Engineering the Alcohol Pocket to Create a Chemically-Activated GIRK Channel

G-protein gated inwardly rectifying potassium (GIRK) channels are implicated in alcohol abuse. Ethanol directly activates GIRKs through interaction with a discrete alcohol-binding pocket that we identified in the cytoplasmic domain. The structural mechanism underlying ethanol-dependent activation, however, is not well understood. Here, we hypothesized that chemically modifying the alcohol pocket with alcohol-like reagents would activate the channel. To examine this, we engineered GIRK2 containing a single cytoplasmic cysteine substitution in the βD-βE domain of the alcohol pocket; S246 is located near the edge of the pocket, and L257 is located within the pocket. All Cys substitutions were introduced into GIRK2c lacking four native Cys residues (GIRK2c(cys-)) and transiently expressed in HEK293T cells. We studied the effect of 2-Hydroxyethyl MTS (MTS-HE, alcohol-like) and two hydrophobic MTS reagents- Benzyl-MTS (MTS-Bn), and 4-hydroxy benzyl-MTS (MTS-Y), on basal (Ba2+-sensitive) and alcohol-activated GIRK currents. MTS-HE (100 μM) increased basal GIRK currents for S246C (34% ± 12%, n=7) and L257C (120% ± 24%, n=7). Similarly, MTS-Bn (10 μM) modification increased basal currents by 74% ± 18% (n=7) for S246C, and by 333% ± 31% (n=4) for L257C. MTS-Y (100 μM) increased the basal currents by 102% ± 25% (n= 5) for L257C but did not affect S246C. In addition, MTS modification decreased MPD-activated current. For L257C, we confirmed that intracellular application of MTS-HE produced a concentration-dependent increase in the time course of channel activation, having a rate constant of 90.6±17.2 M−1s−1 (n=11). Taken together, these data demonstrate that MTS modification of a single amino acid in the alcohol pocket can engage channel activation, and limit the access of bulkier diols (e.g., MPD). These results reveal novel mechanisms for chemical activation of GIRKs and underscore the importance of hydrophobic interactions in alcohol-mediated activation of GIRKs.