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 Ca2+ -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 CaCCinh -A01, niclosamide, MONNA, Ani9 and niflumic acid, to evaluate their possible effect on intracellular Ca2+ . Experimental approach: We recorded cytosolic Ca2+ increase elicited with UTP, ionomycin or IP3 uncaging. Key results: Unexpectedly, we found that all compounds, except for Ani9, markedly decreased intracellular Ca2+ elevation induced by stimuli acting on intracellular Ca2+ 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 CaCCinh -A01. Acute addition of niclosamide directly increased intracellular Ca2+ , an activity consistent with inhibition of the SERCA pump. In contrast to niclosamide, CaCCinh -A01 did not elevate intracellular Ca2+ , 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 Ca2+ handling, which may in part preclude their use as TMEM16A research tools.