title: Homodimeric anoctamin-1, but not homodimeric anoctamin-6, is activated by calcium increases mediated by the P2Y1 and P2X7 receptors.
abstract: The P2X7 receptor (P2X7R) is a ligand-gated ion channel that conducts Na(+), K(+), and Ca(2+) when activated by extracellular ATP. In various cell types, such as secretory epithelia, the P2X7R is co-expressed with Ca(2+)-dependent Cl(-) channels of the TMEM16/anoctamin family. Here, we studied whether the P2X7R and TMEM16A/anoctamin-1 (Ano1) or TMEM16F/anoctamin-6 (Ano6) interact functionally and physically, using oocytes of Xenopus laevis and Ambystoma mexicanum (Axolotl) for heterologous expression. As a control, we co-expressed anoctamin-1 with the P2Y1 receptor (P2Y1R), which induces the release of Ca(2+) from intracellular stores via activating phospholipase C through coupling to Gαq. We found that co-expression of anoctamin-1 with the P2Y1R resulted in a small transient increase in Cl(-) conductance in response to ATP. Co-expression of anoctamin-1 with the P2X7R resulted in a large sustained increase in Cl(-) conductance via Ca(2+) influx through the ATP-opened P2X7R in Xenopus and in Axolotl oocytes, which lack endogenous Ca(2+)-dependent Cl(-) channels. P2Y1R- or P2X7R-mediated stimulation of Ano1 was primarily functional, as demonstrated by the absence of a physically stable interaction between Ano1 and the P2X7R. In the pancreatic cell line AsPC-1, we found the same functional Ca(2+)-dependent interaction of P2X7R and Ano1. The P2X7R-mediated sustained activation of Ano1 may be physiologically relevant to the time course of stimulus-secretion coupling in secretory epithelia. No such increase in Cl(-) conductance could be elicited by activating the P2X7 receptor in either Xenopus oocytes or Axolotl oocytes co-expressing Ano6. The lack of function of Ano6 can, at least in part, be explained by its poor cell-surface expression, resulting from a relatively inefficient exit of the homodimeric Ano6 from the endoplasmic reticulum.
authors.affiliation: Molecular Pharmacology, RWTH Aachen University, Wendlingweg 2, D-52074, Aachen, Germany.
authors.forename: Michaela
authors.initials: M
authors.lastname: Stolz
authors.affiliation: Julius-Bernstein-Institute for Physiology, Martin-Luther-University Halle-Wittenberg, Magdeburger Str. 6, D-06097, Halle/Saale, Germany.
authors.forename: Manuela
authors.initials: M
authors.lastname: Klapperstück
authors.affiliation: Julius-Bernstein-Institute for Physiology, Martin-Luther-University Halle-Wittenberg, Magdeburger Str. 6, D-06097, Halle/Saale, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Kendzierski
authors.affiliation: Molecular Pharmacology, RWTH Aachen University, Wendlingweg 2, D-52074, Aachen, Germany.
authors.forename: Silvia
authors.initials: S
authors.lastname: Detro-Dassen
authors.affiliation: Molecular Pharmacology, RWTH Aachen University, Wendlingweg 2, D-52074, Aachen, Germany.
authors.forename: Anna
authors.initials: A
authors.lastname: Panning
authors.affiliation: Molecular Pharmacology, RWTH Aachen University, Wendlingweg 2, D-52074, Aachen, Germany.
authors.forename: Günther
authors.initials: G
authors.lastname: Schmalzing
authors.affiliation: Julius-Bernstein-Institute for Physiology, Martin-Luther-University Halle-Wittenberg, Magdeburger Str. 6, D-06097, Halle/Saale, Germany. fritz.markwardt@medizin.uni-halle.de.
authors.forename: Fritz
authors.initials: F
authors.lastname: Markwardt
mesh_terms: D000200:Action Potentials; D000558:Ambystoma mexicanum; D000818:Animals; D000075369:Anoctamin-1; D000075368:Anoctamins; D020013:Calcium Signaling; D045744:Cell Line, Tumor; D018118:Chloride Channels / Q000378:metabolism*; D006801:Humans; D051379:Mice; D049294:Phospholipid Transfer Proteins / Q000378:metabolism*; D058486:Receptors, Purinergic P2X7 / Q000378:metabolism*; D058472:Receptors, Purinergic P2Y1 / Q000378:metabolism*; D014981:Xenopus
chemical_list: C531181:ANO1 protein, mouse; C576687:ANO6 protein, mouse; D000075369:Anoctamin-1; D000075368:Anoctamins; D018118:Chloride Channels; D049294:Phospholipid Transfer Proteins; D058486:Receptors, Purinergic P2X7; D058472:Receptors, Purinergic P2Y1
keywords: Anoctamin-1; Anoctamin-6; Intracellular Ca2+; P2X7 receptor; Quaternary structure