title: 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 Ca(2+)-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 Ca(2+) dependent, and whether Ca(2+)-independent scrambling during intrinsic and extrinsic apoptosis is controlled by Ano6. Ca(2+) 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 Ca(2+), 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, Ca(2+)-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 Ca(2+), or Ca(2+) independent by stimulation of Fas receptors; (ii) Ca(2+)-dependent PS induced by Ano6 does not require Cl(-) currents; (iii) Ca(2+)-independent PS does not require Ano6; (iv) Ano6 is necessary for Ca(2+)-dependent PS, but not by increasing intracellular Ca(2+).
authors.affiliation: Institut für Physiologie, Universität Regensburg, Regensburg, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Kmit
authors.forename: R
authors.initials: R
authors.lastname: van Kruchten
authors.forename: J
authors.initials: J
authors.lastname: Ousingsawat
authors.forename: N J A
authors.initials: NJ
authors.lastname: Mattheij
authors.forename: B
authors.initials: B
authors.lastname: Senden-Gijsbers
authors.forename: J W M
authors.initials: JW
authors.lastname: Heemskerk
authors.forename: R
authors.initials: R
authors.lastname: Schreiber
authors.forename: E M
authors.initials: EM
authors.lastname: Bevers
authors.forename: K
authors.initials: K
authors.lastname: Kunzelmann
mesh_terms: D000075368:Anoctamins; D017209:Apoptosis / Q000187:drug effects; D001402:B-Lymphocytes / Q000276:immunology / Q000502:physiology; D001713:Biphenyl Compounds / Q000494:pharmacology; D001778:Blood Coagulation Disorders / Q000503:physiopathology; D002118:Calcium / Q000378:metabolism*; D061207:Calcium Ionophores / Q000494:pharmacology; D018118:Chloride Channels / Q000037:antagonists & inhibitors / Q000378:metabolism; D053222:Fas Ligand Protein / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D017136:Ion Transport / Q000187:drug effects; D015759:Ionomycin / Q000494:pharmacology; D019169:Jurkat Cells; D009596:Nitrophenols / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D049294:Phospholipid Transfer Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D010879:Piperazines / Q000494:pharmacology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology
chemical_list: C501332:ABT-737; C555078:ANO6 protein, human; D000075368:Anoctamins; D001713:Biphenyl Compounds; D061207:Calcium Ionophores; D018118:Chloride Channels; D053222:Fas Ligand Protein; D009596:Nitrophenols; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids; D010879:Piperazines; D034741:RNA, Small Interfering; D013449:Sulfonamides; D015759:Ionomycin; D002118:Calcium