The ROMK1 (Kir1.1) channel activity is predominantly regulated by intracellular pH (pH(i)) and phosphatidylinositol 4,5-bisphosphate (PIP2). Although several residues were reported to be involved in the regulation of p1-1, associated with PIP2 interaction, the detailed molecular mechanism remains unclear. We perform experiments in ROMK1 pH(i)-gating with electrophysiology combined with mutational and structural analysis. In the present study, non basic residues of C-terminal region (S219, N215, I192, L216 and L220) in ROMK1 channels have been found to mediate channel-PIP2 interaction and pH(i) gating. Further, our structural results show these residues with an appropriate distance to interact with membrane PIP2. Meanwhile, a cluster of basic residues (R188, R217 and K218), which was previously discovered regarding the interaction with PIP2, exists in this appropriate distance to discriminate the regulation of channel-PIP2 interaction and pH(i)-gating. This appropriate distance can be observed with high conservation in the Kir channel family. Our results provide insight that an appropriate distance cooperates with the electrostatics interaction of channel-PIP2 to regulate pH(i)-gating. (C) 2016 Elsevier Inc. All rights reserved.