The plasma membrane inner leaflet PI(4,5)P 2 is essential for the activation of proton-activated chloride channels.

Proton-activated chloride (PAC) channels, ubiquitously expressed in tissues, regulate intracellular Cl - levels and cell death following acidosis. However, molecular mechanisms and signaling pathways involved in PAC channel modulation are largely unknown. Herein, we determine that phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] of the plasma membrane inner leaflet is essential for the proton activation of PAC channels. PI(4,5)P 2 depletion by activating phosphatidylinositol 5-phosphatases or G q protein-coupled muscarinic receptors substantially inhibits human PAC currents. In excised inside-out patches, PI(4,5)P 2 application to the cytoplasmic side increases the currents. Structural simulation reveals that the putative PI(4,5)P 2 -binding site is localized within the cytosol in resting state but shifts to the cell membrane's inner surface in an activated state and interacts with inner leaflet PI(4,5)P 2 . Alanine neutralization of basic residues near the membrane-cytosol interface of the transmembrane helice 2 significantly attenuates PAC currents. Overall, our study uncovers a modulatory mechanism of PAC channel through inner membrane PI(4,5)P 2 .