Pharmacological properties of native CaCCs and TMEM16A

Ca²⁺-activated Cl⁻ channels (CaCCs) are voltage-modulated small conductance (∼1-3 pS) chloride channels activated by a rise in intracellular Ca²⁺ concentration ([Ca²⁺]_i) above a threshold of ∼150 nM. CaCCs are expressed in many cell types where they were proposed to exert very specific functions that include cell volume regulation, transepithelial fluid movement, membrane excitability and smooth muscle contraction.¹ In 2008, 3 independent groups of investigators showed that 2 members of the TMEM16 gene family, TMEM16A and B, or Anoctamin1 and 2 (ANO1, ANO2), encoded for CaCCs with biophysical properties similar to those of CaCCs recorded in various cell types.^2–4 Prior to this discovery, assessment of the physiological role of CaCCs in various tissues had almost exclusively relied on the use of pharmacological agents lacking potency and specificity. Similar to native CaCCs,¹ early studies confirmed that CaCCs generated by the expression of ANO1 in mammalian cell lines and amphibian oocytes could be blocked by classical CaCC blockers such as niflumic acid (NFA),^2,4 4,40-diisothiocyanato-stilbene-2,20-disulfonic acid (DIDS),^3,4 5-nitro-2-(3-phenylpropylalanine) benzoate (NPPB),^3,4 and diphenylamine-2-carboxyl acid (DPC).³ Nearly all studies on native and expressed ANO1 up to this date used the Cl⁻ channel blockers mentioned above and others to confirm, along with gene knockdown strategies, the role and function of ANO1 in the tissue of interest. However, no single study focused on the mechanism of action of these inhibitors on expressed ANO1.

In the current issue of Channels, Bradley et al.⁵ filled that gap by providing a comprehensive pharmacological analysis of the effects of 2 classical compounds, NFA and anthracene-9-carboxylic acid (A9C), and 2 of the newer generation of more potent and selective inhibitors developed by high throughput screening of compound libraries,⁶ T16A_Inh-A01 and CaCC_Inh-A01, on ANO1 channels expressed in HEK-293 cells. The choice of NFA and A9C was judicious as the former was considered the most potent and widely used 1^st generation CaCC blocker, and both were shown to produce paradoxical inhibitory and stimulatory effects on native CaCCs. Similar to its effects on native CaCCs, NFA blocked ANO1-induced Ca²⁺-activated Cl⁻ currents (I_ClCa) in a voltage-dependent manner consistent with an open state blocker at positive membrane potentials. Deactivating inward I_ClCa tail currents decayed with a much slower time course in the presence of NFA, a typical effect seen in vascular myocytes⁷ and interpreted to reflect rapid opening and closure of the channel at negative potentials. Enhancement of the inward tail current was also observed for low concentrations of NFA (1 and 3 μM) although a persistent inward I_ClCa was not detected at negative potentials as observed in vascular myocytes.⁷ A9C was shown to cause strong steady-state voltage-dependent block of I_ClCa, and enhancement of the inward I_ClCa tail current in pulmonary artery myocytes,⁸ which were also detected for ANO1-induced CaCCs.⁵ Although these data do not unequivocally prove that the effects of NFA and A9C are produced by an interaction with the pore-forming ANO1 subunit, the nearly complete recapitulation of their complex anomalous effects on ANO1-induced I_ClCa in a mammalian cell line strongly argues in favor of this hypothesis. The lack of voltage-dependence of block by T16A_Inh-A01 had already been reported for expressed ANO1.⁶ The study by Bradley et al.⁵ confirms this initial observation by a detailed analysis on the concentration- and voltage-dependence of the block by T16A_Inh-A01 and its effects on channel kinetics, and provided a similar analysis for CaCC_Inh-A01, which had not been done previously.

The demonstration of voltage-independent block of ANO1 by these 2 compounds is a major step forward in developing better pharmacological tools for the purpose of investigating the physiological and pathophysiological role of CaCCs in various tissues, and for drug development in therapeutics. The results will also be very useful for designing future mechanistic studies examining where the molecules bind (site-directed mutagenesis, cysteine scanning, molecular dynamics simulations, X-ray crystallography, etc.) and how they alter the biophysical properties of the ANO1 channel, which may ultimately provide instrumental information about the 3D structure of the dimeric protein, and its permeation and gating mechanisms.

Many unanswered questions come to mind from the data presented by Bradley et al.⁵ How do these molecules affect the single channel properties of ANO1 channels? Is their interaction with ANO1 affected by intracellular Ca²⁺, and/or the state channel phosphorylation? Is the pharmacology of ANO1 influenced by the expression of alternatively spliced exons known to alter channel gating?²