Two Different Mechanisms of Disinhibition Produced by GABA_AReceptor Mutations Linked to Epilepsy in Humans

Abstract

The first mutations of the GABA(A) receptor channel linked to familial epilepsy in humans were reported recently (Baulac et al., 2001; Wallace et al., 2001). Preliminary functional analysis of alpha1beta2gamma2 GABA(A) receptors expressed in Xenopus oocytes suggested that the gamma2 subunit R43Q mutation abolished current enhancement by the benzodiazepine, diazepam, and that the gamma2 subunit K289M mutation decreased current amplitudes. We used single-channel recording and concentration jump techniques applied to outside out patches to evaluate the impact of these mutations on GABA(A) receptor channel function of the highly conserved rat ortholog subunits expressed in human embryonic kidney cells. When coexpressed with alpha1 and beta3 subunits, no differences were observed between wild-type and mutant GABA(A) receptor current activation rates or rates or extent of desensitization during prolonged (400 msec) GABA application (1 mm). Although deactivation after brief (5 msec) or prolonged (400 msec) GABA application was unaltered by the R43Q mutation, deactivation (a correlate of IPSC duration) was accelerated for the K289M mutation. Faster deactivation was likely a consequence of altered gating, because single-channel openings had shorter mean duration. Interestingly, the R43Q mutation did not alter diazepam potentiation. It did, however, substantially decrease current amplitude, which was not caused by decreased single-channel conductance or open time, suggesting reduced surface expression of functional receptors. The two gamma2 subunit mutations likely produce disinhibition and familial epilepsy by distinct mechanisms, suggesting that maintenance of neuronal inhibition depends not only on the peak amplitude of IPSCs, but also on their time course.

Effect of the α subunit subtype on the macroscopic kinetic properties of recombinant GABAA receptors

“…Our results suggest that either of these changes in subunit expression would slow the deactivation rate. In addition, two GABAR mutations associated with inheritance of epilepsy have been found to accelerate the deactivation rate, also correlating rapid deactivation with conditions of hyperexcitability (Bianchi et al, 2002b;Fisher, 2004a).…”

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.…”

“…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

“…1,[4][5][6][7][8]14 The N40S mutation identified in this study affects a highly conserved Asn at residue 40 of the mature g2 subunit, thus, the mutation is adjacent to the first one of the two high-affinity benzodiazepine-binding domains of the g2 subunit (Lys-41-Trp-82 in the mature g2 subunit). Wallace et al 15 had suggested that R43Q mutation in the benzodiazepine-binding site can attenuate benzodiazepine sensitivity of GABA A receptor. Another study on this mutation was shown to accelerate deactivation of the receptor.…”

“…Several subsequent studies have identified the retention of mutant receptors in the ER 14 and shown that GABRG2 mutations have reduced trafficking either to the membrane surface with relatively normal function 18,19 or to the surface with impaired function. 15,20 It is believed that the main electrophysiological deficit of GABA A receptor resulting from the mutation is due to intracellular trafficking abnormality of channel molecules. Thus, the N40S mutation may result in an aberrant trafficking of the GABA A receptor.…”