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 TMEM16As 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. HIGHLIGHTSO_LIRemoval of the inhibitor 1PBC from TMEM16A induces spontaneous transitions to ion conductive and non-conductive states via bending in TM4 at two different locations. C_LIO_LISimulations suggest that the open state is stabilized by a small hydrophobic network between TM3 and 4 and disrupting this network biases channel closure in electrophysiological recordings. C_LIO_LIA kinetic model of conduction based on energetics from the all-atom MD simulations coupled to continuum calculations gives a linear current-voltage curve consistent with the fully open conformation and removal of 1 Ca2+ switches to an outwardly rectifying state via electrostatic influence on the Cl- energy profile. However, the rectification is too weak to match experiment, but a cooperative model of Ca2+ binding with weak voltage-dependence does match experiment. C_LI