On the Mechanism of Alleviation by Phenobarbital of the Malfunction of an Epilepsy-Linked GABAA Receptor

• Published in: Biochemistry (Easton) 2006-09, Vol.45 (38), p.11632-11641
• Main Authors: Krivoshein, Arcadius V, Hess, George P
• Format: Article
• Language: English
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi061207t

A mechanism for the alleviation of the malfunction of a mutated (γ2K289M) epilepsy-linked γ-aminobutyric acid (GABA) neurotransmitter receptor by phenobarbital is presented. Compared to the wild-type receptor, the GABA-induced current is considerably reduced in the mutated (α1β2γ2K289M) epilepsy-linked GABAA receptor [Baulac, S., Huberfeld, G., Gurfinkel-An, I., Mitropoulou, G., Beranger, A., Prud'homme, J. F., Baulac, M., Brice, A., Bruzzone, R., and LeGuer, E. (2001) Nat. Genet. 28, 46−48]. This is due to an impaired GABA-induced equilibrium between the closed- and open-channel forms of the receptor [Ramakrishnan, L., and Hess, G. P. (2004) Biochemistry 43, 7534−7540]. We report that a barbiturate anticonvulsant, phenobarbital, alleviates the effect of this mutation. Transient kinetic techniques with a millisecond-to-microsecond time resolution and the wild-type and mutated receptors recombinantly expressed in mammalian HEK293T cells were used. The efficacy of phenobarbital in potentiating currents elicited by a saturating concentration of GABA is about 3 times higher for the mutated receptor than for the wild type. The results indicate that phenobarbital alleviates the malfunction of the mutated receptor by increasing its channel-opening equilibrium constant (Φ-1 = k op/k cl) by about an order of magnitude. Phenobarbital changes the channel-opening rate constant (k op) by less than 2-fold but decreases the channel-closing rate constant (k cl) 8-fold. The dissociation constant of GABA is unaffected. The experiments also indicate that at saturating concentrations of GABA the mutated (γ2K289M) form of the α1β2γ2 GABAA receptor is well suited for a rapid and simple screening of positive allosteric modulators of the receptor.