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

• Type of Material: Article
• Authors: Juan A. Contreras-Vite, Universidad Autónoma de San Luis Potosí; Silvia Cruz-Rangel, Universidad Autónoma de San Luis Potosí; José J. De Jesus-Perez, Universidad Autónoma de San Luis Potosí; Iván A. Arechiga Figueroa, Universidad Autónoma de San Luis Potosí; Aldo A. Rodriguez-Menchaca, Universidad Autónoma de San Luis Potosí; Patricia Perez-Cornejo, Universidad Autónoma de San Luis Potosí; Harrison Hartzell Jr., Emory University; Jorge Arreola, Universidad Autónoma de San Luis Potosí
• Language: English
• Date: 2016-07-01
• Publisher: Springer Verlag
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2016, Springer-Verlag Berlin Heidelberg.
• Final Published Version (URL): http://dx.doi.org/10.1007/s00424-016-1830-9
• Title of Journal or Parent Work: Pflügers Archiv European Journal of Physiology
• Volume: 468
• Issue: 7
• Start Page: 1241
• End Page: 1257
• Grant/Funding Information: This work was supported by grants 219949 (CONACyT, Mexico) to JA. HCH is supported by grants from the National Institutes of Health GM60448, AR067786, and EY11482. JAC-V and JJ De J-P are recipients of a Graduate Student Fellowship from CONACyT, Mexico (229968 and 234820).; SCR is a recipient of a Postdoctoral Fellowship from CONACyT, Mexico (256034-290807).
• Keywords: INACTIVATION; Gating; VOLTAGE-DEPENDENCE; GATE; Life Sciences & Biomedicine; Chloride channel; Science & Technology; Kinetics; Physiology; XENOPUS OOCYTES; EPITHELIAL-CELLS; GLAND ACINAR-CELLS; CA2+-ACTIVATED CL-CHANNEL; Mathematical modelling; SINGLE-CHANNEL; Permeation; CALCIUM; Patch clamp; ANION PERMEATION
• Research Categories: Biology, Physiology; Biology, Cell

Abstract

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.