Mechanism of potentiation of a dysfunctional epilepsy-linked mutated GABA(A) receptor by a neurosteroid (3alpha, 21-dihydroxy-5alpha-pregnan-20-one): transient kinetic investigations.
The malfunction of a mutated GABA(A) receptor (alpha1beta2gamma2L(K289M)) in an inheritable form of epilepsy (GEFS+, generalized epilepsy with febrile seizures plus) in humans [Baulac, S., Huberfeld, G., Gourfinkel-An, I., Mitropoulou, G., Beranger, A., Prud'homme, J. F., Baulac, M., Brice, A., Bruzzone, R., and LeGuern, E. (2001) Nat. Genet. 28, 46-48] has been accounted for by a 5-fold decrease in the channel-opening equilibrium of the mutated receptor compared to the wild type [Ramakrishnan, L., and Hess, G. P. (2004) Biochemistry 43, 7534-7540]. Here we describe the mechanism by which the neurosteroid 3alpha, 21-dihydroxy-5alpha-pregnan-20-one (5alpha-THDOC) alleviates this malfunction of the mutated receptor transiently expressed in HEK293 cells. Two rapid reaction techniques, the cell-flow and the laser-pulse photolysis methods, were used in combination with whole-cell current recordings. 150-muM 5alpha-THDOC does not affect the rate constant for channel opening (k(op)) of approximately 250 s(-1) but does decrease the rate constant for channel closing (k(cl)) from 121 +/- 11 s(-1) to 56 +/- 21 s(-1). This results in an increase in the channel-opening equilibrium constant ((Phi(-1) = k(op)/k(cl)) by a factor of about 2, leading to about 50% alleviation of the malfunction of the inheritable mutated (alpha1beta2gamma2L(K289M)) GABA(A) receptor linked to GEFS+.
Ramakrishnan
Latha
L
Department of Molecular Biology and Genetics, 216 Biotechnology Building, Cornell University, Ithaca, New York 14853-2703, USA.
Hess
George P
GP
D002478:Cells, Cultured; D003900:Desoxycorticosterone / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D004827:Epilepsy / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009154:Mutation; D018377:Neurotransmitter Agents / Q000737:chemistry / Q000494:pharmacology; D010782:Photolysis; D011963:Receptors, GABA-A / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection
D018377:Neurotransmitter Agents; D011963:Receptors, GABA-A; D003900:Desoxycorticosterone; C009413:tetrahydrodeoxycorticosterone