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+.
Highlights
Endogenous or overexpressed anoctamins produce chloride (Cl À ) or cation currents.[1,2,3,4,5,6,7,8] This putative family of Cl À channels demonstrate variable Ca2 þ sensitivity and are activated during cell swelling or by apoptotic stimuli.[9,10,11,12] Anoctamins are unique as they do not show any obvious homology to other ion channels
It was recently shown that Ano[6] is an essential component of outwardly rectifying Cl À channel (ORCC) in Jurkat T lymphocytes that is activated by Fas receptor stimulation.[9]
The present data suggest that strong increase in intracellular Ca2 þ by ionomycin in Jurkat cells activates a whole-cell Cl À current, along with a nonselective cation current (Supplementary Figures 2E,F)
Summary
Endogenous or overexpressed anoctamins produce chloride (Cl À ) or cation currents.[1,2,3,4,5,6,7,8] This putative family of Cl À channels demonstrate variable Ca2 þ sensitivity and are activated during cell swelling or by apoptotic stimuli.[9,10,11,12] Anoctamins are unique as they do not show any obvious homology to other ion channels. Anoctamins show a high degree of sequence similarity, for the putative pore region.[7] We were able to demonstrate that anoctamin 6 (Ano6) produces an outwardly rectifying Cl À channel (ORCC) in Jurkat lymphocytes and epithelial cells, which is activated during stimulation of the proapoptotic Fas receptor or by the pro-apoptotic compound staurosporine.[9]. We and others have shown that phosphatidylserine exposed at the surface of platelets has an essential role in the process of blood coagulation.[15,16] Recent findings indicate that two different patients with the so-called Scott syndrome, a quite rare congenital bleeding disorder, have missense mutations in the TMEM16F gene.[14,17] Defective function of this gene can explain why Scott platelets and other hematopoietic cells are unable to perform Ca2 þ -dependent phosphatidylserine exposure and, are impaired in supporting blood coagulation.[16] Here, we analyzed Ano6-dependent Cl À currents in immortalized B lymphocytes from the two Scott patients with characterized TMEM16F mutations, and compared these with their ability to PS under conditions of Ca2 þ activation and apoptosis
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