Allosteric coupling between PIP₂ and Ca²⁺ binding sites gates TMEM16A channels

TMEM16A channels conduct Ca²⁺-activated Cl^- currents that underlie essential physiological processes including epithelial secretion, smooth muscle contraction, and sensory transduction. Channel activation requires both intracellular Ca²⁺ and the signaling phospholipid phosphatidylinositol 4,5-bisphosphate (PIP₂), yet the molecular basis of this dual regulation has remained unclear. Using gating molecular-dynamics simulations and structure-guided electrophysiology, we show that PIP₂ and Ca²⁺ cooperatively gate TMEM16A through an allosterically coupled electrostatic network centered on the α4 helix. Specific PIP₂ headgroup phosphate interactions are essential for coupling Ca²⁺ binding to channel opening, while the PIP₂ acyl chains engage hydrophobic surfaces of the helix to stabilize the open conformation. Disrupting either component of this lipid-protein interface reduces apparent PIP₂ affinity and impairs activation, whereas long-chain PIP₂ fully restores wild-type activity. These interactions act in concert with Ca²⁺-dependent structural rearrangements that widen the conduction pathway and enable Cl^- permeation. Our findings establish that both the headgroup phosphates and acyl chains of PIP₂ play indispensable and complementary roles in TMEM16A gating. This mechanism defines a cooperative lipid-ion activation process that provides a general framework for understanding phosphoinositide regulation of ion channels and offers opportunities for structure-based design of TMEM16A modulators.