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Additional Supporting Information can be found in the online version of this article.

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ANA_22010_sm_SupFig1.tif4242KSupplementary Figure 1. Characterization of KCNK5-transfected HEK293 cells. A) Immunocytochemical stainings reveal a clear expression of K2P5.1 on transfected HEK cells (right side, cell nuclei counterstaining with DAPI). Omittance of primary antibody reveals no positive signal (negative control; left side). B) By western blotting, K2P5.1 protein expression (∼ 60 kDa) was detected in human T cells (T cells) and untransfected HEK cells (HEK). Signal intensity was increased for transfected HEK cells (HEK + TASK2) while T cells from KCNK5−/− mice (negative signal) showed no signal at 60 kDa. C) Current density (pA/pF) of K2P5.1 currents was significantly elevated in transfected (HEK+; n = 20; p = 0.0003) in comparison to untransfected HEK cells (HEK-; n = 6). D) Typical K2P5.1 currents were elicited by depolarizing 500 ms steps from −80 mV to 40 mV (inset). Sensitive currents were obtained by graphical substraction of control currents and currents after K2P5.1 modulation. E) Effects of different blockers on KCNK5-transfected HEK cells (n = 4-8; control; anandamide 30 μM, 5 ± 8%; charybdotoxin 10 nM, 26 ± 5%; ShK 10 nM, 4 ± 5%; maurotoxin 100 nM, 18 ± 4%; kaliotoxin 1 nM, 19 ± 3% and TRAM-34 100 nM, 30 ± 6%. ** p < 0.05 F) Computer modeling quantifies quinidine, ShK and TRAM effects on the single component level. Left and middle panel show overlay plots with contrasted measured currents (light gray traces) and modeled results (black traces). The applied voltage clamp protocol started at −120 mV and reached 40 mV after 200 msec. Left panel: With respect to control currents (solid line) a reduction of ITASK2 to 10% and Ileak to 60% matches the measured quinidine effect (dashed line). Middle panel: Reducing IKv1.3 to 10% and Ileak to 75% of the control values (upper solid line) suffices to mimic the effect of ShK application (dashed line). The combination of ShK and TRAM (lower solid line) required IC, ITASK2, IKv1.3 and Ileak to be set to 5%, 70%, 10% and 50% respectively. Right panel: Current reduction was calculated according to the maximum current values of each experiment.
ANA_22010_sm_SupFig2.tif3218KSupplementary Figure 2. Properties of different human T cell subtypes. A) Flow cytometry for CCR7 and CD45RA was used to dissect naive, TCM and TEM CD4+ T cells after MACS isolation. B) Electrophysiological recordings on freshly isolated T cell subtypes (current reduction after application of quinidine 20 μM; n = 3− 5). C) Chronically activated CD4+ T lymphocytes develop into TEM. Freshly isolated T lymphocytes (d 0) were stained for CCR7 and CD45RA (left side) and show predominantly a naïve phenotype (CCR7+CD45RA+). Long time stimulated CD4+ T lymphocytes (day 35) are depicted on the right side, showing enrichment of TEM (CCR7−CD45RA−).
ANA_22010_sm_SupFig3.tif1343KSupplementary Figure 3. Influence of the pH value on K2P5.1 upregulation upon stimulation. The left panel shows the delta pH value of the T cell medium in comparison to day 0 during 5 days of stimulation (decrease from pH 7.2 to 6.6). The right panel shows the difference between K2P5.1 upregulation in pH adjusted medium (every day to pH 7.2) and pH-decreasing medium in %, indicating that stable pH values result in a stronger upregulation of K2P5.1 (about +30% over 5 days). One representative example out of three is shown.
ANA_22010_sm_SupFig4.tif3983KSupplementary Figure 4. Supporting data on MS patients' characteristics. A) Delta ct values of patients with a stable disease course (relapsing-remitting, no therapy, stable), acute relapses (active) and healthy controls (control) is shown. Corresponding quantification can be found in Fig 4 A. B) K2P5.1 expression of patients with RRMS receiving disease-modifying therapies were investigated (control, healthy donors; stable, no therapy (CD4: n = 12, CD8: n = 11); glatiramer acetate (CD4: n = 12, CD8: n = 13), mitoxantrone (CD4: n = 14, CD8: n = 14), betainterferons (CD4: n = 12, CD8: n = 12)) which did not differ significantly from untreated stable MS patients, respectively. C) K2P5.1 expression on CD8+ and CD4+ cells of patients with NMO compared to healthy controls.
ANA_22010_sm_SupFig5.tif3278KSupplementary Figure 5. Characterization of the role of KCa3.1 and KV1.3 on human T cells. A) + B) Expression levels of KCa3.1 (A) and KV1.3 (B) were investigated in T cells from the peripheral blood of MS patients (stable versus active disease versus controls). C) + D) Correlation between ΔΔct expression levels of K2P5.1 versus KCa3.1 (C) and K2P5.1 versus KV1.3 (D) in individual patients. Only weak correlation levels were found (K2P5.1/KCa3.1 R = 0.31; K2P5.1/KV1.3 R = 0.11). E) + F) MACS-isolated CD4, CD8, naive T cells, TEM and TCM (all from the same healthy donor) were assessed for expression levels of KCa3.1 (C) and KV1.3 (D) freshly after isolation and after 2 days of in vitro stimulation (n = 5−8; CD3/CD28 beads; cell to bead ratio = 2:1).
ANA_22010_sm_SupMaterialandMethods.doc30KSupplementary Materials and Methods
ANA_22010_sm_OnlineSupplementalMaterial.doc22KOnline supplemental material
ANA_22010_sm_SupTabs.doc49KSupplementary Tables

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