Ca(2+)-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 Ca(2+)-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 Ca(2+) diffusing from the patch pipette, released from intracellular stores through activation of a G-protein-coupled receptor, or photoreleased from caged Ca(2+) inside the cell. In inside-out membrane patches, a current was rapidly activated by bath application of controlled Ca(2+) concentrations, indicating that mTMEM16B is directly gated by Ca(2+). Both in the whole-cell and in the inside-out configurations, the Ca(2+)-induced current was anion selective, blocked by the Cl(-) channel blocker niflumic acid, and displayed a Ca(2+)-dependent rectification. In inside-out patches, Ca(2+) concentration for half-maximal current activation decreased from 4.9 microM at -50 mV to 3.3 microM 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 Ca(2+) 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 Ca(2+)-activated Cl(-) channel when expressed in HEK 293T cells.

TMEM16B induces chloride currents activated by calcium in mammalian cells

DIBATTISTA, Michele;
2009-01-01

Abstract

Ca(2+)-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 Ca(2+)-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 Ca(2+) diffusing from the patch pipette, released from intracellular stores through activation of a G-protein-coupled receptor, or photoreleased from caged Ca(2+) inside the cell. In inside-out membrane patches, a current was rapidly activated by bath application of controlled Ca(2+) concentrations, indicating that mTMEM16B is directly gated by Ca(2+). Both in the whole-cell and in the inside-out configurations, the Ca(2+)-induced current was anion selective, blocked by the Cl(-) channel blocker niflumic acid, and displayed a Ca(2+)-dependent rectification. In inside-out patches, Ca(2+) concentration for half-maximal current activation decreased from 4.9 microM at -50 mV to 3.3 microM 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 Ca(2+) 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 Ca(2+)-activated Cl(-) channel when expressed in HEK 293T cells.
File in questo prodotto:
Non ci sono file associati a questo prodotto.

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11586/178678
 Attenzione

Attenzione! I dati visualizzati non sono stati sottoposti a validazione da parte dell'ateneo

Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus 188
  • ???jsp.display-item.citation.isi??? 169
social impact