Mechanism of Potentiation of a Dysfunctional Epilepsy-Linked Mutated GABAA Receptor by a Neurosteroid (3α, 21-Dihydroxy-5α-pregnan-20-one): Transient Kinetic Investigations

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• 作者：Latha Ramakrishnan ; George P. Hess
• 刊名：Biochemistry
• 出版年：2010
• 出版时间：September 14, 2010
• 年：2010
• 卷：49
• 期：36
• 页码：7892-7901
• 全文大小：916K
• 年卷期：v.49,no.36(September 14, 2010)
• ISSN：1520-4995

文摘

The malfunction of a mutated GABA_A receptor (α1β2γ2L^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 3α, 21-dihydroxy-5α-pregnan-20-one (5α-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-μM 5α-THDOC does not affect the rate constant for channel opening (k_op) of 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 ((Φ⁻¹ = k_op/k_cl) by a factor of about 2, leading to about 50% alleviation of the malfunction of the inheritable mutated (α1β2γ2L^K289M) GABA_A receptor linked to GEFS+.