Received for publication July 7, 2006.
Revised November 14, 2006.
Accepted for publication November 15, 2006.

Agonist-, Antagonist-, and Benzodiazepine-Induced Structural Changes in the ₁Met¹¹³-Leu¹³² Region of the GABA_A Receptor

Abstract

The structural basis by which agonists, antagonists and allosteric modulators exert their distinct actions on ligand-gated ion channels is poorly understood. We used the substituted cysteine accessibility method to probe the structure of the GABA_A receptor in the presence of ligands that elicit different pharmacological effects. Residues in the ₁Met¹¹³-Leu¹³² region of the GABA binding site were individually mutated to cysteine and expressed with wild-type ₂ and ₂ subunits in Xenopus laevis oocytes. Using electrophysiology, we determined the rates of reaction of N-biotinaminoethyl methanethiosulfonate (MTSEA-biotin) with the introduced cysteines in the resting (unliganded) state and compared them to rates determined in the presence of GABA (agonist), SR-95531 (antagonist), pentobarbital (allosteric modulator) and flurazepam (allosteric modulator). ₁N115C, ₁L117C, ₁T129C, and ₁R131C line the GABA binding pocket because MTSEA-biotin modification of these residues decreased the amount of current elicited by GABA, and the rates/extents of modification were decreased both by GABA and SR-95531. Reaction rates of some substituted cysteines were different depending on the ligand indicating that barbiturate- and GABA-induced channel gating, antagonist binding, and benzodiazepine receptor modulation induce specific structural rearrangements. Chemical reactivity of ₁E122C decreased in response to channel gating induced by either GABA or pentobarbital but was unaltered by SR-95531 binding, whereas ₁L127C reactivity was decreased by agonist and antagonist binding but not affected by pentobarbital-induced gating. Furthermore, ₁E122C, ₁L127C, and ₁R131C changed accessibility in response to flurazepam, providing structural evidence that residues in and near the GABA binding site move in response to benzodiazepine modulation.

Key words: GABAA, GABAC, Molecular dynamics, Func. analysis receptor/ion channel mutants, Barbiturates, Benzodiazepines

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