TMEM16F Forms a Ca2+-Activated Cation Channel Required for Lipid Scrambling in Platelets during Blood Coagulation

Abstract

SUMMARY Collapse of membrane lipid asymmetry is a hallmark of blood coagulation. TMEM16F of the TMEM16 family that includes TMEM16A/B Ca2+-activated Cl− channels (CaCCs) is linked to Scott syndrome with deficient Ca2+-dependent lipid scrambling. We generated TMEM16F knockout mice that exhibit bleeding defects and protection in an arterial thrombosis model associated with platelet deficiency in Ca2+-dependent phosphatidylserine exposure and procoagulant activity and lack a Ca2+-activated cation current in the platelet precursor megakaryocytes. Heterologous expression of TMEM16F generates a small-conductance Ca2+-activated nonselective cation (SCAN) current with subpicosiemens single-channel conductance rather than a CaCC. TMEM16F-SCAN channels permeate both monovalent and divalent cations, including Ca2+, and exhibit synergistic gating by Ca2+ and voltage. We further pinpointed a residue in the putative pore region important for the cation versus anion selectivity of TMEM16F-SCAN and TMEM16A-CaCC channels. This study thus identifies a Ca2+-activated channel permeable to Ca2+ and critical for Ca2+-dependent scramblase activity during blood coagulation.

Selective serotonin reuptake inhibitors facilitate ANO6 (TMEM16F) current activation and phosphatidylserine exposure

“…ANO1 has been investigated most extensively, but ANO6 has also been a focus of recent studies. Although it is unclear whether ANO6 acts as a CaCC [13,39,44], a small conductance Ca 2+ -activated nonselective cation channel (SCAN) [49], or a component of the outwardly rectifying Cl − channel [28], it is established as a key regulator of Ca 2+ -dependent phosphatidylserine (PS) scramblase activity [42], which is important for triggering blood coagulation and is impaired in patients with Scott syndrome, a rare congenital bleeding disorder [41]. Recently, it has also been reported that Cl − entry through Cl − channels, possibly ANO6, is required for membrane hyperpolarization, which maintains the driving force for Ca 2+ transport and triggers full phosphatidylserine exposure in platelets [16].…”

Cryo-EM structures of the TMEM16A calcium-activated chloride channel

“…3a–c). Besides R511 on TM3 and K599 on the TM5-TM6 loop important for anion selectivity 25 , and K584 on TM5 that partially accounts for the selectivity for anions over cations 28 (Fig. 3b), we tested alanine substitutions of 24 other pore-lining residues including N542 and D550 on TM4, N587 and V595 on TM5, Q705 and F712 on TM7, and S635 on TM6 (Fig.…”

Extracellular chloride is required for efficient platelet aggregation

“…This may also explain why anion channels have previously received much less attention than cation channels. It is worth noting that anion channels have been associated with cystic fibrosis, bleeding phenotypes, and inflammatory conditions [12, 13, 15, 26, 27] and may represent valuable therapeutic targets, as demonstrated by clinical use of CFTR modulators [28]. Further work will be required to investigate the contribution(s) by the cohort of platelet anion channels during platelet activation.…”

“…Proteomic [10] and transcriptomic [11] analyses have since identified numerous anion channels that may be expressed by platelets. Of these, functional expressions of CLIC1 (Intracellular Cl − channel-1) [12], TMEM16F [13], and pannexin-1 [14] have been confirmed. Indicative of a hemostatic role for anion channels, CLIC1- and TMEM16F-deficient mice have associated platelet-related bleeding phenotypes [12, 13].…”

“…Of these, functional expressions of CLIC1 (Intracellular Cl − channel-1) [12], TMEM16F [13], and pannexin-1 [14] have been confirmed. Indicative of a hemostatic role for anion channels, CLIC1- and TMEM16F-deficient mice have associated platelet-related bleeding phenotypes [12, 13]. Additionally, pannexin-1 channels have been shown to amplify platelet activation responses to threshold agonist concentrations [14].…”