Phosphatidylinositol 4,5-Bisphosphate (PIP2) Regulate KCNQ3 K+ Channels Via Multiple Sites Of Action

Phosphatidylinositol 4,5-bisphosphate (PIP2) regulates the function of many ion channels, including M-type (KCNQ1-5, Kv7) K+ channels; however the molecular mechanisms involved in this regulation remain unclear. To identify the sites of action on KCNQ3 channels, we used as our baseline the A315T pore mutant (KCNQ3T) that increases channel currents without modifying the apparent affinity of PIP2 and performed extensive mutagenesis in regions that have been suggested to be involved in PIP2 interactions among the KCNQ family. Using the zebrafish (Danio rerio) voltage-sensitive phosphatase to deplete PIP2 as a probe for apparent affinity of the channels, we found that PIP2 modulates KCNQ channel function through four different domains. 1) the A-B helix linker that we previously identified as important for both KCNQ2 and KCNQ3, 2) the junction between S6 and the A helix (S6Jx), 3) the S2-S3 linker and 4) the S4-S5 linker. We found that PIP2 interactions within these domains were not coupled to the voltage dependence of activation. Extensive homology modeling and docking simulations between the wild-type or mutant KCNQ3 channels and PIP2, correlated with the experimental data. Our results indicate that PIP2 modulates KCNQ3 channel function by interacting synergistically with a minimum of four cytoplasmic domains.