Inwardly rectifying K+ (Kir) channels set the resting membrane potential and regulate cellular excitability. The activity of Kir channels depends critically on the phospholipid PIP2. The molecular mechanism by which PIP2 regulates Kir channel gating is poorly understood. Here, we utilized a combination of computational and electrophysiological approaches to discern structural elements involved in regulating the PIP2-induced gating kinetics of Kir2 channels. We identify a novel role for the cytosolic GH loop. Mutations that directly or indirectly affect GH loop flexibility (e.g. V223L, E272G, D292G) increase both the on- and especially the off-gating kinetics. These effects are consistent with a model in which competing interactions between the CD and GH loops for the N terminus regulate the gating of the intracellular G loop gate. [http://dx.doi.org/10.1074/jbc.M112.418640] The article was published as: An HL;Lv SQ;Li JW;Meng XY;Zhan Y;Cui M;Long M;Zhang HL;Logothetis DE. The cytosolic GH loop regulates the phosphatidylinositol 4,5-bisphosphate-induced gating kinetics of Kir2 channels. JOURNAL OF BIOLOGICAL CHEMISTRY, 287(50):42278-42287(2012)