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Juni Banerjee, Ang Li, Chi Ting Leung, Kim Peterson-Yantorno, W Daniel Stamer, Mortimer Civan; Regulatory Roles of Anoctamin-6 in Human Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1981.
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© ARVO (1962-2015); The Authors (2016-present)
Cell volume of trabecular meshwork (TM) cells is linked with aqueous humor outflow resistance. TM-cell volume regulation depends on swelling-activated Cl- channels (ICl,swell) of unknown molecular identity. ICl,swell in some cells has been reported modulated by anoctamins. Most tissues express anoctamins Ano1-2, which are Ca2+-activated Cl- channels (CaCCs), and Ano6, which is a scramblase, but the full functions and sites of this protein family are uncertain. We are testing if anoctamins regulate TM cell volume regulation.
Explant-derived human TM (HTM) cells, transformed normal human TM5 and glaucomatous GM3 TM cells and HEK293 cells were studied. Gene expression was measured by reverse-transcription PCR (RT-PCR) and real-time PCR (qPCR), protein product by western blots, membrane currrents by whole-cell ruptured-patch recording, cell volume by electronic cell sizing and Ca2+ concentration by fura-2.
All cells strongly expressed Ano6. Ano1 was present in HEK293 cells, very much less in HTM cells and undetected in GM3 and TM cells (N=3). Expression of Ano1 by qPCR was much lower (<0.1%) than that of Ano6 in HTM cells. RT-PCR detected Ano2 only in GM3 and HEK293 cells but not in HTM and TM5 cells (N=3). Increasing intracellular Ca2+ with 5 μM ionomycin strongly activated CaCCs in HTM (N=10), TM5 (N=3) and GM3 (N=5) cells. The nonspecific CaCC inhibitor tannic acid (TA, 100 μM) blocked these currents by ca. 80% in all TM cells (N=3 each). HTM CaCC was also reduced (52±6% inhibition, N=4) by the nonselective CaCC inhibitor, CaCCinh-AO1 (50 μM) but not by the selective inhibitor of Ano1-2 channels, T16Ainh-A01 (20 μM), (N=3). ICl,swell and the regulatory volume decrease (RVD) following hypotonic swelling were also inhibited by non-selective CaCC blockers (TA > CaCCinh-AO1) and not by the selective T16Ainh-A01. SiRNA knockdown (80%) of Ano6 inhibited CaCCs of TM5 cells (N=3) and the RVD of HTM (N=3) and TM5 (N=4) cells.
Normal human TM cells display undetectable/minor expression of Ano1-2 documented to underlie CaCCs. Nevertheless, non-selective CaCC blockers inhibit CaCCs, ICl,swell and the RVD of TM cells and knockdown of highly expressed Ano6 inhibits CaCCs and the RVD of TM cells. We suggest that Ano6 may regulate both CaCC and ICl,swell through its scramblase activity, modulating signal transduction.
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