Functional characterization of a novel PIP2-independent GIRK2 channel gating mechanism

G protein-gated inwardly rectifying potassium (GIRK) channels regulate cellular excitability in the heart and brain. GIRK channels have emerged as a candidate drug target for modulating activity in the brain and treating diseases such as epilepsy or alcoholism. However, molecular and structural determinants of gating regulation of GIRK channels by various ligands are not fully understood. Two regions have been identified as important molecular determinants of PIP2 and Gβ gating: the inner helix bundle crossing (HBC) formed by the M2 TM domain, and the G loop in the cytoplasmic terminal domain (CTD). Previously, we determined the cryoEM structure of GIRK2 in complex with PIP2 and the cholesterol analog CHS. This structure identified the interaction of one residue, R92, with both cholesterol and PIP2, suggesting an important role in channel gating. To further investigate the role of R92 in channel gating, we introduced aromatic residues at this locus. Substitution with aromatic residues (i.e., Y and F) showed elevated basal activity in electrophysiological studies. Channels containing these mutations were then purified, reconstituted into proteoliposomes, and functionally characterized using a fluorescence-based flux assay. We found that both mutations increased basal flux, even in the absence of PIP2. The R92Y channels had an increase in PIP2 affinity while the R92F channel was completely PIP2-insensitive. These results suggest a PIP2-independent gating mode introduced via mutation of this critical residue, R92 to aromatic residues. Further structural and functional characterization of these GIRK2 mutants will provide clarity on GIRK2 channel gating and a potential framework for rational drug design of GIRK channels.