Stepwise Dissociation of an Inner Gate Controls Pore Opening in the Calcium-activated Chloride Channel TMEM16A

Abstract

TMEM16A is a Ca2+-activated Cl- channel that plays key roles in diverse physiological processes. This channel functions as a homodimer with each subunit containing an independent pore and closely apposed Ca2+ binding sites. Recent cryo-electron microscopy structures of TMEM16A have revealed a conformational change involving the rearrangement of a pore-lining helix (α6) upon Ca2+ binding in a process that precedes pore activation. We have further shown that an electrostatic gating mechanism operates in TMEM16A where bound Ca2+ ions lower the energy barriers for anion conduction via direct Coulombic interactions. However, despite the described advances, the location of the gate that prevents ion conduction in the close conformation has remained elusive. Here, we investigated conformational gating of TMEM16A using electrophysiology and spectral analysis. Systematic mutagenesis of pore-lining helices identifies a specific cluster of hydrophobic residues at the inner pore that closes the anion conduction path in the closed state. The stability of the cluster increases with hydrophobic volume but correlates inversely with increasing hydrophilicity, consistent with a gate region where water and ions are excluded when closed. We analysed the power spectra of alanine mutants at the constriction and found that the loss of hydrophobic volume results in enhanced pore opening at equilibrium. The presence of three Lorentzian components in the spectra suggests an intermediate gating step that relays α6 activation to the opening of the pore. Combined with analysis of multiple double-mutant cycles, our results reveal a pore opening mechanism that involves stepwise dissociation of the inner hydrophobic gate and the pairwise interactions underlying these conformational transitions.