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J Physiol 594.11 (2016) pp 2785–2786

JOURNAL CLUB

TMEM16F: function from (iso)form Kathryn Acheson Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK

The Journal of Physiology

Email: [email protected]

Ion channels are transmembrane proteins that form aqueous pores allowing charged ions to cross the oily, hydrophobic environment of the plasma membrane. A plethora of ion channels have been identified and physiologists have grouped these channels into families depending on genetic, structural and functional features. Therefore, channels belonging to the same family usually share molecular features and physiological roles. It is beginning to emerge that the recently identified TMEM16/anoctamin channels represent an exception: TMEM16 proteins possess different, and in some cases multiple, functions. Ten TMEM16 proteins have been identified, denoted by the letters A–J (with I missing). The founding member of the TMEM16 family, TMEM16A, was recognised to function as a Ca2+ -activated chloride channel (CaCC) in 2008. The prime role of TMEM16A-encoded CaCCs is to couple intracellular Ca2+ signalling with cell excitability. According to a recently reported X-ray structure, TMEM16 proteins should have 10 transmembrane domains, with cytosolic N- and C-termini, and form homodimers (Brunner et al. 2014). Another member of the TMEM16 family, TMEM16B, also forms CaCCs. Therefore, it was a surprise that the TMEM16F member possessed a distinct cellular function: phospholipid scrambling (Suzuki et al. 2010). Since this original observation, other members of the TMEM16 family (TMEM16C/D/G/J), were also found to serve as scramblases. Phospholipid scrambling refers to Ca2+ -dependent externalisation of inner membrane phospholipids, such as phosphatidylserine (PS). This function is triggered by a rise in intracellular free Ca2+ ([Ca2+ ]i ), and can initiate many important cellular responses including blood coagulation and apoptosis. In the

context of blood coagulation, exposure of PS to the outer leaflet of erythrocytes facilitates the binding of cofactors such as prothrombinase, required for conversion of prothrombin to thrombin. The importance of phospholipid scramblase in blood coagulation is illustrated by the fact that mutations in TMEM16F lead to Scott syndrome, an inherited bleeding disorder in man. Intriguingly, TMEM16F also functions as an ion channel. The electrophysiological features of TMEM16F, however, are not fully defined, with reports in the literature being divergent and even contradictory at times. Experimental evidence indicated that TMEM16F can be gated open in response to changes in cell volume, membrane depolarisation and/or an increase in [Ca2+ ]i . These studies also suggested that TMEM16F channels mediated anionic currents. Conversely, TMEM16F has also been described as a small-conductance Ca2+ -activated non-selective cation channel (SCAN) (Yang et al. 2012). Understanding the physiological roles, gating mechanism(s) and ionic selectivity of TMEM16F is a highly topical and active area of research. In unprecedented studies recently published in The Journal of Physiology, Scudieri et al. (2015) demonstrate that alternative splicing could explain some of the seemingly divergent TMEM16F findings reported in the literature. Alternative splicing allows the expression of one gene to result in multiple protein isoforms. For example, various alternatively spliced isoforms of TMEM16A exist, which were characterised by differences in channel gating and ion permeability. In the present study cellular localisation, expression levels and electrophysiological properties of various TMEM16F splice variants have been explored. The authors initiated the study using bioinformatics to identify TMEM16F splice variants in available DNA sequence repositories. Five variants were identified, and termed variant 1–5 (V1–V5). Sequence alignment revealed that two of the variants, V3 and V4, were identical. V1, V2 and V5 were predicted to have 10 transmembrane domains with intracellular Nand C-termini. Splice variants affected either the N- or C-terminus of TMEM16F.

 C 2016 The Authors. The Journal of Physiology  C 2016 The Physiological Society

Specifically, relative to V1, V2 and V5 consisted of a 28 amino acid deletion and a 21 amino acid insertion at the N-terminus, respectively. In contrast, V3 differed from V1 at the C-terminus: an insertion of 19 amino acids formed an additional, eleventh, transmembrane domain and an extracellular C-terminus. In order to gain an understanding of possible physiological roles for individual TMEM16F splice variants, the expression levels of all variants were assessed in a range of human tissues, using real time quantitative PCR (RT-qPCR) transcript amplification. Comparable levels of V1 and V5 mRNA were detected in most tissues, whilst V2 expression was more specifically confined to smooth muscles. No transcripts for V3 were detected in any of the tissues under examination. Immunofluorescence and Western blotting were employed to explore cellular localisation of all splice variants. When V1, V2 and V5 TMEM16F proteins were heterologously expressed in the human embryonic kidney (HEK) cell line, they appear to be localised on the cell membrane. In contrast, V3 remained in intracellular compartments and was expressed at extremely low levels, consistent with the finding that V3 mRNA transcripts in human tissues were below detectable levels. The sensitivity of TMEM16A and TMEM16B in response to changes in [Ca2+ ]i have been comprehensively characterised. Scudieri et al. (2015) set out to define the Ca2+ sensitivity of various TMEM16F splice variants. The individual splice variants were expressed in HEK cells, and channel activity was assessed by measuring I− fluxes via genetically encoded halide-sensitive yellow fluorescent proteins (HS-YFP). Ionomycin was used to raise [Ca2+ ]i to 5 µM. Under these conditions, cells expressing V1, V2 and V5 proteins appeared to take up I− . In contrast, no I− fluxes were detected in cells expressing the V3 protein, possibly because the unusual C-terminus may cause altered intracellular trafficking and instability. To establish the exact Ca2+ sensitivity of TMEM16F splice variants, whole-cell patch-clamp electrophysiology was used. Small endogenous currents were present in non-transfected cells. However, these were completely abolished by

