RESUMO
The TMEM16A-mediated Ca2+-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 Ca2+. 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-ß-cyclodextrin M-ßCD) or restoration (with M-ßCD+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-ßCD alone transiently increases TMEM16A activity and dampens rundown whereas M-ßCD+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-ßCD, M-ßCD+cholesterol, oleic, or docosahexaenoic acids. Thus, we propose that lipids and fatty acids regulate TMEM16A channels through a membrane-delimited protein-lipid interaction.
Assuntos
Anoctamina-1/metabolismo , Sinalização do Cálcio/fisiologia , Membrana Celular/metabolismo , Colesterol/metabolismo , Ácidos Graxos/metabolismo , Proteínas de Neoplasias/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Anoctamina-1/genética , Cálcio/metabolismo , Membrana Celular/genética , Colesterol/genética , Ácidos Graxos/genética , Células HEK293 , Humanos , Proteínas de Neoplasias/genética , Fosfatidilinositol 4,5-Difosfato/genéticaRESUMO
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.