Calcium-activated and apoptotic phospholipid scrambling induced by Ano6 can occur independently of Ano6 ion currents

Abstract

Immune cells and platelets maintain plasma membrane phospholipid asymmetry. Upon activation, this asymmetry is disrupted by phospholipid scrambling (PS), which is a major step during activation of immune cells, hemostasis and apoptosis. Anoctamin 6 (Ano6; TMEM16F) causes chloride (Cl−) and cation currents and is required for Ca2+-dependent PS. It is defective in blood cells from patients with Scott syndrome, a rare bleeding disorder. We examined if Cl− currents and PS are related, whether both processes are Ca2+ dependent, and whether Ca2+-independent scrambling during intrinsic and extrinsic apoptosis is controlled by Ano6. Ca2+ increase by ionomycin activated Ano6 Cl− currents and PS in normal lymphocytes, but not in B-lymphocytes from two different patients with Scott syndrome. Fas ligand (FasL) did not increase intracellular Ca2+, but activated Cl− currents in normal but not in Scott lymphocytes. Whole-cell currents were inhibited by Cl− channel blockers and by siRNA knockdown of Ano6. In contrast, intrinsic mitochondrial apoptosis by ABT-737 did not induce Cl− currents in lymphocytes. PS was not inhibited by blockers of Ano6 or removal of Cl− ions. Remarkably, Ca2+-independent scrambling due to extrinsic (FasL) or intrinsic (ABT-737) apoptosis was unchanged in Scott cells. We conclude that: (i) Ano6 Cl− currents are activated by increase in cytosolic Ca2+, or Ca2+ independent by stimulation of Fas receptors; (ii) Ca2+-dependent PS induced by Ano6 does not require Cl− currents; (iii) Ca2+-independent PS does not require Ano6; (iv) Ano6 is necessary for Ca2+-dependent PS, but not by increasing intracellular Ca2+.

Role of anoctamins in cancer and apoptosis

“…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]. Moreover, we demonstrated recently that Ano6 is activated during cell swelling and by pro-apoptotic stimuli and therefore contributes to both RVD as well as apoptotic volume decrease [5,55,56].…”

“…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].…”

“…A splice form of Ano6 was identified that was associated with metastatic capability of mammary cancers in mouse and was related to poor prognosis of patients with breast cancer [54]. Notably, Ano6 has recently been associated with membrane phospholipid scrambling and cell shrinkage and therefore seems to be correlated to apoptosis rather than proliferation and cancer [55][56][57][58][59]. The distribution of lipids in the outer and inner leaflets of plasma membranes is asymmetrical: while phosphatidylcholine is mainly found in the outer leaflet, phosphatidylserine is present in the inner surface.…”

Endosomal Trafficking Defects Can Induce Calcium-Dependent Azole Tolerance in Candida albicans

“…Following incubation at 37°C for 1 h, cells were pelleted and washed three times with PBS. Fluorescence was determined by dual excitation at 340 nm and 380 nm with a single emission at 505 nm in a Cytation 5 plate reader (BioTek Instruments, Inc.), with relative Ca 2ϩ levels being expressed as the ratio of the fluorescence intensity upon excitation at 380 nm (F 380 ) to the F 340 (25).…”

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

“…78 VRAC is known to play an important role in this process but also ANO proteins appear to play a role in AVD/apoptotic cell death in several cell types investigated. 45,150,151 The amplicon 11q13 contains the Ano1 gene as well as well as a Fas-Associated protein with Death Domain (FADD) gene, i.e., a gene associated with apoptosis, suggesting a coupling between the regulation of ANO1 and cell death. Not much is published about ANO1 and apoptosis.…”