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The epilepsy associated GABAA receptor γ2R43Q mutation increases sensitivity to Zn2+ inhibition

B. Bennetts,1 P.J. Walsh,1 K.S. Tan,1 B.A. Cromer,2 A.L. Clarke,2 S. Petrou2 and M.W. Parker,1,3 1St. Vincent's Institute of Medical Research, 9 Princes St., Fitzroy, VIC 3065, Australia, 2Howard Florey Institute, Florey Neurosciences Institutes, University of Melbourne, VIC 3010, Australia and 3Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, 30 Flemington Road, Parkville, VIC 3010, Australia.

GABAA receptors mediate rapid inhibitory signalling in the central nervous system, and mutations in various subunits of these pentameric receptors are associated with epilepsy. Receptors composed of α and β subunits are highly sensitive to inhibition by extracellular Zn2+ ions; however incorporation of a γ subunit disrupts two of three known Zn2+ binding sites and greatly reduces Zn2+ sensitivity. Here we demonstrate that the epilepsy associated γ2(R43Q) mutation greatly increases the susceptibility of heterologously expressed receptors to Zn2+ inhibition while preserving functional characteristics underpinned by presence of the γ2 subunit. α1β2γ2 receptors are believed to contain a single N-terminal Zn2+ binding site. Mutation of residues contributed to this site by the α subunit ameliorated the effect of γ2(R43Q) on Zn2+ sensitivity, indicating that γ2(R43Q) allosterically affects Zn2+ binding or affects signal transduction, rather than directly interacting with Zn2+. This assertion was bolstered by the increased Zn2+ sensitivity of mutations predicted, by molecular modelling, to interact with γ2(R43Q). We also examined other epilepsy-associated γ2 mutations, γ2(K289M) and γ2(R139G), and found that they did not substantially increase sensitivity to Zn2+ inhibition. Increased Zn2+ sensitivity may be physiologically important in hippocampal neurones, where synaptic Zn2+ reaches high enough levels to modulate GABAergic signalling, and may represent a novel mechanism underlying the increased occurrence of febrile seizures reported in patients harbouring the γ2(R43Q) mutation.