Revealing the activation pathway for TMEM16A chloride channels from macroscopic currents and kinetic models

• Published in: Pflügers Archiv Vol. 468; no. 7; pp. 1241 - 1257
• Main Authors: Contreras-Vite, Juan A., Cruz-Rangel, Silvia, De Jesús-Pérez, José J., Figueroa, Iván A. Aréchiga, Rodríguez-Menchaca, Aldo A., Pérez-Cornejo, Patricia, Hartzell, H. Criss, Arreola, Jorge
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.07.2016; Springer Nature B.V
• Subjects: Animals; Anions - metabolism; Anoctamin-1; Biomedical and Life Sciences; Biomedicine; Calcium - metabolism; Cell Biology; Cell Line; Chloride Channels - metabolism; Chlorides - metabolism; HEK293 Cells; Human Physiology; Humans; Ion Channel Gating - physiology; Ion Channels; Kinetics; Mice; Molecular Medicine; Muscle Contraction - physiology; Myocytes, Smooth Muscle - metabolism; Neoplasm Proteins - metabolism; Neurosciences; Receptors; Receptors and Transporters; Chloride channel; Patch clamp; Mathematical modelling; Kinetics; Gating; Permeation
• ISSN: 0031-6768; 1432-2013
• DOI: 10.1007/s00424-016-1830-9

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.