title: On the mechanism of alleviation by phenobarbital of the malfunction of an epilepsy-linked GABA(A) receptor.
abstract: A mechanism for the alleviation of the malfunction of a mutated (gamma2(K289M)) epilepsy-linked gamma-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 (alpha1beta2gamma2(K289M)) epilepsy-linked GABA(A) 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 (phi(-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 (gamma2(K289M)) form of the alpha1beta2gamma2 GABA(A) receptor is well suited for a rapid and simple screening of positive allosteric modulators of the receptor.
authors.affiliation: Department of Molecular Biology and Genetics, 216 Biotechnology Building, Cornell University, Ithaca, New York 14853-2703, USA.
authors.forename: Arcadius V
authors.initials: AV
authors.lastname: Krivoshein
authors.forename: George P
authors.initials: GP
authors.lastname: Hess
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects; D000818:Animals; D002478:Cells, Cultured; D004357:Drug Synergism; D004827:Epilepsy / Q000378:metabolism* / Q000503:physiopathology; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects; D007700:Kinetics; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010634:Phenobarbital / Q000494:pharmacology*; D010782:Photolysis; D051381:Rats; D011963:Receptors, GABA-A / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
chemical_list: D050505:Mutant Proteins; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; D005680:gamma-Aminobutyric Acid; D010634:Phenobarbital