Abstracts

Upon binding its specific neurotransmitter, the [gamma]-aminobutyric acid[sub]A[/sub] (GABA[sub]A[/sub]) receptor forms transient anion-conducting transmembrane channels that counteract excitatory cation-conducting receptor channels. In epilepsy the GABA[sub]A[/sub] receptor functions inefficiently (Schwartzkroin, P.A. and Wheal, H.V. (1984) [italic]Electrophysiology of Epilepsy[/italic], Academic Press, New York). This is believed to lead to neuronal hyperexcitability and seizure susceptibility. Baulac [italic]et al[/italic]. ((2001) [italic]Nature Genetics[/italic] , 46-48) reported a single mutation (K289M) in the [gamma][sub]2[/sub]-subunit of the GABA[sub]A[/sub] receptor in a form of human epilepsy. Electrophysiological recordings from [italic]Xenopus laevis[/italic] oocytes expressing [alpha][sub]1[/sub][beta][sub]2[/sub][gamma][sub]2K289M[/sub] GABA[sub]A[/sub] receptors revealed smaller amplitude currents relative to the wild-type receptor at equal concentrations of GABA. We introduced the same mutation in the rat GABA[sub]A[/sub] receptor that now exhibits the same dysfunction when expressed in HEK293 (Human Embryonic Kidney) cells. The goal of this research is to determine the functional consequences of this K289M mutation, linked to human epilepsy, of the [gamma][sub]2[/sub]-subunit of the GABA[sub]A[/sub] receptor expressed in HEK293 cells.
Site-directed mutagenesis (Stratagene, La Jolla, CA) was used to introduce the K289M mutation in the rat [gamma][sub]2[/sub]-subunit of the GABA[sub]A[/sub] receptor. The single-channel current-recording technique is used to determine the channel conductance and mean channel-open time ([tau][sub]o[/sub]) of the [gamma]2[sub]K289M[/sub] mutated and the wild-type GABA[sub]A[/sub] receptors. Additionally, two transient kinetic methods (reviewed Hess, G.P. and Grewer, C. (1998) [italic]Methods Enzymol. [/italic], 443-473), the laser-pulse photolysis (LaPP) and cell-flow techniques with 0.05 and 10 ms time resolution respectively, are used to investigate other possible differences in the mechanism between the normal and the mutated GABA[sub]A[/sub] receptor.
The ligand dissociation constant ([italic]K[/italic][sub]1[/sub]) we evaluated from the GABA-dose response curve shows that the ligand (GABA) affinity for the wild-type receptor is 35 [plusmn] 9 [mu]M; for the K289M mutated receptor it is 60 [plusmn] 19 [mu]M. This indicates that decreased ligand affinity does not account for the decrease in the whole-cell current observed in the case of the [alpha][sub]1Ala322Asp[/sub][beta][sub]2[/sub][gamma][sub]2 [/sub]mutated (Cossette, P. [italic]et al[/italic]. (2002) [italic]Nature Genetics [/italic], 184-189) GABA[sub]A[/sub] receptor. In our experiments the channel-opening ([italic]k[sub]op[/sub][/italic]) and -closing ([italic]k[sub]cl[/sub][/italic]) rate constants of the wild-type and the [gamma][sub]2K289M[/sub] mutated GABA[sub]A[/sub] receptors are being determined by the LaPP technique. These experiments indicate that while [italic]k[sub]cl[/sub] [/italic]remains unchanged [italic]k[sub]op[/sub] [/italic]decreases. Consequently, the channel-opening equilibrium constant ([italic]k[sub]op[/sub][/italic]/[italic] k[sub]cl[/sub][/italic]) is decreased in the epileptic receptor.
Our results indicate that a decrease in the channel-opening equilibrium constant, determined by the LaPP technique, decreases the function of the inhibitory GABA[sub]A[/sub] receptor when the K289M mutation is present in the [gamma][sub]2[/sub]-subunit.