Analysis of inhibitors of the anoctamin‐1 chloride channel (transmembrane member 16A, TMEM16A) reveals indirect mechanisms involving alterations in calcium signalling

Background and Purpose Pharmacological inhibitors of TMEM16A (ANO1), a Ca 2+ ‐activated Cl − channel, are important tools of research and possible therapeutic agents acting on smooth muscle, airway epithelia and cancer cells. We tested a panel of TMEM16A inhibitors, including CaCC inh ‐A01, niclosamide, MONNA, Ani9 and niflumic acid, to evaluate their possible effect on intracellular Ca 2+ . Experimental Approach We recorded cytosolic Ca 2+ increase elicited with UTP, ionomycin or IP 3 uncaging. Key Results Unexpectedly, we found that all compounds, except for Ani9, markedly decreased intracellular Ca 2+ elevation induced by stimuli acting on intracellular Ca 2+ stores. These effects were similarly observed in cells with and without TMEM16A expression. We investigated in more detail the mechanism of action of niclosamide and CaCC inh ‐A01. Acute addition of niclosamide directly increased intracellular Ca 2+ , an activity consistent with inhibition of the SERCA pump. In contrast to niclosamide, CaCC inh ‐A01 did not elevate intracellular Ca 2+ , thus implying a different mechanism of action, possibly a block of inositol triphosphate receptors. Conclusions and Implications Most TMEM16A inhibitors are endowed with indirect effects mediated by alteration of intracellular Ca 2+ handling, which may in part preclude their use as TMEM16A research tools.