Ca 2+-activated Cl − channels (CaCCs) are exceptionally well adapted to subserve diverse physiological roles, from epithelial fluid transport to sensory transduction, because their gating is cooperatively controlled by the interplay between ionotropic and metabotropic signals. A molecular understanding of the dual regulation of CaCCs by voltage and Ca 2+ has recently become possible with the discovery that Ano1 (TMEM16a) − channels (CaCCs) play manifold roles in cell physiology (1, 2), including epithelial secretion (3, 4), sensory transduction and adaptation (5-8), regulation of smooth muscle contraction (9), control of neuronal and cardiac excitability (10), and nociception (11). This myriad of functions has attracted attention for more than 25 years (12, 13), but a lack of consensus regarding their molecular composition has stymied a mechanistic understanding of their gating. Recently, two members of the TMEM16/anoctamin family (Ano1 and Ano2) were identified as CaCC channels (14-16) and shown to be essential for salivary exocrine secretion (14,17,18), gut slow-wave activity (18, 19), tracheal secretion (18,20,21), and olfactory transduction (5-7).Ano1 and Ano2 are well suited for their diverse roles because they are dually gated by voltage (V m ) and intracellular Ca 2+ concentration ([Ca 2+ ] i ), so that their activity is tuned by the interplay between metabotropic and ionotropic inputs (14,16,(22)(23)(24) and depolarization. In the absence of Ca 2+ , no current was evident at V m between −100 mV and +100 mV, but as [Ca 2+ ] i was increased, an outward current was activated by depolarization and deactivated by hyperpolarization (Fig. 1 A-D and F). As [Ca 2+ ] i was increased, outward rectification (Fig. 1F) and the fraction of total current exhibiting time-dependence (Fig. 1E) were reduced. V m -dependent activation of Ano1 was evaluated by plotting normalized conductance versus V m (G/G max vs. V m curves; Fig. 1G). The data were well fit by the Boltzmann equation,where G/G max is normalized conductance; z is the equivalent gating charge associated with voltage-dependent channel opening; V 0.5 is the membrane potential (V m ) where G/G max is halfmaximal and is related to the conformational energy associated with voltage-independent channel opening; and F/RT = 0.039 mV −1 . At 1 μM Ca 2+ , V 0.5 was 64 ± 0.9 mV (Fig. 1G, black squares); doubling [Ca 2+ ] to 2 μM shifted the G/G max vs. V m curve to the left by −145 mV (Fig. 1G, red circles) with no significant effect on z. Because Ca 2+ shifts the G/G max vs. V m curves so dramatically, a complete G/G max vs. V m curve could be recorded for only a narrow range of [Ca 2+ ] i . For these [Ca 2+ ], z was not obviously Ca 2+ -dependent (z = 0.40-0.46). This indicates that Ca 2+ does not change the V m sensitivity (z) of the Ano1 channel, but rather shifts V 0.5 , the energy associated with V m -independent gating.Although these data may imply that Ano1 is a simple ligandgated channel, Ano1 is actually more complicated, because Ca 2+ gating is st...

The newly discovered Ca 2+ -activated Cl − channel (CaCC), Anoctamin 1 (Ano1 or TMEM16A), has been implicated in vital physiological functions including epithelial fluid secretion, gut motility, and smooth muscle tone. Overexpression of Ano1 in HEK cells or Xenopus oocytes is sufficient to generate Ca 2+ -activated Cl − currents, but the details of channel composition and the regulatory factors that control channel biology are incompletely understood. We used a highly sensitive quantitative SILAC proteomics approach to obtain insights into stoichiometric protein networks associated with the Ano1 channel. These studies provide a comprehensive footprint of putative Ano1 regulatory networks. We find that Ano1 associates with the signaling/scaffolding proteins ezrin, radixin, moesin, and RhoA, which link the plasma membrane to the cytoskeleton with very high stoichiometry. Ano1, ezrin, and moesin/radixin colocalize apically in salivary gland epithelial cells, and overexpression of moesin and Ano1 in HEK cells alters the subcellular localization of both proteins. Moreover, interfering RNA for moesin modifies Ano1 current without affecting its surface expression level. Another network associated with Ano1 includes the SNARE and SM proteins VAMP3, syntaxins 2 and -4, and syntaxin-binding proteins munc18b and munc18c, which are integral to translocation of vesicles to the plasma membrane. A number of other regulatory proteins, including GTPases, Ca 2+ -binding proteins, kinases, and lipid-interacting proteins are enriched in the Ano1 complex. These data provide stoichiometrically prioritized information about mechanisms regulating Ano1 function and trafficking to polarized domains of the plasma membrane.calcium | interactome | cross-linker | apical targeting

