Stoichiometry-dependent gating and structural pharmacology of cardiac GIRK1/4 channels

Cardiac G protein-gated inwardly rectifying potassium-K ⁺ (GIRK) channels, formed by GIRK1/4 heterotetramers or GIRK4 homotetramers, are crucial regulators of heart rate, and their dysfunction is associated with atrial fibrillation. Despite their therapeutic potential as anti-arrhythmic targets, development of selective GIRK modulators has been limited by an incomplete understanding of their structures, gating mechanisms, and pharmacology. Here, we report high-resolution structures of the GIRK4 homotetramer and of GIRK1/4 heterotetramers, revealing that they form in unexpected 1:3 and 2:2 assembly stoichiometries that we corroborate using cell-based single molecule microscopy. Electrophysiological assays and molecular dynamics simulations further show that incorporation of one or two GIRK1 subunits into GIRK4-containing channels progressively opens the helix bundle crossing (HBC) gate and increases basal channel activity. Additional structures of GIRK1/4 complexes with ML297, a small-molecule ligand selective for GIRK1-containing channels, reveal distinct GIRK1-centered intersubunit binding environments. Remarkably, ML297 inhibits GIRK1/4 channels with a 1:3 stoichiometry but potentiates channels with a 2:2 stoichiometry, establishing a direct link between subunit composition, ligand-binding environment, and pharmacological efficacy. Together, these findings provide a structural and mechanistic framework for the rational design of ligands with enhanced selectivity among cardiac and neuronal GIRK channel heterotetramers.