-Aminobutyric Acid (GABA) and Pentobarbital Induce Different Conformational Rearrangements in the GABAA Receptor

-Aminobutyric acid (GABA) binding to GABAA receptors (GABAARs) triggers conformational movements in the 1 and 2 pre-M1 regions that are associated with channel gating. At high concentrations, the barbiturate pentobarbital opens GABAAR channels with similar conductances as GABA, suggesting that their open state structures are alike. Little, however, is known about the structural rearrangements induced by barbiturates. Here, we examined whether pentobarbital activation triggers movements in the GABAAR pre-M1 regions. 1 2 GABAARs containing cysteine substitutions in the pre-M1 1 (K219C, K221C) and 2 (K213C, K215C) subunits were expressed in Xenopus oocytes and analyzed using two-electrode voltage clamp. The cysteine substitutions had little to no effect on GABA and pentobarbital EC50 values. Tethering chemically diverse thiol-reactive methanethiosulfonate reagents onto 1K219C and 1K221C affected GABAand pentobarbital-activated currents differently, suggesting that the pre-M1 structural elements important for GABA and pentobarbital current activation are distinct. Moreover, pentobarbital altered the rates of cysteine modification by methanethiosulfonate reagents differently than GABA. For 1K221C 2 receptors, pentobarbital decreased the rate of cysteine modification whereas GABA had no effect. For 1 2K215C receptors, pentobarbital had no effect whereas GABA increased the modification rate. The competitive GABA antagonist SR-95531 and a low, non-activating concentration of pentobarbital did not alter their modification rates, suggesting that the GABAand pentobarbital-mediated changes in rates reflect gating movements. Overall, the data indicate that the pre-M1 region is involved in both GABAand pentobarbital-mediated gating transitions. Pentobarbital, however, triggers different movements in this region than GABA, suggesting their activation mechanisms differ. Ligand-gated ion channels (LGICs)2 are integral membrane proteins that mediate fast synaptic transmission between cells in the brain and at the neuromuscular junction. The type A -aminobutyric acid receptor (GABAAR) is themain inhibitory LGIC in the brain and is the target for a wide range of therapeutic agents such as benzodiazepines, barbiturates, and anesthetics. Barbiturates, such as pentobarbital (PB), have three distinct effects on GABAAR activity. At low concentrations, PB modulates GABA-mediated Cl current (IGABA). At higher concentrations, PB directly activates the GABAAR in the absence of GABA, and at still higher concentrations, PB blocks channel activity (1). Little is known, however, about the structural rearrangements underlying these functional effects. Single channel studies frommouse spinal neurons (2–4) and from rat hippocampal neurons (5) have shown that currents evoked by PB are similar in conductance as those evoked by GABA, suggesting that the open state structures stabilized by PB binding are similar to those stabilized by GABA. However, GABA and PB bind to distinct sites on the GABAAR (Fig. 1). The GABA binding site is located at the interfaces of the 1 and 2 subunits in the extracellular domain,whereas the PB/general anesthetics binding site(s) are believed to be located 50 Å below the GABA binding site in a water-accessible pocket located between the four transmembrane helices (M1–4) of the receptor (Fig. 1). Mutational analyses as well as photolabeling studies have identified positions in the GABAAR transmembrane helices that are important for mediating the effects of PB/anesthetics, with a proposed binding pocket involving residues in M1 ( 1M236), M2 ( 2N265), and M3 ( 2M286) (6–8). The structural machinery associated with coupling agonist binding to channel gating in theCys-loop family of LGICs likely involves distributed movements of, and interactions between, several discrete domains. Recent evidence suggests that binding of neurotransmitter in the extracellular domain triggers a series of molecular motions (conformational wave) that initiates in the ligand binding pocket, followed by movements in Loop 2, Loop 7 (Cys-loop), the pre-M1 region, the M2-M3 linker, and finally the transmembrane domains to gate the channel (9–11). Because PBand GABA-activated channel open state structures are alike (3, 12) but PB and GABA bind to different sites, we were interested in determining whether gating motions induced by PB are similar to those induced by GABA. * This work was supported, in whole or in part, by National Institutes of Health Grant NS34727 from NINDS (to C. C.). This work was also supported by the Diversity Program in Neuroscience of the American Psychological Association (to J. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom correspondence should be addressed: University of WisconsinMadison, 601 Science Dr., Madison, WI 53711. Tel.: 608-265-5863; E-mail: [email protected]. 2 The abbreviations used are: LGIC, ligand-gated ion channel; GABAAR, -aminobutyric acid type A receptor; PB, pentobarbital; MTS, methanethiosulfonate; MTS, MTS-ethyltrimethylammonium; MTSES, MTS-ethylsulfonate. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 22, pp. 15250 –15257, May 30, 2008 © 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.