Potassium ion conduction through open potassium channels is essential to control of membrane potentials in all cells. To elucidate the open conformation and hence the mechanism of K + ion conduction in the classic inward rectifier Kir2.2, we introduced a negative charge (G178D) at the crossing point of the inner helix bundle, the location of ligand-dependent gating. This “forced open” mutation generated channels that were active even in the complete absence of phosphatidylinositol-4,5-bisphosphate (PIP 2 ), an otherwise essential ligand for Kir channel opening. Crystal structures were obtained at a resolution of 3.6 Å without PIP 2 bound, or 2.8 Å in complex with PIP 2 . The latter revealed a slight widening at the helix bundle crossing (HBC) through backbone movement. MD simulations showed that subsequent spontaneous wetting of the pore through the HBC gate region allowed K + ion movement across the HBC and conduction through the channel. Further simulations reveal atomistic details of the opening process and highlight the role of pore-lining acidic residues in K + conduction through Kir2 channels.

Inward rectifier potassium (Kir) channel activity is controlled by plasma membrane lipids. Phosphatidylinositol-4,5-bisphosphate (PIP 2 ) binding to a primary site is required for opening of classic inward rectifier Kir2.1 and Kir2.2 channels, but interaction of bulk anionic phospholipid (PL − ) with a distinct second site is required for high PIP 2 sensitivity. Here we show that introduction of a lipid-partitioning tryptophan at the second site (K62W) generates high PIP 2 sensitivity, even in the absence of PL − . Furthermore, high-resolution x-ray crystal structures of Kir2.2[K62W], with or without added PIP 2 (2.8- and 2.0-Å resolution, respectively), reveal tight tethering of the C-terminal domain (CTD) to the transmembrane domain (TMD) in each condition. Our results suggest a refined model for phospholipid gating in which PL − binding at the second site pulls the CTD toward the membrane, inducing the formation of the high-affinity primary PIP 2 site and explaining the positive allostery between PL − binding and PIP 2 sensitivity.