title: Extracellular protons enable activation of the calcium-dependent chloride channel TMEM16A.
abstract: The calcium-activated chloride channel TMEM16A provides a pathway for chloride ion movements that are key in preventing polyspermy, allowing fluid secretion, controlling blood pressure, and enabling gastrointestinal activity. TMEM16A is opened by voltage-dependent calcium binding and regulated by permeant anions and intracellular protons. Here we show that a low proton concentration reduces TMEM16A activity while maximum activation is obtained when the external proton concentration is high. In addition, protonation conditions determine the open probability of TMEM16A without changing its calcium sensitivity. External glutamic acid 623 (E623) is key for TMEM16A's ability to respond to external protons. At physiological pH, E623 is un-protonated and TMEM16A is activated when intracellular calcium increases; however, under acidic conditions E623 is partially protonated and works synergistically with intracellular calcium to activate the channel. These findings are critical for understanding physiological and pathological processes that involve changes in pH and chloride flux via TMEM16A.
  
  
  
  Transmembrane protein 16A (TMEM16A), also known as ANO1, the pore-forming subunit of a Ca2+-dependent Cl-channel (CaCC), is activated by direct, voltage-dependent, binding of intracellular Ca2+. Endogenous CaCCs are regulated by extracellular protons; however, the molecular basis of such regulation remains unidentified. Here, we evaluated the effects of different extracellular proton concentrations ([H+]o) on mouse TMEM16A expressed in HEK-293 cells using whole-cell and inside-out patch-clamp recordings. We found that increasing the [H+]ofrom 10-10to 10-5.5m caused a progressive increase in the chloride current (ICl) that is described by titration of a protonatable site with pK = 7.3. Protons regulate TMEM16A in a voltage-independent manner, regardless of channel state (open or closed), and without altering its apparent Ca2+sensitivity. Noise analysis showed that protons regulate TMEM16A by tuning its open probability without modifying the single channel current. We found a robust reduction of the proton effect at high [Ca2+]i. To identify protonation targets we mutated all extracellular glutamate and histidine residues and 4 of 11 aspartates. Most mutants were sensitive to protons. However, mutation that substituted glutamic acid (E) for glutamine (Q) at amino acid position 623 (E623Q) displayed a titration curve shifted to the left relative to wild type channels and the IClwas nearly insensitive to proton concentrations between 10-5.5and 10-9.0m. Additionally, IClof the mutant containing an aspartic acid (D) to asparagine (N) substitution at position 405 (D405N) mutant was partially inhibited by a proton concentration of 10-5.5m, but 10-9.0m produced the same effect as in wild type. Based on our findings we propose that external protons titrate glutamic acid 623, which enables voltage activation of TMEM16A at non-saturating [Ca2+]i.
authors.affiliation: Physics Institute, Universidad Autónoma de San Luis Potosí, Ave. Dr. Manuel Nava #6, 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, 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
authors.initials: IA
authors.lastname: Aréchiga-Figueroa
authors.affiliation: Department of Physiology and Biophysics, 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 and Biophysics, 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, San Luis Potosí, SLP, 78290, México.
authors.forename: Jorge
authors.identifier: 0000-0002-5354-3709
authors.initials: J
authors.lastname: Arreola
mesh_terms: D000075369:Anoctamin-1; D002118:Calcium / Q000502:physiology; D018118:Chloride Channels / Q000235:genetics / Q000502:physiology*; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D011522:Protons
chemical_list: C531181:ANO1 protein, mouse; D000075369:Anoctamin-1; D018118:Chloride Channels; D011522:Protons; D002118:Calcium
keywords: TMEM16A; calcium sensitivity; channel regulation; mutation; open probability; patch clamp; protons