Orthosteric interactions with PIP ₂ activate TMEM16A channels

TMEM16A channels pass Ca ²⁺ -activated Cl ^- currents that drive a plethora of fundamental physiological processes. TMEM16A channels are activated by a rise in intracellular Ca ²⁺ levels but also require interactions with the signaling phospholipid phosphatidylinositol 4,5-bisphosphate (PIP ₂ ) to gate open. Although PIP ₂ is essential for the activity of many types of ion channels, its precise binding site and role in channel gating remain poorly understood in most cases, limiting efforts to study channel dynamics and design targeted modulators. In this study, we identify the PIP ₂ binding interactions that govern TMEM16A gating and permeation. Using a combination of gating molecular dynamics (GMD) simulations and electrophysiological assays, we reveal how the α4 helix of TMEM16A interacts with both the phosphate headgroups and acyl chains of PIP ₂ to open an electrostatic ring within the channel and stabilize the extracellular opening of the Cl ^- conduction pathway. These findings provide key insights into the dynamic role of PIP ₂ in membrane protein function and shed light on the activation mechanism of TMEM16A. This work establishes a framework for rational targeting of TMEM16A in drug development, with potential therapeutic applications in a variety of diseases.