Conformational changes upon pore blocker removal reveal conductive states of TMEM16A

TMEM16A is a Ca²⁺-activated anion channel that provides direct electrical feedback to the plasma membrane in response to intracellular Ca²⁺. Its conductive state remains unresolved, leaving questions about gating, Cl^- permeation, and modulation by Ca²⁺, depolarization, and lipids. To investigate the open state, we performed molecular dynamics simulations of TMEM16A bound to the putative open-state blocker 1PBC. After inhibitor removal, the putative, pore-lining helix TM4 developed kinks at two sites: an upper site that opens the pore for Cl^- permeation, and a deeper site causing constriction. A conserved hydrophobic network between TM3 and TM4 persisted in most open structures but separated during extreme dilation, allowing lipids to transiently block the pore. Patch-clamp recordings indicated that the intact network promotes activation. Further simulations yielded >60 Cl^- permeation events and a single-channel conductance matching experiments. Additional electrostatic and kinetic modeling indicated that TMEM16A's transition from outward-rectification to Ohmic conductance with increasing Ca²⁺ results from a weak voltage dependence of Ca²⁺ binding, which act cooperatively to open the pore.