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The Journal of Neuroscience, July 15, 2001, 21(14):4977-4986

A -Strand in the ₂ Subunit Lines the Benzodiazepine Binding Site of the GABA_A Receptor: Structural Rearrangements Detected during Channel Gating

Jeremy A. Teissére¹ and Cynthia Czajkowski^{1, 2}

¹ Neuroscience Training Program and ² Department of Physiology, University of Wisconsin, Madison, Wisconsin 53706

Benzodiazepines (BZDs) exert their effects in the CNS by binding to a modulatory site on GABA_A receptors. Individual amino acids have been implicated in BZD recognition and modulation of the GABA_A receptor, but the secondary structure of the amino acids contributing to the BZD binding site has not been elucidated. In this report we used the substituted cysteine accessibility method to understand the structural dynamics of a region of the GABA_A receptor implicated in BZD binding, ₂Y72-₂Y83. Each residue within this region was mutated to cysteine and expressed with wild-type ₁ and ₂ subunits in Xenopus oocytes. Methanethiosulfonate (MTS) reagents were used to modify covalently the engineered cysteines, and the subsequent effects on BZD modulation of the receptor were monitored functionally by two-electrode voltage clamp. We identified an alternating pattern of accessibility to sulfhydryl modification, indicating that the region ₂T73-₂T81 adopts a -strand conformation. By monitoring the ability of BZD ligands to impede the covalent modification of accessible cysteines, we also identified two residues within this region, ₂A79 and ₂T81, that line the BZD binding site. Sulfhydryl modification of ₂A79C or ₂T81C allosterically shifts the GABA EC₅₀ of the receptor, suggesting that certain MTS compounds may act as tethered agonists at the BZD binding site. Last, we present structural evidence that a portion of the BZD binding site undergoes a conformational change in response to GABA binding and channel gating (opening and desensitization). These data represent an important step in understanding allosteric communication in ligand-gated ion channels.

Key words: benzodiazepine; binding site; allostery; ligand-gated ion channel; GABA; GABA_A receptor; substituted cysteine accessibility method; Xenopus oocytes; secondary structure

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Copyright © 2001 Society for Neuroscience  0270-6474/01/21144977-10$05.00/0

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