title: Revealing the activation pathway for TMEM16A chloride channels from macroscopic currents and kinetic models.
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) ICl 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 ICl activation displays fast and slow kinetics. To explain the contribution of Vm, Ca(2+) and Cl(-) to gating, we developed a 12-state Markov chain model. This model explains TMEM16A activation as a sequential, direct, and Vm-dependent binding of two Ca(2+) ions coupled to a Vm-dependent binding of an external Cl(-) ion, with Vm-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 Vm dependence of activation.
authors.affiliation: Physics Institute, Universidad Autónoma de San Luis Potosí, Ave. Dr. Manuel Nava #6, Zona Universitaria, San Luis Potosí, SLP 78290, México.
authors.forename: Juan A
authors.initials: JA
authors.lastname: Contreras-Vite
authors.affiliation: Physics Institute, Universidad Autónoma de San Luis Potosí, Ave. Dr. Manuel Nava #6, Zona Universitaria, San Luis Potosí, SLP 78290, México.
authors.forename: Silvia
authors.initials: S
authors.lastname: Cruz-Rangel
authors.affiliation: Physics Institute, Universidad Autónoma de San Luis Potosí, Ave. Dr. Manuel Nava #6, Zona Universitaria, San Luis Potosí, SLP 78290, México.
authors.forename: José J
authors.initials: JJ
authors.lastname: De Jesús-Pérez
authors.affiliation: CONACYT - Universidad Autónoma de San Luis Potosí School of Medicine, Ave. V. Carranza 2405, San Luis Potosí, SLP 78290, México.
authors.forename: Iván A Aréchiga
authors.initials: IAA
authors.lastname: Figueroa
authors.affiliation: Department of Physiology, Universidad Autónoma de San Luis Potosí School of Medicine, Ave. V. Carranza 2405, San Luis Potosí, SLP 78290, México.
authors.forename: Aldo A
authors.initials: AA
authors.lastname: Rodríguez-Menchaca
authors.affiliation: Department of Physiology, Universidad Autónoma de San Luis Potosí School of Medicine, Ave. V. Carranza 2405, San Luis Potosí, SLP 78290, México.
authors.forename: Patricia
authors.initials: P
authors.lastname: Pérez-Cornejo
authors.affiliation: Department of Cell Biology, Emory University School of Medicine, Atlanta, GA, 30322, USA.
authors.forename: H Criss
authors.initials: HC
authors.lastname: Hartzell
authors.affiliation: Physics Institute, Universidad Autónoma de San Luis Potosí, Ave. Dr. Manuel Nava #6, Zona Universitaria, San Luis Potosí, SLP 78290, México. arreola@dec1.ifisica.uaslp.mx.
authors.forename: Jorge
authors.initials: J
authors.lastname: Arreola
mesh_terms: D000818:Animals; D000838:Anions / Q000378:metabolism; D000075369:Anoctamin-1; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D018118:Chloride Channels / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology; D007700:Kinetics; D051379:Mice; D009119:Muscle Contraction / Q000502:physiology; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism*
chemical_list: C476213:ANO1 protein, human; D000838:Anions; D000075369:Anoctamin-1; D018118:Chloride Channels; D002712:Chlorides; D009363:Neoplasm Proteins; D002118:Calcium
keywords: Chloride channel; Gating; Kinetics; Mathematical modelling; Patch clamp; Permeation