title: An asymmetric contribution to gamma-aminobutyric type A receptor function of a conserved lysine within TM2-3 of alpha1, beta2, and gamma2 subunits.
abstract: Mutations that impair the expression and/or function of gamma-aminobutyric acid type A (GABAA) receptors can lead to epilepsy. The familial epilepsy gamma2(K289M) mutation affects a basic residue conserved in the TM2-3 linker of most GABAA subunits. We investigated the effect on expression and function of the Lys --> Met mutation in mouse alpha1(K278M), beta2(K274M), and gamma2(K289M) subunits. Compared with cells expressing wild-type and alpha1beta2gamma2(K289M) receptors, cells expressing alpha1(K278M)beta2gamma2 and alpha1beta2(K274M)gamma2 receptors exhibited reduced agonist-evoked current density and reduced GABA potency, with no change in single channel conductance. The low current density of alpha1beta2(K274M)gamma2 receptors coincided with reduced surface expression. By contrast the surface expression of alpha1(K278M)beta2gamma2 receptors was similar to wild-type and alpha1beta2gamma2(K289M) receptors suggesting that the alpha1(K278M) impairs function. In keeping with this interpretation GABA-activated channels mediated by alpha1(K278M)beta2gamma2 receptors had brief open times. To a lesser extent gamma2(K289M) also reduced mean open time, whereas beta2(K274M) had no effect. We used propofol as an alternative GABAA receptor agonist to test whether the functional deficits of mutant subunits were specific to GABA activation. Propofol was less potent as an activator of alpha1(K278M)beta2gamma2 receptors. By contrast, neither beta2(K274M) nor gamma2(K289M) affected the potency of propofol. The beta2(K274M) construct was unique in that it reduced the efficacy of propofol activation relative to GABA. These data suggest that the alpha1 subunit Lys-278 residue plays a pivotal role in channel gating that is not dependent on occupancy of the GABA binding site. Moreover, the conserved TM2-3 loop lysine has an asymmetric function in different GABAA subunits.
authors.affiliation: Department of Pharmacology & Physiology, The George Washington University, Washington, D. C. 20037; Department of Anesthesiology & Critical Care Medicine, The George Washington University, Washington, D. C. 20037. Electronic address: phmtgh@gwumc.edu.
authors.forename: Tim G
authors.initials: TG
authors.lastname: Hales
authors.affiliation: Department of Pharmacology & Physiology, The George Washington University, Washington, D. C. 20037.
authors.forename: Tarek Z
authors.initials: TZ
authors.lastname: Deeb
authors.affiliation: Department of Pharmacology & Neuroscience, Ninewells Medical School, University of Dundee, Dundee DD1 9SY, Scotland, United Kingdom.
authors.forename: Haiyan
authors.initials: H
authors.lastname: Tang
authors.affiliation: Department of Pharmacology & Neuroscience, Ninewells Medical School, University of Dundee, Dundee DD1 9SY, Scotland, United Kingdom.
authors.forename: Karen A
authors.initials: KA
authors.lastname: Bollan
authors.affiliation: Department of Pharmacology & Neuroscience, Ninewells Medical School, University of Dundee, Dundee DD1 9SY, Scotland, United Kingdom.
authors.forename: Dale P
authors.initials: DP
authors.lastname: King
authors.affiliation: Department of Pharmacology & Neuroscience, Ninewells Medical School, University of Dundee, Dundee DD1 9SY, Scotland, United Kingdom.
authors.forename: Sara J
authors.initials: SJ
authors.lastname: Johnson
authors.affiliation: Department of Pharmacology & Neuroscience, Ninewells Medical School, University of Dundee, Dundee DD1 9SY, Scotland, United Kingdom.
authors.forename: Christopher N
authors.initials: CN
authors.lastname: Connolly
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D006801:Humans; D008239:Lysine / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D011963:Receptors, GABA-A / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D011963:Receptors, GABA-A; D008239:Lysine