Control of TMEM16A by INO ‐4995 and other inositolphosphates

Background And Purpose Ca 2+ ‐dependent C l − secretion ( CaCC ) in airways and other tissues is due to activation of the C l − channel TMEM16A (anoctamin 1). Earlier studies suggested that C a 2+ ‐activated C l − channels are regulated by membrane lipid inositol phosphates, and that 1‐ O ‐octyl‐2‐ O ‐butyryl‐myo‐inositol 3,4,5,6‐tetrakisphosphate octakis(propionoxymethyl) ester ( INO ‐4995) augments CaCC . Here we examined whether TMEM16A is the target for INO ‐4995 and if the channel is regulated by inositol phosphates. Experimental Approach The effects of INO ‐4995 on CaCC were examined in overexpressing HEK 293, colonic and primary airway epithelial cells as well as X enopus oocytes. We used patch clamping, double electrode voltage clamp and U ssing chamber techniques. Key Results We found that INO ‐4995 directly activates a TMEM16A whole cell conductance of 6.1 ± 0.9  nS pF –1 in overexpressing cells. The tetrakisphosphates I ns(3,4,5,6) P 4 or I ns(1,3,4,5) P 4 and enzymes controlling levels of I ns P 4 or PIP 2 and PIP 3 had no effects on the magnitude or kinetics of TMEM16A currents. In contrast in X enopus oocytes, human airways and colonic cells, which all express TMEM16A endogenously, C l − currents were not acutely activated by INO ‐4995. However incubation with INO ‐4995 augmented 1.6‐ to 4‐fold TMEM16A ‐dependent C l − currents activated by ionomycin or ATP , while intracellular C a 2+ signals were not affected. The potentiating effect of INO ‐4995 on transient ATP ‐activated TMEM16A ‐currents in cystic fibrosis ( CF) airways was twice of that observed in non‐ CF airways. Conclusions And Implications These data indicate that TMEM16A is the target for INO ‐4995, although the mode of action appears different for overexpressed and endogenous channels. INO ‐4995 may be useful for the treatment of CF lung disease.