Abstract
The anion channel TMEM16A is activated by intracellular Ca2+ in a highly cooperative process. By combining electrophysiology and autocorrelation analysis, we investigated the mechanism of channel activation and the concurrent rearrangement of the gate in the narrow part of the pore. Features in the fluctuation characteristics of steady-state current indicate the sampling of intermediate conformations that are successively occupied during gating. The initial step is related to conformational changes induced by Ca2+ binding, which is ensued by rearrangements that open the pore. Mutations in the gate shift the equilibrium of transitions in a manner consistent with a progressive destabilization of this region during pore opening. We come up with a mechanism of channel activation where the binding of Ca2+ induces conformational changes in the protein that, in a sequential manner, propagate from the binding site and couple to the gate in the narrow pore to allow ion permeation.
Highlights
The anion channel TMEM16A is activated by intracellular Ca2+ in a highly cooperative process
We show that the fluctuation characteristics of steady-state current in TMEM16A is consistent with the sampling of intermediate conformations that are successively occupied during gating
Our results suggest a mechanism of channel activation where the binding of Ca2+ induces conformational changes in the protein that, in a sequential manner, propagate from the binding site and couple to the gate in the narrow pore to allow ion permeation
Summary
The anion channel TMEM16A is activated by intracellular Ca2+ in a highly cooperative process. We come up with a mechanism of channel activation where the binding of Ca2+ induces conformational changes in the protein that, in a sequential manner, propagate from the binding site and couple to the gate in the narrow pore to allow ion permeation. Whereas major factors controlling ion flow have been identified in previous studies and a general model for activation was proposed[14], the detailed sequence of events and the existence of intermediates in the gating process, which together define the activation mechanism, are still elusive This process would ideally be characterized by single-channel analysis, such studies are prohibited by the low conductance of TMEM16A10,15. Our results suggest a mechanism of channel activation where the binding of Ca2+ induces conformational changes in the protein that, in a sequential manner, propagate from the binding site and couple to the gate in the narrow pore to allow ion permeation
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