Anoctamin 1 (ANO1)/TMEM16A is a Cl<sup>−</sup> channel activated by intracellular Ca<sup>2+</sup> mediating numerous physiological functions. However, little is known of the ANO1 activation mechanism by Ca<sup>2+</sup>. Here, we demonstrate that two helices, “reference” and “Ca<sup>2+</sup> sensor” helices in the third intracellular loop face each other with opposite charges. The two helices interact directly in a Ca<sup>2+</sup>-dependent manner. Positively and negatively charged residues in the two helices are essential for Ca<sup>2+</sup>-dependent activation because neutralization of these charges change the Ca<sup>2+</sup> sensitivity. We now predict that the Ca<sup>2+</sup> sensor helix attaches to the reference helix in the resting state, and as intracellular Ca<sup>2+</sup> rises, Ca<sup>2+</sup> acts on the sensor helix, which repels it from the reference helix. This Ca<sup>2+</sup>-dependent push-pull conformational change would be a key electromechanical movement for gating the ANO1 channel. Because chemical activation of ANO1 is viewed as an alternative means of rescuing cystic fibrosis, understanding its gating mechanism would be useful in developing novel treatments for cystic fibrosis.
by
Juan A. Contreras-Vite;
Silvia Cruz-Rangel;
José J. De Jesus-Perez;
Iván A. Arechiga Figueroa;
Aldo A. Rodriguez-Menchaca;
Patricia Perez-Cornejo;
Harrison Hartzell Jr.;
Jorge Arreola
TMEM16A (ANO1), the pore-forming subunit of calcium-activated chloride channels, regulates several physiological and pathophysiological processes such as smooth muscle contraction, cardiac and neuronal excitability, salivary secretion, tumour growth and cancer progression. Gating of TMEM16A is complex because it involves the interplay between increases in intracellular calcium concentration ([Ca 2+ ] i ), membrane depolarization, extracellular Cl − or permeant anions and intracellular protons. Our goal here was to understand how these variables regulate TMEM16A gating and to explain four observations. (a) TMEM16A is activated by voltage in the absence of intracellular Ca 2+ . (b) The Cl − conductance is decreased after reducing extracellular Cl − concentration ([Cl − ] o ). (c) I Cl is regulated by physiological concentrations of [Cl − ] o . (d) In cells dialyzed with 0.2 μM [Ca 2+ ] i , Cl − has a bimodal effect: at [Cl − ] o < 30 mM TMEM16A current activates with a monoexponential time course, but above 30 mM, [Cl − ] o I Cl activation displays fast and slow kinetics. To explain the contribution of V m , Ca 2+ and Cl − to gating, we developed a 12-state Markov chain model. This model explains TMEM16A activation as a sequential, direct, and V m -dependent binding of two Ca 2+ ions coupled to a V m -dependent binding of an external Cl − ion, with V m -dependent transitions between states. Our model predicts that extracellular Cl − does not alter the apparent Ca 2+ affinity of TMEM16A, which we corroborated experimentally. Rather, extracellular Cl − acts by stabilizing the open configuration induced by Ca 2+ and by contributing to the V m dependence of activation.