Identification of an Inhibitory Alcohol Binding Site in GABAaRho1

We examined why alcohols inhibit the γ-aminobutyric acid type A rho1 receptor (GABAaRrho1) function whereas alcohols potentiate other GABAaR subtypes. We did so by introducing mutations in the second transmembrane helix in GABAaRrho1. Homology models of rho1 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; whereas L3′ and L277(TM1) were facing the neighboring subunit. We studied ethanol and hexanol effects on GABA responses using two-voltage clamp electrophysiology in Xenopus laevis oocytes. The 6′ mutations showed changes in ethanol and hexanol effects: small changes (T6′M), increased inhibition (T6′V), and small potentiation (T6′Y and T6′F). The 5′ mutations produced increased hexanol inhibition. Other mutations produced smaller (3′ and 9′) or no changes (2′ and L277) on alcohol effects. These results suggest an inhibitory alcohol binding site near the 6′ position By testing ethanol through octanol on single and double mutated rho1 receptors [rho1(I15′S), rho1(T6′Y) and rho1(T6′Y,I15′S)], we further characterized the inhibitory alcohol pocket in the wild-type ρ1 receptor that can only bind relatively short-chain alcohols, and is eliminated by introducing Y in the 6’ position. Replacing the 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 ρ1 receptor is determined by the sum of its actions on inhibitory and potentiating sites.