TMEM16F is a component of a Ca²⁺-activated Cl⁻ channel but not a volume-sensitive outwardly rectifying Cl⁻ channel

Abstract

TMEM16 (transmembrane protein 16) proteins, which possess eight putative transmembrane domains with intracellular NH2- and COOH-terminal tails, are thought to comprise a Cl− channel family. The function of TMEM16F, a member of the TMEM16 family, has been greatly controversial. In the present study, we performed whole cell patch-clamp recordings to investigate the function of human TMEM16F. In TMEM16F-transfected HEK293T cells but not TMEM16K- and mock-transfected cells, activation of membrane currents with strong outward rectification was found to be induced by application of a Ca2+ ionophore, ionomycin, or by an increase in the intracellular free Ca2+ concentration. The free Ca2+ concentration for half-maximal activation of TMEM16F currents was 9.6 μM, which is distinctly higher than that for TMEM16A/B currents. The outwardly rectifying current-voltage relationship for TMEM16F currents was not changed by an increase in the intracellular Ca2+ level, in contrast to TMEM16A/B currents. The Ca2+-activated TMEM16F currents were anion selective, because replacing Cl− with aspartate− in the bathing solution without changing cation concentrations caused a positive shift of the reversal potential. The anion selectivity sequence of the TMEM16F channel was I− > Br− > Cl− > F− > aspartate−. Niflumic acid, a Ca2+-activated Cl− channel blocker, inhibited the TMEM16F-dependent Cl− currents. Neither overexpression nor knockdown of TMEM16F affected volume-sensitive outwardly rectifying Cl− channel (VSOR) currents activated by osmotic swelling or apoptotic stimulation. These results demonstrate that human TMEM16F is an essential component of a Ca2+-activated Cl− channel with a Ca2+ sensitivity that is distinct from that of TMEM16A/B and that it is not related to VSOR activity.

Anoctamin 6 Contributes to Cl− Secretion in Accessory Cholera Enterotoxin (Ace)-stimulated Diarrhea

“…This is consistent with prior reports of its secretory activity in animal models (5,17,39). Using human epithelial colorectal adenocarcinoma cells, Caco-2 which possessed the machinery for Cl Ϫ transport, we showed that Ace acutely stimulated Cl Ϫ secretion.…”

“…Each member is predicted to have multiple isoforms being simultaneously expressed in a given cell (15). Apart from ANO1 and ANO2, ANO6 was also recently shown to be a Ca 2ϩ -activated Cl Ϫ channel with delayed Ca 2ϩ activation (16,17). Despite numerous reports on ANO as a Cl Ϫ channel, the contribution of specific member(s) of this protein family to Cl Ϫ secretion in diarrhea remains obscure.…”

Role of anoctamins in cancer and apoptosis

“…These results came as a surprise, since Ano6 has been characterized as a Ca 2þ -activated Cl 2 channel. Moreover, Ca 2þ -activated Cl 2 currents were also observed after overexpression of Ano4, 7 and 9 [6,55,56,61,62]. However, Cl 2 channel and scramblase activity were shown to be independent [55].…”

“…It was also shown to be activated during cell swelling, probably secondary to an increase in intracellular Ca 2þ [5]. While some detected a role of Ca 2þ -dependent anoctamins, such as Ano1 or Ano6, to volume regulation, others did not [5,6]. Upregulation of endogenous Ca 2þ -activated Cl 2 channels in proliferating cells has been observed recently, but the role of these channels for proliferation has remained unclear [7,8].…”

Role of volume-regulated and calcium-activated anion channels in cell volume homeostasis, cancer and drug resistance

“…On the other hand, Okada and coworkers found that ANO6 K D has no effect on staurosporine induced AVD. 154 Hence, the role of ANO6 in AVD/apoptosis seems to vary among cell types.…”