A discrete alcohol pocket involved in GIRK channel activation

Abstract

Ethanol modifies neural activity in the brain by modulating ion channels. Ethanol activates G protein-gated inwardly rectifying K+ channels, but the molecular mechanism is not well understood. Here, we used a crystal structure of a mouse inward rectifier containing a bound alcohol and structure-based mutagenesis to probe a putative alcohol-binding pocket located in the cytoplasmic domains of GIRK channels. Substitutions with bulkier side-chains in the alcohol-binding pocket reduced or eliminated activation by alcohols. By contrast, alcohols inhibited constitutively open channels, such as IRK1 or GIRK2 that binds PIP2 strongly. Mutations in the hydrophobic alcohol-binding pocket of these channels had no effect on alcohol-dependent inhibition, suggesting an alternate site is involved in inhibition. Comparison of high-resolution structures of inwardly rectifying K+ channels suggests a model for activation of GIRK channels utilizing this hydrophobic alcohol-binding pocket. These results provide a tool for developing therapeutic compounds that could mitigate the effects of alcohol.

Molecular mechanism underlying ethanol activation of G-protein–gated inwardly rectifying potassium channels

“…The proximity of alcohol pocket and Gβγ binding site, which both involve the βL-βM loop previously implicated in GIRK activation (3,25), may explain the partial overlap in these activation pathways. Possibly, Gβγ induces a conformational wave, similar to that in ligand-gated ion channels (50), which propagates to the PIP 2 binding site through the alcohol pocket.…”

“…We engineered GIRK2 (or Kir3.2) with a single thiol-reactive cysteine (Cys) at or near the alcohol pocket, a region comprising the βD-βE and βL-βM loops and N-terminal domain (Fig. 1A) (3). First, four native Cys residues in the cytoplasmic domain were replaced with thiol-unreactive residues to create a "Cys-less" GIRK2 (referred to as GIRK2*).…”

“…1F). However, the inability to use saturating concentrations of ethanol precluded measuring the EC 50 (3)(4)(5).…”

“…Although the neural circuits underlying such addictive disorders are becoming better understood (1,2), little is known about the molecular mechanisms underlying ethanol's interaction with specific target proteins, such as ion channels. G-proteingated inwardly rectifying K + (GIRK or Kir3) channels are activated by concentrations of ethanol relevant to human consumption (18 mM ethanol or 0.08% blood alcohol level) (3)(4)(5) and have been found to play a key role in alcohol-related disorders (6)(7)(8)(9). For example, mice lacking GIRK2 (or Kir3.2) channels self-administer more ethanol and fail to develop conditioned place preference for ethanol, compared with wild-type (WT) littermates (6,10).…”

“…Structural views of putative alcohol-binding pockets are emerging (3,16,17), but the molecular details and chemical rules governing the interaction of alcohol with these specific alcohol-binding pockets remain elusive.…”