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Olaf Strauss, Simon Schoeberl, Vladimir Milenkovic, Susanne Keckeis, Raphael Lange, Nadine Reichart; Anoctamin-4 forms a Ca2+-dependent non-selective cation channel in the retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2349.
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© ARVO (1962-2015); The Authors (2016-present)
Anoctamins (TMEM16) represent a family of transmembranal proteins functioning as both Ca2+-dependent ion channels and scramblases. Whereas the anoctamins ANO1 and ANO2 have been identified as Ca2+-dependent Cl channels the ion channel function of other anoctamins is not clear. The retinal pigment (RPE) is known to express Ca2+-dependent non-selective cation channels and ANO4. Purpose of the study is to clarify the molecular identity of this channel, likely as ANO4.
Heterologeous expression of ANO4 in HEK293 cells and analysis of membrane currents by patch-clamp whole-cell recordings. Modulation of ANO4 structure by side-directed mutagenesis. siRNA knock-down of ANO4 in ARPE-19cells. Immunohistochemistry to show ANO4 expression in the mouse retina and in cultured cells.<br />
HEK293 cells expressing ANO4 show Ca2+-dependent non-selective cation currents which were sensitive to niflumic acid and were showing a weak Eisenmann V selectivity for monovalent cations. Using sequence homology analysis we searched for conserved negatively charged amino acids in ANO4 which are positively charged amino acids in the confirmed Cl channels ANO1 and ANO2. We identified in the assumed pore region a cluster of positively charged amino acids in ANO1/ANO2 where at the same position ANO4 carries a negatively charged amino acid (E775) but no other charged amino acids. In order to demonstrate that the membrane currents in transfected cells result from ion channel pore activity of ANO4 we generated two mutants: in one variant amino acid E775 was changed into a non-charged one (E775G) and in s second variant into a positively charged one (E775R). The variant E775G did not lead to increased membrane conductance and the E775R variant lead to Ca2+-dependent Cl channel currents indicating that E775 is the major determinant for ion selectivity. In the mouse retina we found ANO4 heterologeously expressed in the RPE as well as in ARPE-19 cells. ARPE-19 cells show the activity of a Ca2+-dependent non-selective cation channel which was reduced by siRNA knock-down against ANO4.<br />
Using heterologeous expression and side-directed mutagenesis we identified ANO4 as a bonafide Ca2+-dependent non-selective cation channel. ANO4 is also responsible to generate the Ca2+-dependent non-selective cation channel currents in the RPE.<br />
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