Identification of an Inhibitory Alcohol Binding Site in GABAA ρ1 Receptors

Alcohols inhibit gamma-aminobutyric acid type A rho 1 receptor function. After introducing mutations in several positions of the second transmembrane helix in rho 1, we studied the effects of ethanol and hexanol on GABA responses using two-electrode voltage damp electrophysiology in Xenopus laevis oocytes. The 6' mutations produced the following effects on ethanol and hexanol responses: small increase or no change (T6'M), increased inhibition (T6'V), and small potentiation (T6'Y and T6'F). The S' mutations produced mainly increases in hexanol inhibition. Other mutations produced small (3' and 9') or no changes (2' and L277 in the first transmembrane domain) in alcohol effects. These results suggest an inhibitory alcohol binding site near the 6' position. Homology models of rho 1 receptors based on the X-ray structure of GluCl showed that the 2', 5', 6', and 9' residues were easily accessible from the ion pore, with 5' and 6' residues from neighboring subunits facing each other; L3' and L277 also faced the neighboring subunit. We tested ethanol through octanol on single and double mutated rho 1 receptors [rho 1(I15'S), rho 1(T6'Y), and rho 1(T6'Y,I15'S)] to further characterize the inhibitory alcohol pocket in the wild-type rho 1 receptor. The pocket can only bind relatively short-chain alcohols and is eliminated by introducing Y in the 6' position. Replacing the bulky 15' residue with a smaller side chain introduced a potentiating binding site, more sensitive to long-chain than to short chain alcohols. In conclusion, the net alcohol effect on the rho 1 receptor is determined by the sum of its actions on inhibitory and potentiating sites.