γ-Aminobutyric Acid (GABA) and Pentobarbital Induce Different Conformational Rearrangements in the GABAA Receptor α1 and β2 Pre-M1 Regions

Abstract

␥-Aminobutyric acid (GABA) binding to GABA A receptors (GABA A Rs) triggers conformational movements in the ␣ 1 and ␤ 2 pre-M1 regions that are associated with channel gating. At high concentrations, the barbiturate pentobarbital opens GABA A R 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 GABA A R pre-M1 regions. ␣ 1 ␤ 2 GABA A Rs 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 EC 50 values. Tethering chemically diverse thiol-reactive methanethiosulfonate reagents onto ␣ 1 K219C and ␣ 1 K221C affected GABA-and 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 ␣ 1 K221C␤ 2 receptors, pentobarbital decreased the rate of cysteine modification whereas GABA had no effect. For ␣ 1 ␤ 2 K215C 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 GABA-and pentobarbital-mediated changes in rates reflect gating movements. Overall, the data indicate that the pre-M1 region is involved in both GABA-and 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 (GABA A R) is the main 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 GABA A R activity. At low concentrations, PB modulates GABA-mediated Cl Ϫ current (I GABA ). At higher concentrations, PB directly activates the GABA A R 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 from mouse 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 GABA A...

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