Endogenous phosphatidylinositol 4,5-bisphosphate facilitates activation of heterologously expressed TMEM16A via functionally coupled binding sites.

The Ca²⁺-activated Cl⁻ channel TMEM16A translates cytosolic Ca²⁺ signals into transmembrane Cl⁻ fluxes, supporting essential physiological functions across diverse tissues. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a phosphoinositide enriched in the inner leaflet of the plasma membrane, has emerged as a key regulator of TMEM16A. However, the role of endogenous PI(4,5)P2 and the specific binding sites mediating its regulatory effects remain poorly understood. Here, using electrophysiology, voltage-sensitive 5-phosphatases, site-directed mutagenesis, and molecular dynamics simulations, we show that endogenous PI(4,5)P2 facilitates TMEM16A activation by lowering the apparent gating energy by ∼0.89 kcal/mol at physiological Ca²⁺ concentrations. This energetic contribution remains consistent across Ca²⁺-dependent activation states, within experimental resolution. Cumulative disruption of previously identified PI(4,5)P2 binding sites 1 or 3 revealed that Site 1 promotes pore opening, whereas Site 3 functions in a manner consistent with a modulation of the apparent gating energy without detectable effects on pore function. Simultaneous disruption of both sites produced no additional change in apparent gating energy, suggesting that Sites 1 and 3 act as a functional unit that defines the energetic minimum required for channel opening. Residue R484 serves as a structural bridge coupling both Sites under endogenous PI(4,5)P2 conditions. Together, these findings support a two-site model in which endogenous PI(4,5)P2 shapes the energetic landscape, thereby facilitating TMEM16A pore opening. These findings contribute to understanding the role of PI(4,5)P2 as a central endogenous modulator of TMEM16A.