DOI: 10.1113/JP271980

2786 treatment with siRNA directed against TMEM16F, indicating that some endogenous TMEM16F was expressed in HEK cells. Patch-clamp analysis revealed that splice variants differ in their Ca2+ sensitivity. Specifically, [Ca2+ ]i of 2 µM was sufficient to activate V2 and V5 currents, whereas [Ca2+ ]i of 5 µM was required for V1 channel activation. For all variants 20 µM [Ca2+ ]i induced maximal currents. Ionic selectivity of TMEM16F channels is not fully defined. Scudieri et al. performed ion substitution experiments and found that TMEM16F presented only minimal preference for Cl− over Na+ . This almost equal permeability for these ions can explain some of the seemingly contradictory results in the literature. For example, some studies reporting TMEM16F cationic currents involved electrophysiological recordings obtained in the absence of Cl− ; under these conditions cationic current could be observed (Yang et al. 2012). The authors next examined the scramblase activity of TMEM16F splice variants. An annexin V binding assay was used, due to the selectivity of annexin V for externalised PS. Heterologous expression of V1, V2 and V5 in HEK cells resulted in cell scramblase activity in response to an increase in [Ca2+ ]i , achieved via exposure to ionomycin. Propidium iodide staining indicated that no membrane disruption occurred as a result of ionomycin treatment. The structural features involved in ion permeation and phospholipid scrambling in TMEM16F are not fully defined. Whether the ion and lipid transportation pathways are functionally coupled is unclear. Scudieri et al. (2015) started to address these important points by studying the functional consequences of single amino acid substitutions in cloned TMEM16F. Two mutations were selected from the literature. Substitution of glutamine 559 to lysine (Q559K) was considered as it reduced TMEM16F cation permeability (Yang et al. 2012), while substitution of aspartate 409 with glycine (D409G) reportedly increased constitutive scramblase activity (Segawa et al. 2011). Channel and scramblase

Journal Club activities were tested in these two mutants with the rationale that if these activities occurred in conjunction, alteration of one function would affect the other. Whole-cell currents mediated by the Q559K mutant were indistinguishable from currents recorded from non-transfected cells. Notably, the scramblase activity of the mutant channel remained unaltered. TMEM16F proteins carrying the gain-of-function mutation for scramblase activity (D409G) presented altered channel properties (Ca2+ sensitivity was enhanced by 5-fold). It could be envisaged that an enhanced Ca2+ sensitivity may affect both ion channel and scramblase functions. However, in the presence of 1 µM ionomycin, which raised [Ca2+ ]i by 50 nM, scramblase activity was increased by a factor of 2. Conversely, whole-cell currents in 100 nM [Ca2+ ]i were very small and remained unaltered. In the presence of 10 µM ionomycin, a condition that led to an increase in [Ca2+ ]i to 5 µM, scramblase activity was unaltered, while whole-cell currents were increased by 20%. In conclusion, this research provides important insights into TMEM16F both as a channel and a scramblase. V1, V2 and V5 protein expression levels correlated to ion channel and scramblase activity. Phospholipid scrambling was concluded to be the main function of TMEM16F, as this activity occurred at low [Ca2+ ]i . Ion transport required significantly higher levels of [Ca2+ ]i , above normal resting levels. Thus, ion flux and phospholipid translocation may not occur simultaneously as these functions are triggered by different [Ca2+ ]i . In contrast to this suggestion is the observation that in a fungal TMEM16F homologue, direct coupling between ion flux and phospholipid scrambling may indeed occur (Malvezzi et al. 2013). Notably, however, Brunner et al. (2014) did not detect ion channel activity in another fungal TMEM16 protein which nevertheless worked as a scramblase. There is no doubt that much of future research effort will include investigations into whether ions and phospholipid heads

J Physiol 594.11

translocate through the same or distinct pathways, and whether the two functions could be modulated separately by drugs. From a systems physiology point of view, we still need to know the precise functional roles of TMEM16F splice variants in the multiple tissues and organs in which they are expressed. There are clearly exciting times ahead in TMEM16F research. References Brunner JD, Lim NK, Schenck S, Duerst A & Dutzler R (2014). X-ray structure of a calcium-activated TMEM16 lipid scramblase. Nature 516, 207–212. Malvezzi M, Chalat M, Janjusevic R, Picollo A, Terashima H, Menon AK & Accardi A (2013). Ca2+ -dependent phospholipid scrambling by a reconstituted TMEM16 ion channel. Nat Commun 4, 2367. Scudieri P, Caci E, Venturini A, Sondo E, Pianigiani G, Marchetti C, Ravazzolo R, Pagani F & Galietta LJV (2015). Ion channel and lipid scramblase activity associated with expression of TMEM16F/ANO6 isoforms. J Physiol 593, 3829–3848. Segawa K, Suzuki J & Nagata S (2011). Constitutive exposure of phosphatidylserine on viable cells. Proc Natl Acad Sci USA 108, 19246–19251. Suzuki J, Umeda M, Sims PJ & Nagata S (2010). Calcium-dependent phospholipid scrambling by TMEM16F. Nature 468, 834–838. Yang H, Kim A, David T, Palmer D, Jin T, Tien J, Huang F, Cheng T, Coughlin SR, Jan YN & Jan LY (2012). TMEM16F forms a Ca2+ -activated cation channel required for lipid scrambling in platelets during blood coagulation. Cell 151, 111–122.

Additional information Competing interests

None declared.

Acknowledgements

I would like to thank Dr Paolo Tammaro for his constructive review of this manuscript. KA is a BHF funded PhD student (grant number FS/15/68/32042).

 C 2016 The Authors. The Journal of Physiology  C 2016 The Physiological Society

TMEM16F: function from (iso)form.

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