Voltage-Dependent Protonation of the Calcium Pocket Enable Activation of the Calcium-Activated Chloride Channel Anoctamin-1 (TMEM16A)

Sci Rep. 2020 Apr 20;10(1):6644. doi: 10.1038/s41598-020-62860-9.


Anoctamin-1 (ANO1 or TMEM16A) is a homo-dimeric Ca2+-activated Cl- channel responsible for essential physiological processes. Each monomer harbours a pore and a Ca2+-binding pocket; the voltage-dependent binding of two intracellular Ca2+ ions to the pocket gates the pore. However, in the absence of intracellular Ca2+ voltage activates TMEM16A by an unknown mechanism. Here we show voltage-activated anion currents that are outwardly rectifying, time-independent with fast or absent tail currents that are inhibited by tannic and anthracene-9-carboxylic acids. Since intracellular protons compete with Ca2+ for binding sites in the pocket, we hypothesized that voltage-dependent titration of these sites would induce gating. Indeed intracellular acidification enabled activation of TMEM16A by voltage-dependent protonation, which enhanced the open probability of the channel. Mutating Glu/Asp residues in the Ca2+-binding pocket to glutamine (to resemble a permanent protonated Glu) yielded channels that were easier to activate at physiological pH. Notably, the response of these mutants to intracellular acidification was diminished and became voltage-independent. Thus, voltage-dependent protonation of glutamate/aspartate residues (Glu/Asp) located in the Ca2+-binding pocket underlines TMEM16A activation in the absence of intracellular Ca2+.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Action Potentials / drug effects
  • Action Potentials / physiology
  • Animals
  • Anoctamin-1 / antagonists & inhibitors
  • Anoctamin-1 / genetics
  • Anoctamin-1 / metabolism*
  • Anthracenes / pharmacology
  • Calcium / metabolism*
  • Cations, Divalent
  • Chlorides / metabolism*
  • Genes, Reporter
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • HEK293 Cells
  • Humans
  • Ion Channel Gating / drug effects
  • Ion Transport / drug effects
  • Mice
  • Mutation
  • Patch-Clamp Techniques
  • Plasmids / chemistry
  • Plasmids / metabolism
  • Protons
  • Recombinant Fusion Proteins / genetics*
  • Recombinant Fusion Proteins / metabolism
  • Structure-Activity Relationship
  • Tannins / pharmacology
  • Transfection


  • ANO1 protein, mouse
  • Anoctamin-1
  • Anthracenes
  • Cations, Divalent
  • Chlorides
  • Protons
  • Recombinant Fusion Proteins
  • Tannins
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • 9-anthroic acid
  • Calcium