PT  - JOURNAL ARTICLE
AU  - Teissére, Jeremy A.
AU  - Czajkowski, Cynthia
TI  - A β-Strand in the γ<sub>2</sub> Subunit Lines the Benzodiazepine Binding Site of the GABA<sub>A</sub> Receptor: Structural Rearrangements Detected during Channel Gating
AID  - 10.1523/JNEUROSCI.21-14-04977.2001
DP  - 2001 Jul 15
TA  - The Journal of Neuroscience
PG  - 4977--4986
VI  - 21
IP  - 14
4099  - http://www.jneurosci.org/content/21/14/4977.short
4100  - http://www.jneurosci.org/content/21/14/4977.full
SO  - J. Neurosci.2001 Jul 15; 21
AB  - Benzodiazepines (BZDs) exert their effects in the CNS by binding to a modulatory site on GABAA receptors. Individual amino acids have been implicated in BZD recognition and modulation of the GABAA 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 GABAA receptor implicated in BZD binding, γ2Y72–γ2Y83. Each residue within this region was mutated to cysteine and expressed with wild-type α1 and β2 subunits inXenopus 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 γ2T73–γ2T81 adopts a β-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, γ2A79 and γ2T81, that line the BZD binding site. Sulfhydryl modification of γ2A79C or γ2T81C 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.