Inwardly rectifying potassium (Kir) channels regulate multiple tissues. All Kir channels require interaction of phosphatidyl-4,5-bisphosphate (PIP2) at a crystallographically identified binding site, but an additional nonspecific secondary anionic phospholipid (PL(-)) is required to generate high PIP2 sensitivity of Kir2 channel gating. The PL(-)-binding site and mechanism are yet to be elucidated. Here we report docking simulations that identify a putative PL(-)-binding site, adjacent to the PIP2-binding site, generated by two lysine residues from neighbouring subunits. When either lysine is mutated to cysteine (K64C and K219C), channel activity is significantly decreased in cells and in reconstituted liposomes. Directly tethering K64C to the membrane by modification with decyl-MTS generates high PIP2 sensitivity in liposomes, even in the complete absence of PL(-)s. The results provide a coherent molecular mechanism whereby PL(-) interaction with a discrete binding site results in a conformational change that stabilizes the high-affinity PIP2 activatory site.