TMEM16B induces chloride currents activated by calcium in mammalian cells
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Ca2+-activated Cl− channels play important physiological roles in various cell types, but their molecular identity is still unclear. Recently, members of the protein family named transmembrane 16 (TMEM16) have been suggested to function as Ca2+-activated Cl− channels. Here, we report the functional properties of mouse TMEM16B (mTMEM16B) expressed in human embryonic kidney (HEK) 293T cells, measured both in the whole-cell configuration and in inside-out excised patches. In whole cell, a current induced by mTMEM16B was activated by intracellular Ca2+ diffusing from the patch pipette, released from intracellular stores through activation of a G-protein-coupled receptor, or photoreleased from caged Ca2+ inside the cell. In inside-out membrane patches, a current was rapidly activated by bath application of controlled Ca2+ concentrations, indicating that mTMEM16B is directly gated by Ca2+. Both in the whole-cell and in the inside-out configurations, the Ca2+-induced current was anion selective, blocked by the Cl− channel blocker niflumic acid, and displayed a Ca2+-dependent rectification. In inside-out patches, Ca2+ concentration for half-maximal current activation decreased from 4.9 µM at −50 mV to 3.3 µM at +50 mV, while the Hill coefficient was >2. In inside-out patches, currents showed a reversible current decrease at −50 mV in the presence of a constant high Ca2+ concentration and, moreover, an irreversible rundown, not observed in whole-cell recordings, indicating that some unknown modulator was lost upon patch excision. Our results demonstrate that mTMEM16B functions as a Ca2+-activated Cl− channel when expressed in HEK 293T cells.
KeywordsCalcium signaling Calcium sensitivity Calcium-activated chloride current Anion channel Caged calcium Chloride current
We thank Lara Masten, Giulia Betto, and Fulvio Celsi for their help in some experiments. This study was supported by grants from the Italian Ministry of Research (MIUR) and from the Italian Institute of Technology.
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