Unique Anionic Phospholipid Binding Site and Gating Mechanism in Kir2.1 Inward Rectifier Channels

Abstract

Inwardly rectifying potassium (Kir) channels regulate cell excitability and potassium homeostasis in multiple tissues. All Kir channels absolutely require interaction of phosphatidyl-4,5-bisphosphate (PIP2) with a crystallographically identified binding site, but an additional non-specific secondary anionic phospholipid (PL(-)) is required to generate high PIP2 sensitivity of Kir2 channel gating, but the PL(-) binding site and mechanism are yet to be elucidated. We used docking simulations to identify a putative PL(-) binding site, adjacent to the PIP2 binding site, generated by two lysine residues from neighboring subunits. When either lysine is mutated to cysteine (K64C, K219C), channel activity is significantly decreased in cell membranes and in reconstituted liposomes. By directly tethering the residue to the membrane, modification of the K64C mutant with decyl MTS generates high PIP2 sensitivity in liposomes, even in the complete absence of PL(-)s. The results provide a coherent molecular mechanism for the secondary anionic lipid requirement of Kir2 channel activity: PL(-) interaction with a discrete binding site results in a conformational change that pulls the cytosolic Kir domain closer to the membrane, thereby stabilizing the high-affinity PIP2 activatory site.