Regulatory Roles of Anoctamin-6 in Human Trabecular Meshwork Cells

PURPOSE: Trabecular meshwork (TM) cell volume is a determinant of aqueous humor outflow resistance, and thereby IOP. Regulation of TM cell volume depends on chloride ion (Cl(−)) release through swelling-activated channels (I(Cl,Swell)), whose pore is formed by LRRC8 proteins. Chloride ion release through swelling-activated channels has been reported to be regulated by calcium-activated anoctamins, but this finding is controversial. Particularly uncertain has been the effect of anoctamin Ano6, reported as a Ca(2+)-activated Cl(−) (CaCC) or cation channel in other cells. The current study tested whether anoctamin activity modifies volume regulation of primary TM cell cultures and cell lines. METHODS: Gene expression was studied with quantitative PCR, supplemented by reverse-transcriptase PCR and Western immunoblots. Currents were measured by ruptured whole-cell patch clamping and volume by electronic cell sizing. RESULTS: Primary TM cell cultures and the TM5 and GTM3 cell lines expressed Ano6 3 to 4 orders of magnitude higher than the other anoctamin CaCCs (Ano1 and Ano2). Ionomycin increased cell Ca(2+) and activated macroscopic currents conforming to CaCCs in other cells, but displayed significantly more positive mean reversal potentials (+5 to +12 mV) than those displayed by I(Cl,Swell) (−14 to −21 mV) in the same cells. Nonselective CaCC inhibitors (tannic acid>CaCC(inh−)A01) and transient Ano6 knockdown strongly inhibited ionomycin-activated currents, I(Cl,Swell) and the regulatory volume response to hyposmotic swelling. CONCLUSIONS: Ionomycin activates CaCCs associated with net cation movement in TM cells. These currents, I(Cl,Swell), and cell volume are regulated by Ano6. The findings suggest a novel clinically-relevant approach for altering cell volume, and thereby outflow resistance, by targeting Ano6.