title: Control of TMEM16A by INO-4995 and other inositolphosphates.
abstract: OBJECTIVE
  Ca(2+)-dependent Cl(-) secretion (CaCC) in airways and other tissues is due to activation of the Cl(-) channel TMEM16A (anoctamin 1). Earlier studies suggested that Ca(2+) -activated Cl(-) channels are regulated by membrane lipid inositol phosphates, and that 1-O-octyl-2-O-butyryl-myo-inositol 3,4,5,6-tetrakisphosphate octakis(propionoxymethyl) ester (INO-4995) augments CaCC. Here we examined whether TMEM16A is the target for INO-4995 and if the channel is regulated by inositol phosphates.
  
  
  METHODS
  The effects of INO-4995 on CaCC were examined in overexpressing HEK293, colonic and primary airway epithelial cells as well as Xenopus oocytes. We used patch clamping, double electrode voltage clamp and Ussing chamber techniques.
  
  
  RESULTS
  We found that INO-4995 directly activates a TMEM16A whole cell conductance of 6.1 ± 0.9 nS pF(-1) in overexpressing cells. The tetrakisphosphates Ins(3,4,5,6)P(4) or Ins(1,3,4,5)P(4) and enzymes controlling levels of InsP(4) or PIP(2) and PIP(3) had no effects on the magnitude or kinetics of TMEM16A currents. In contrast in Xenopus oocytes, human airways and colonic cells, which all express TMEM16A endogenously, Cl(-) currents were not acutely activated by INO-4995. However incubation with INO-4995 augmented 1.6- to 4-fold TMEM16A-dependent Cl(-) currents activated by ionomycin or ATP, while intracellular Ca(2+) signals were not affected. The potentiating effect of INO-4995 on transient ATP-activated TMEM16A-currents in cystic fibrosis (CF) airways was twice of that observed in non-CF airways.
  
  
  CONCLUSIONS
  These data indicate that TMEM16A is the target for INO-4995, although the mode of action appears different for overexpressed and endogenous channels. INO-4995 may be useful for the treatment of CF lung disease.
authors.affiliation: Institut für Physiologie, Universität Regensburg, Germany.
authors.forename: Yuemin
authors.initials: Y
authors.lastname: Tian
authors.forename: Rainer
authors.initials: R
authors.lastname: Schreiber
authors.forename: Podchanart
authors.initials: P
authors.lastname: Wanitchakool
authors.forename: Patthara
authors.initials: P
authors.lastname: Kongsuphol
authors.forename: Marisa
authors.initials: M
authors.lastname: Sousa
authors.forename: Inna
authors.initials: I
authors.lastname: Uliyakina
authors.forename: Marta
authors.initials: M
authors.lastname: Palma
authors.forename: Diana
authors.initials: D
authors.lastname: Faria
authors.forename: Alexis E
authors.initials: AE
authors.lastname: Traynor-Kaplan
authors.forename: José I
authors.initials: JI
authors.lastname: Fragata
authors.forename: Margarida D
authors.initials: MD
authors.lastname: Amaral
authors.forename: Karl
authors.initials: K
authors.lastname: Kunzelmann
mesh_terms: D000818:Animals; D000075369:Anoctamin-1; D001980:Bronchi / Q000166:cytology; D002478:Cells, Cultured; D018118:Chloride Channels / Q000187:drug effects* / Q000378:metabolism*; D003550:Cystic Fibrosis; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D057809:HEK293 Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism / Q000494:pharmacology*; D015759:Ionomycin / Q000494:pharmacology; D009363:Neoplasm Proteins / Q000187:drug effects* / Q000378:metabolism; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D018408:Patch-Clamp Techniques; D014981:Xenopus
chemical_list: C476213:ANO1 protein, human; D000075369:Anoctamin-1; D018118:Chloride Channels; C503977:INO-4995; D007295:Inositol Phosphates; D009363:Neoplasm Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D015759:Ionomycin