Phosphatidylinositol 4,5-bisphosphate (PIP₂) regulates KCNQ3 K channels by interacting with four cytoplasmic channel domains

Phosphatidylinositol 4,5-bisphosphate (PIP₂) in the plasma membrane regulates the function of many ion channels, including M-type (potassium voltage-gated channel subfamily Q member (KCNQ), K_v7) K channels; however, the molecular mechanisms involved remain unclear. To this end, we here focused on the KCNQ3 subtype that has the highest apparent affinity for PIP₂ and performed extensive mutagenesis in regions suggested to be involved in PIP₂ interactions among the KCNQ family. Using perforated patch-clamp recordings of het-erologously transfected tissue culture cells, total internal reflection fluorescence microscopy, and the zebrafish (Danio rerio) voltage-sensitive phosphatase to deplete PIP₂ as a probe, we found that PIP₂ regulates KCNQ3 channels 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, 3) the S2–S3 linker, and 4) the S4 –S5 linker. We also found that the apparent strength of PIP₂ interactions within any of these domains was not coupled to the voltage dependence of channel activation. Extensive homology modeling and docking simulations with the WT or mutant KCNQ3 channels and PIP₂ were consistent with the experimental data. Our results indicate that PIP₂ modulates KCNQ3 channel function by interacting synergistically with a minimum of four cytoplasmic domains.