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

Jose Mercado,Cynthia Czajkowski

doi:10.1074/jbc.m708638200

Jose Mercado, Cynthia Czajkowski

Open Access

https://doi.org/10.1074/jbc.m708638200

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Abstract

γ-Aminobutyric acid (GABA) binding to GABAA receptors (GABAARs) triggers conformational movements in the α1 and β2 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. α1β2 GABAARs containing cysteine substitutions in the pre-M1 α1 (K219C, K221C) and β2 (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 α1K219C and α1K221C 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 α1K221Cβ2 receptors, pentobarbital decreased the rate of cysteine modification whereas GABA had no effect. For α1β2K215C 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.

Highlights

Single channel studies from mouse spinal neurons [2,3,4] 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
We previously demonstrated that the ␣1 and ␤2 pre-M1 centration of PB, Imax is the maximum amplitude of current, regions of the GABAA receptors (GABAARs), which connect the extracellular EC50 is the concentration of PB that evokes half-maximal domain of each subunit with the transmembrane domain, response, [A] is the agonist concentration, and n is the Hill undergo structural rearrangements during GABA activation coefficient
The Monod-Wyman-Changeux allosteric theory has been used with great success to model Ligand-gated ion channels (LGICs) gating behavior [19]

Summary

Introduction

Single channel studies from mouse spinal neurons [2,3,4] 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. B, summary of the effects of a 2-min application of 2 mM MTSEA-biotin (top), MTSETϩ (middle), or MTSESϪ (bottom) on GABA (EC50) -activated currents (IGABA) (previously reported in Ref. 13) and PB (EC50) -activated currents (IPB) from ␣1␤2 and mutant receptors.

Results

Conclusion