The molecular nature of CaCC has been uncertain with CLCA, TWEETY and BEST genes having been considered as likely candidates [7,11,15]. It is now accepted that CLCA expression products are unlikely to form channels per se and probably function as cell adhesion proteins, or are secreted [20]. Similarly, TWEETY gene products do not recapitulate the properties of endogenous CaCC. The bestrophins encoded by genes BEST1-4 have a topology more consistent with ion channels [11] and form chloride channels that are activated by physiological concentrations of Ca²⁺, but whether such activation is direct is not known [11]. However, currents generated by bestrophin over-expression do not resemble native CaCC currents. The evidence for and against bestrophin proteins forming CaCC is critically reviewed by Duran et al. [7]. Recently, a new gene family, TMEM16 (anoctamin) consisting of 10 members (TMEM16A-K; anoctamin 1-10) has been identified and there is firm evidence that some of these members form chloride channels [6,12].

Many of the listed TMEM16A channel blockers are recognised as being unselective [2,8]. Ani9 demonstrates selectivity for TMEM16A versus TMEM16B [23] and in contrast to several of the other blockers, has no effects on intracellular Ca²⁺ signalling in human airway epithelial cells [4]. In addition, niclosamide has been widely claimed as a TMEM16A blocker [17]. However recent reports demonstrate that niclosamide does not block TMEM16A but interferes with intracellular Ca²⁺ signalling [4,8]. Studies have also revealed that with intracellular Ca²⁺ levels tightly buffered, niclosamide can potentiate TMEM16A activity [4,14]. Blockade of I_Cl(Ca) by niflumic acid, DIDS and 9-anthroic acid is voltage-dependent whereas block by NPPB is voltage-independent [10]. Extracellular niflumic acid; DCDPC and 9-anthroic acid (but not DIDS) exert a complex effect upon I_Cl(Ca) in vascular smooth muscle, enhancing and inhibiting inwardly and outwardly directed currents in a manner dependent upon [Ca²⁺]_i (see [13] for summary). Considerable crossover in pharmacology with large conductance Ca²⁺-activated K⁺ channels also exists (see [9] for overview). Two novel compounds, CaCCinh-A01 and CaCCinh-B01 have been identified as blockers of calcium-activated chloride channels in T84 human intestinal epithelial cells [5]. Significantly, other novel compounds totally block currents mediated by TMEM116A, but have only a modest effect upon total current mediated by CaCC native to T84 cells or human bronchial epithelial cells, suggesting that TMEM16A is not the predominant CaCC in such cells [18]. CaMKII modulates CaCC in a tissue dependent manner (reviewed by [10,13]). CaMKII inhibitors block activation of I_Cl(Ca) in T84 cells but have no effect in parotid acinar cells. In tracheal and arterial smooth muscle cells, but not portal vein myocytes, inhibition of CaMKII reduces inactivation of I_Cl(Ca). Intracellular Ins(3,4,5,6)P₄ may act as an endogenous negative regulator of CaCC channels activated by Ca²⁺, or CaMKII. Smooth muscle CaCC are also regulated positively by Ca²⁺-dependent phosphatase, calcineurin (see [13] for summary).

TMEM16A (anoctamin 1; Ano 1) produces Ca²⁺-activated Cl^- currents with kinetics similar to native CaCC currents recorded from different cell types [3,21-22,24]. Knockdown of TMEM16A greatly reduces currents mediated by calcium-activated chloride channels in submandibular gland cells [24] and smooth muscle cells from pulmonary artery [16]. In TMEM16A^(-/-) mice Ca²⁺-dependent Cl^-secretion by several epithelia is reduced [19,21]. In TMEM16B^(-/-) mice Ca-activated Cl^- currents in the main olfactory epithelium (MOE) and in the vomeronasal organ are virtually absent [1].

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