Relationship between TMEM16A/anoctamin 1 and LRRC8A.

TMEM16A/anoctamin 1/ANO1 and VRAC/LRRC8 are independent chloride channels activated either by increase in intracellular Ca(2+) or cell swelling, respectively. In previous studies, we observed overlapping properties for both types of channels. (i) TMEM16A/ANO1 and LRRC8 are inhibited by identical compounds, (ii) the volume-regulated anion channel VRAC requires compartmentalized Ca(2+) increase to be fully activated, (iii) anoctamins are activated by cell swelling, (iv) both channels have a role for apoptotic cell death, (v) both channels are possibly located in lipid rafts/caveolae like structures, and (vi) VRAC and anoctamin 1 currents are not additive when each are fully activated. In the present study, we demonstrate in different cell types that loss of LRRC8A expression not only inhibited VRAC, but also attenuated Ca(2+) activated Cl(-) currents. Moreover, expression of LRRC8A enhanced Ca(2+) activated Cl(-) currents, and both LRRC8A and ANO1 could be coimmunoprecipitated. We found that LRRC8A becomes accessible to biotinylation upon exposure to hypotonic bath solution, while membrane capacitance was not enhanced. When intracellular Ca(2+) was increased in ANO1-expressing cells, the membrane capacitance was enhanced and increased binding of FM4-64 to the membrane was observed. As this was not seen in cells lacking ANO1 expression, a role of ANO1 for exocytosis was suggested. We propose that ANO1 and LRRC8A are activated in parallel. Thus, ionomycin or purinergic stimulation will not only activate ANO1 but also LRRC8 currents. Cell swelling will not only activate LRRC8/VRAC, but also stimulate ANO1 currents by enhancing compartmentalized Ca(2+) increase and/or through swelling induced autocrine release of ATP.