Mouse parotid acinar cells express P2X 4 and P2X 7 receptors (mP2X 4 R and mP2X 7 R) whose physiological function remains undetermined. Here we show that mP2X 4 R expressed in HEK-293 cells do not allow the passage of tetraethylammonium (TEA + ) and promote little, if any, ethidium bromide (EtBr) uptake when stimulated with ATP or BzATP. In contrast, mP2X 7 R generates slowly decaying TEA + current, sustained Na + current and promotes robust EtBr uptake. However, ATP-activated TEA + current from acinar cells was unlike that generated by mP2X 7 R or mP2X 4 R. Functional interactions between mP2X 4 R and mP2X 7 R were investigated in HEK cells co-transfected with different mP2X 4 : mP2X 7 cDNA ratios and using solutions containing either TEA + or Na + ions. Co-expressed channels generated a TEA + current that displayed faster decay during ATP stimulation than mP2X 7 R alone. Moreover, cells transfected with a 2 : 1 cDNA ratio displayed decaying kinetics similar to those observed in acinar cells. Concentration-response curves in Na + -containing solutions were constructed for heterologously expressed mP2X 4 R, mP2X 7 R and mP2X 4 R:mP2X 7 R co-expressions as well as acinar cells. The EC 50 values determined were 11, 220, 434 and 442 μm, respectively. Na + currents generated by expressing mP2X 4 R or mP2X 7 R alone were potentiated by ivermectin (IVM). In contrast, IVM potentiation in acinar cells and HEK cells co-expressing P2X 4 and P2X 7 (1 : 1 or 2 : 1 cDNA ratios) was seen only when the ATP concentration was lowered from 5 to 0.03 mm. Taken together our observations indicate a functional interaction between murine P2X 7 and P2X 4 receptors. Such interaction might occur in acinar cells to shape the response to extracellular ATP in salivary epithelia.

The TMEM16A-mediated Ca-activated Cl current drives several important physiological functions. Membrane lipids regulate ion channels and transporters but their influence on members of the TMEM16 family is poorly understood. Here we have studied the regulation of TMEM16A by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), cholesterol, and fatty acids using patch clamp, biochemistry and fluorescence microscopy. We found that depletion of membrane PI(4,5)P2 causes a decline in TMEM16A current that is independent of cytoskeleton, but is partially prevented by removing intracellular Ca. On the other hand, supplying PI(4,5)P2 to inside-out patches attenuated channel rundown and/or partially rescued activity after channel rundown. Also, depletion (with methyl-β-cyclodextrin M-βCD) or restoration (with M-βCD+cholesterol) of membrane cholesterol slows down the current decay observed after reduction of PI(4,5)P2. Neither depletion nor restoration of cholesterol change PI(4,5)P2 content. However, M-βCD alone transiently increases TMEM16A activity and dampens rundown whereas M-βCD+cholesterol increases channel rundown. Thus, PI(4,5)P2 is required for TMEM16A function while cholesterol directly and indirectly via a PI(4,5)P2-independent mechanism regulate channel function. Stearic, arachidonic, oleic, docosahexaenoic, and eicosapentaenoic fatty acids as well as methyl stearate inhibit TMEM16A in a dose- and voltage-dependent manner. Phosphatidylserine, a phospholipid whose hydrocarbon tails contain stearic and oleic acids also inhibits TMEM16A. Finally, we show that TMEM16A remains in the plasma membrane after treatment with M-βCD, M-βCD+cholesterol, oleic, or docosahexaenoic acids. Thus, we propose that lipids and fatty acids regulate TMEM16A channels through a membrane-delimited protein-lipid interaction.

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