Abstract
Anoctamin 6 (ANO6) is a member of the recently identified TMEM16/anoctamin protein family comprising Ca2+-activated Cl− channels that generate outward-rectifying ionic currents in response to intracellular Ca2+ increase. ANO6 is also essential for Ca2+-dependent phospholipid scrambling required for blood coagulation. Selective serotonin reuptake inhibitors (SSRIs)—fluoxetine, sertraline, and paroxetine—that are used for the treatment of major depressive disorders can increase the risk of upper gastrointestinal bleeding after chronic treatment. However, at the earlier stage of intake, which is 1–7 days after the treatment, the possibility of blood coagulation might also increase, but transiently. Therefore, in this study, we investigated whether therapeutic SSRI concentrations affected the Cl− current or phospholipid scrambling activity of ANO6 by assessing ANO6 currents (I ANO6), phosphatidylserine (PS) exposure, and platelet aggregation. In the whole-cell patch mode, SSRIs facilitated Ca2+-dependent activation of IANO6 in ANO6-transfected cells, as evidenced by a significant decrease in the delay of IANO6 generation. On the other hand, in the inside-out patch clamp configuration, SSRIs showed an inhibitory effect on ANO6 currents, suggesting that SSRIs activate ANO6 via an indirect mechanism in intact cells. SSRIs also facilitated Ca2+-dependent PS exposure and α-thrombin-induced platelet aggregation. These results indicate that SSRIs at clinically relevant concentrations promote Ca2+-dependent activation of ANO6, which may have potential clinical implications such as the underlying mechanism of SSRI-induced adverse drug reactions.
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Acknowledgments
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2011-0014404, to J.H.N) and by grants 2013R1A3A2042197 and 2007-0056092 from the National Research Foundation, the Ministry of Science, ICT and Future Planning, Korea (to M.G.L).
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Hyun Jong Kim and Ikhyun Jun contributed equally to this work.
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Supplementary Fig. 1
Ca2+-dependent activation kinetics of hANO1. a-b Typical IANO1 traces in the whole-cell patch clamp with symmetrical NMDG-Cl (150 mM) and 200 nM free Ca2+ in the pipette solution and the related I-V curves in hANO1-overexpressing HEK293T cells (n = 6). I-V relationship was assessed for each event by voltage ramps from -100 mV to +100 mV at the rate (dV/dt) = 0.2 V/s. Note that IANO1 was generated immediately after the start of the whole-cell recording. (DOC 199 kb)
Supplementary Fig. 2
ANO6 expression and whole-cell patch clamp in mock-transfected HEK293T cells. a Comparison of ANO6 expression between mock- and ANO6-transfected HEK293T cells by immunoblotting analysis. ANO6 expression was detected only after long exposure of the membranes. b Representative traces of mock-transfected HEK293T cells in the whole-cell patch clamp with 10 μM free Ca2+ pipette solution. Note that IANO6 was not generated during the recording time (over 20 min). c Relative I-V relationship. (DOC 766 kb)
Supplementary Fig. 3
Inhibitory effects of SSRIs on ANO1 activation in HEK293T cells. IANO1 was inhibited by SSRIs in both whole-cell and inside-out patch clamps. The whole-cell patch clamp recording from hANO1-expressing HEK293T cells was performed using symmetrical (150 mM) NMDG-Cl (bath and pipette); the pipette solution contained 200 nM Ca2+ for ANO1 activation. For the inside-out patch clamp recording, the patches were exposed to 300 nM Ca2+, with symmetrical (150 mM) NMDG-Cl (left-side panels). I-V relationship was obtained by voltage ramps from −100 to +100 mV at the rate (dV/dt) = 0.2 V/s; the holding potential was 0 mV (right-side panels). Representative traces of fluoxetine (F), sertraline (S), and paroxetine (P) on IANO1 in inside-out (a, d, e) and whole-cell (b, d) patch clamp recording. The data are the results of three independent experiments. (DOC 175 kb)
Supplementary Fig. 4
Fluoxetine potentiates ANO6-like currents in PANC-1 cells. a Representative traces showing the effect of 300 μM fluoxetine on IANO6-like current traces with 3 μM Ca2+ pipette solution. b Mean I-V relationship curve obtained before fluoxetine treatment and during the application of the peak current after fluoxetine treatment. Data are presented as means ± SE (n = 7). (DOC 175 kb)
Supplementary Fig. 5
Fluoxetine at 100 μM induces PS exposure in both mock- and ANO6-transfected HEK293T cells. PS exposure of mock-transfected (a) or ANO6-transfected (b)HEK293T cells pre-treated with 100 μM fluoxetine for 10 min and then treated with of 1 μM ionomycin was detected by flow cytometry using Annexin V-FITC staining. Note that PS exposure occurred in both mock- and ANO6-transfected HEK293T cells (right panels). (DOC 774 kb)
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Kim, H.J., Jun, I., Yoon, J.S. et al. Selective serotonin reuptake inhibitors facilitate ANO6 (TMEM16F) current activation and phosphatidylserine exposure. Pflugers Arch - Eur J Physiol 467, 2243–2256 (2015). https://doi.org/10.1007/s00424-015-1692-6
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DOI: https://doi.org/10.1007/s00424-015-1692-6