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

Kim, Hyun Jong; Jun, Ikhyun; Yoon, Jae Seok; Jung, Jinsei; Kim, Yung Kyu; Kim, Woo Kyung; Kim, Byung Joo; Song, Jaewoo; Kim, Sung Joon; Nam, Joo Hyun; Lee, Min Goo

doi:10.1007/s00424-015-1692-6

Cited by 14 publications

(21 citation statements)

References 51 publications

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“…By comparing the properties of TMEM16F isoforms having plasma membrane localization, we found interesting differences in terms of time course of activation and Ca 2+ sensitivity. Regarding the first parameter, we found that TMEM16F-dependent membrane currents developed with a delay of minutes from the beginning of whole-cell recordings, a behaviour that has been also described in three other studies (Grubb et al 2013;Shimizu et al 2013;Kim et al 2015). The time necessary to reach the peak current in cells expressing TMEM16F-V1 and V5 was about 3-4 min, a time similar but slightly shorter than that observed by others (Grubb et al 2013;Shimizu et al 2013;Kim et al 2015).…”

Section: Discussionsupporting

confidence: 86%

Ion channel and lipid scramblase activity associated with expression of TMEM16F/ANO6 isoforms

Scudieri

Caci

Venturini

et al. 2015

The Journal of Physiology

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Key pointsr TMEM16F is a membrane protein with possible dual function as an ion channel and a phospholipid scramblase.r The properties of ion channels associated with TMEM16F and the link between ion channel and scramblase activity are a matter of debate.r We studied the properties of four isoforms of TMEM16F generated by alternative splicing. r Upregulation of three TMEM16F isoforms or silencing of endogenous TMEM16F increased and decreased, respectively, both scramblase and channel activities.r Introduction of an activating mutation in TMEM16F sequence caused a marked increase in phosphatidylserine scrambling and in ion transport indicating direct involvement of the protein in both functions.Abstract TMEM16F, also known as ANO6, is a membrane protein that has been associated with phospholipid scramblase and ion channel activity. However, the characteristics of TMEM16F-dependent channels, particularly the ion selectivity, are a matter of debate. Furthermore, the direct involvement of TMEM16F in phospholipid scrambling has been questioned. We studied the properties of different TMEM16F variants generated by alternative splicing. Using whole-cell patch-clamp recordings, we found that V1, V2 and V5 variants generated membrane currents activated by very high (micromolar) intracellular Ca 2+ concentrations and positive membrane potentials. These variants showed different degrees of Ca 2+ sensitivity and kinetics of activation but similar ion permeability, characterized by a slight selectivity for Cl − over Na + . A fourth variant (V3) showing a unique carboxy-terminus was devoid of activity, in agreement with its intracellular localization. We also measured scramblase activity using the binding of annexin V to detect phosphatidylserine on the cell surface. V1, V2 and V5 variants were associated with calcium-dependent phosphatidylserine externalization. Interestingly, introduction of an activating mutation, D409G, produced a marked increase in the apparent Ca 2+ sensitivity of TMEM16F-dependent channels. In parallel, this mutation also enhanced the extent of phosphatidylserine externalization that occurred even under resting conditions. These results support the conclusion that TMEM16F proteins are directly involved in dual activity, as a phospholipid scramblase and as an ion channel.

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Section: Discussionsupporting

confidence: 86%

Ion channel and lipid scramblase activity associated with expression of TMEM16F/ANO6 isoforms

Scudieri

Caci

Venturini

et al. 2015

The Journal of Physiology

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“…Furthermore, contrarily to TMEM16F [ 32 ], TMEM16E showed low basal ion transport activity at highly depolarized potentials in the absence of cytosolic Ca 2+ . It is also noteworthy that TMEM16E-mediated currents were observed immediately after the establishment of the whole-cell configuration (even at low [Ca 2+ ]), differently to TMEM16F-dependent currents, which consistently presented an activation delay of several minutes [ 12 , 18 , 32 , 33 , 41 ], indicative of negative regulation by a putative cytosolic factor in heterologous expression systems.…”

Section: Discussionmentioning

confidence: 99%

Gain of function of TMEM16E/ANO5 scrambling activity caused by a mutation associated with gnathodiaphyseal dysplasia

Zanni

Gradogna

Scholz‐Starke

et al. 2017

Cell. Mol. Life Sci.

