title: Anoctamin2 (TMEM16B) forms the Ca2+-activated Cl-channel in the retinal pigment epithelium.
abstract: Chloride channels (Cl channels) play an essential role for the retinal pigment epithelium (RPE). They provide a plasma membrane conductance for Cl-important for transepithelial transport and volume regulation. Ca2+-dependent chloride channels (CaCC) in the RPE were found to adapt Cl-transport to specific needs by increasing intracellular free Ca2+. Although a variety of Cl channels have been identified in the RPE, the molecular identity of the CaCC remains controversial. Sagittal sections of mouse retina were stained against anoctamin2 (Ano2) and analyzed by confocal microscopy. Membrane currents from ARPE-19 cells and primary murine RPE cells were recorded in the whole-cell configuration of the patch-clamp technique. Expression of Ano2 was assessed via immunocytochemistry, PCR and western-blot and down-regulated via siRNA approaches. In the mouse retina, Ano2 was found in the basolateral membrane of the RPE. In primary mouse RPE cells, Ano2 was localized predominantly in the cell membrane. Ano2 mRNA and protein were also detected in rat and primate RPE as well as ARPE-19 cells. Whole-cell currents were elicited by increasing intracellular free Ca2+via ATP application. These currents were identified as Cl-currents by their reversal potential and blocker sensitivity. Knock-down of Ano2 by siRNA decreased both the Ca2+dependent chloride conductance and protein expression of Ano2. The biophysical and pharmacological properties of CaCC in ARPE-19 and primary mouse RPE cells resemble those described in previous publications using RPE cells from different species. The siRNA knock-down suggests that Ano2 contributes to Ca2+-dependent chloride conductance in the RPE.
authors.affiliation: Experimental Ophthalmology, Department of Ophthalmology, Charité University Medicine Berlin, Berlin, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Keckeis
authors.affiliation: Experimental Ophthalmology, Department of Ophthalmology, Charité University Medicine Berlin, Berlin, Germany.
authors.forename: Nadine
authors.initials: N
authors.lastname: Reichhart
authors.affiliation: Experimental Ophthalmology, Department of Ophthalmology, Charité University Medicine Berlin, Berlin, Germany.
authors.forename: Christophe
authors.initials: C
authors.lastname: Roubeix
authors.affiliation: Experimental Ophthalmology, Department of Ophthalmology, Charité University Medicine Berlin, Berlin, Germany. Electronic address: olaf.strauss@charite.de.
authors.forename: Olaf
authors.initials: O
authors.lastname: Strauß
mesh_terms: D000818:Animals; D000075368:Anoctamins; D001692:Biological Transport; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D018118:Chloride Channels / Q000096:biosynthesis / Q000235:genetics*; D005786:Gene Expression Regulation; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D018408:Patch-Clamp Techniques; D034741:RNA, Small Interfering / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D055213:Retinal Pigment Epithelium / Q000166:cytology* / Q000378:metabolism*
chemical_list: C532161:ANO2 protein, mouse; D000075368:Anoctamins; D018118:Chloride Channels; D034741:RNA, Small Interfering; D002118:Calcium
keywords: ATP; Ano2; Ca(2+)-dependent chloride channels; Chloride; RPE; anoctamin2