the basis of several models of epilepsy (4). Several recently discovered GABA A mutations that reduce inhibition accompany hereditary forms of epilepsy (5-14). To date, the most thoroughly investigated of these are ␥2(R43Q) and ␥2(K289M), both associated with febrile seizures. Two mechanisms could account for reduced inhibition caused by these mutations: impaired receptor expression and/or function. The ␥2(R43Q) mutation alters receptor kinetics (13,14), and this may contribute to inhibitory deficits. The mutation also reduces receptor biogenesis (15-18). Likewise ␥2(K289M) also impairs receptor function and expression (5,14,18).The N-terminal ␥2 subunit Arg-43 residue is conserved across GABA A receptor subunits and systematic Arg 3 Gln mutation in ␣1, ␤2, and ␥2 uncovered a general role for the argi...

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

“…In heterologous expression systems, the K289M mutation has been shown to reduce the amplitude (Baulac et al, 2001;Ramakrishnan and Hess, 2004) and accelerate the kinetics of GABA currents (Bianchi et al, 2002;Hales et al, 2006) and to reduce membrane expression of the ␥2 subunit (Kang et al, 2006). We found no change in surface expression, synaptic or extrasynaptic clustering or coaggregation with ␣ subunits in hippocampal neurons.…”

“…This is consistent with a recent modeling study showing that, although the K289 residue forms part of the channel lining (O'Mara et al, 2005), the K to M mutation does not change channel conductance and so is unlikely to affect current density. However mIPSCs decayed more quickly, consistent with reduced mean open time and accelerated deactivation observed in heterologous cells (Bianchi et al, 2002;Hales et al, 2006). Because our experiments were conducted on neurons expressing endogenous, wild-type GABRG2, we conclude that acceleration of synaptic GABA currents is a dominant effect of the K289M mutation.…”

“…Functional studies in heterologous systems have provided ambiguous results. The K289M mutation affects a conserved residue in the second extracellular domain of ␥2 and was first shown to reduce the amplitude of GABA currents in oocytes (Baulac et al, 2001), but studies in HEK cells demonstrate an accelerated deactivation and unchanged amplitude (Bianchi et al, 2002;Hales et al, 2006). The R43Q mutation, in the N-terminal domain, is suggested to abolish benzodiazepine sensitivity (Wallace et al, 2001), accelerate deactivation or decrease GABA currents by reducing membrane expression (Bianchi et al, 2002;Hales et al, 2006).…”

Inverse Effects on Gating and Modulation Caused by a Mutation in the M2-M3 Linker of the GABAA Receptor γ Subunit

“…Specifically, electrostatic interactions between residues in the M2-M3 linker and these N-terminal loops are proposed to couple agonist binding with channel gating at the pore. In support of this idea, GABA A Rs containing ␥2(K289M), a naturally occurring M2-M3 linker mutation found in patients with epilepsy (GEFS ϩ ; Baulac et al, 2001), show decreased channel open times in saturating concentrations of GABA Hales et al, 2006).…”

Tonically Active GABAA Receptors in Hippocampal Pyramidal Neurons Exhibit Constitutive GABA-Independent Gating

“…Single-channel currents recorded from outside-out patches were low-pass-filtered at 1 kHz (Bessel characteristics) and digitized at 10 kHz for acquisition onto the hard drive of a Pentium personal computer as described previously (Hales et al, 2006). GABA (1 M) and/or gabazine (20 M) was applied directly to patches by local pressure ejection, as was picrotoxin (100 M).…”