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Mutations in the human TMEM16E (ANO5) gene are associated both with the bone disease gnathodiaphyseal dysplasia (GDD; OMIM: 166260) and muscle dystrophies (OMIM: 611307, 613319). However, the physiological function of TMEM16E has remained unclear. We show here that human TMEM16E, when overexpressed in mammalian cell lines, displayed partial plasma membrane localization and gave rise to phospholipid scrambling (PLS) as well as non-selective ionic currents with slow time-dependent activation at highly depolarized membrane potentials. While the activity of wild-type TMEM16E depended on elevated cytosolic Ca2+ levels, a mutant form carrying the GDD-causing T513I substitution showed PLS and large time-dependent ion currents even at low cytosolic Ca2+ concentrations. Contrarily, mutation of the homologous position in the Ca2+-activated Cl− channel TMEM16B paralog hardly affected its function. In summary, these data provide the first direct demonstration of Ca2+-dependent PLS activity for TMEM16E and suggest a gain-of-function phenotype related to a GDD mutation.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-017-2704-9) contains supplementary material, which is available to authorized users.

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“…How ANO9 can upregulate and activate EGFR remains a question. Most ANO proteins are located at the cell surface and some (e.g., ANO1, ANO2, ANO6) have CaCC activity ( Kim et al , 2015 ; Pedemonte and Galietta, 2014 ). Although ANO9 is expressed at the plasma membrane ( Figure 1E–F ), it did not show CaCC activity ( Supplementary Figure 5 ), indicating that ANO9-mediated EGFR activation is not associated with CaCC activity.…”

Section: Discussionmentioning

confidence: 99%

“…The mammalian-expressible plasmids for ANO5 were purchased from GeneCopoeia (Rockville, MD, USA; Clone ID: EX-T8509-M02). The mammalian expression plasmids for ANO1 and ANO6 have been previously described ( Kim et al , 2015 ; Jun et al , 2016 ). The coding regions of ANO1 , ANO5 , and ANO6 were subcloned into the pCMV-Myc-N vector with an N-terminal Myc tag.…”

Section: Methodsmentioning

confidence: 99%

“…Since ANO1/TMEM16A was identified as a Ca 2+ -activated Cl − channel (CaCC) in 2008 ( Caputo et al , 2008 ; Schroeder et al , 2008 ; Yang et al , 2008 ), the biological roles and clinical implications of other ANO/TMEM16 proteins have been described. For example, ANO2/TMEM16B and ANO6/TMEM16F were also found to possess CaCC activity ( Pedemonte and Galietta, 2014 ; Kim et al , 2015 ), and ANO3/TMEM16C facilitates sodium-activated potassium currents in primary sensory neurons of rats and regulates pain processing ( Huang et al , 2013 ). Recessive mutations of ANO5/TMEM16E cause proximal limb girdle muscular dystrophy and distal non-dysferlin Miyoshi muscular dystrophy ( Bolduc et al , 2010 ).…”

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confidence: 99%

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ANO9/TMEM16J promotes tumourigenesis via EGFR and is a novel therapeutic target for pancreatic cancer

et al. 2017

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Background:Anoctamin (ANO)/transmembrane member 16 (TMEM16) proteins mediate diverse physiological and pathophysiological functions including cancer cell proliferation. The present study aimed to identify the role of ANOs in pancreatic cancer.Methods:In an initial screen of ANOs, ANO9/TMEM16J was overexpressed in pancreatic cancer cells, and its role in the pathogenesis of pancreatic cancer was evaluated using an integrated in vitro and in vivo approach. To determine clinical relevance of the experimental findings, the prognostic value of ANO9 was evaluated in patients with pancreatic cancer.Results:The ANO9 mRNA and protein levels were increased in pancreatic cancer-derived cells. Exogenous expression of ANO9 in PANC-1 cells significantly increased cell proliferation in cell cultures and in mice. In contrast, knockdown of ANO9 in AsPC-1, BxPC-3, and Capan-2 cells strongly inhibited cell proliferation. Mechanistic analysis suggested that physical association of ANO9 with epidermal growth factor receptor (EGFR) underlies ANO9-induced cell proliferation. Knockdown of ANO9 augmented the effects of the EGFR inhibitor and the cytotoxic agent on pancreatic cancer cell proliferation. In addition, high ANO9 expression is a poor prognostic factor in patients with pancreatic cancer.Conclusions:The ANO9/TMEM16J appears to be a clinically useful prognostic marker for pancreatic cancer and a potential therapeutic target.

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

Cited by 14 publications

References 51 publications

Ion channel and lipid scramblase activity associated with expression of TMEM16F/ANO6 isoforms

Ion channel and lipid scramblase activity associated with expression of TMEM16F/ANO6 isoforms

Gain of function of TMEM16E/ANO5 scrambling activity caused by a mutation associated with gnathodiaphyseal dysplasia

ANO9/TMEM16J promotes tumourigenesis via EGFR and is a novel therapeutic target for pancreatic cancer

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