Journal: Annals of Translational Medicine

Article Title: Homocysteine alters vasoreactivity of human internal mammary artery by affecting the K Ca channel family

doi: 10.21037/atm-20-6821

Figure Lengend Snippet: Effect of homocysteine (Hcy) on the relaxant response of internal mammary artery (IMA) to KCa channel activators. Hcy exposure significantly suppressed the BKCa channel activator NS1619-induced relaxation (A) whereas showed no influence on the IKCa/SKCa channel activator NS309-induced response (B). n=6. *P<0.05, ***P<0.001 vs. control.

Article Snippet: Cumulative dose-response curves were then established for (I) BK Ca channel activator NS1619 (−9–−4.5 LogM) (MedChemExpress, Cat#HY-12496), or (II) IK Ca /SK Ca channel activator NS309 (−9–−4.5 LogM) (MedChemExpress, Cat#HY-15416), or (III) acetylcholine (−10–−4.5LogM) (Sigma Aldrich, Cat#A6625) when the contraction reached a plateau.

Techniques: Control

Journal: Nature Chemical Biology

Article Title: Selective phosphorylation modulates the PIP2 sensitivity of the CaM–SK channel complex

doi: 10.1038/nchembio.1592

Figure Lengend Snippet: Figure 6 | CaM T79 phosphorylation facilitates modulation of SK2 by PIP2 hydrolysis triggered by stimulation of a GPCR, hMR1. (a) Activation of hMR1 inhibits the SK2 channel, and such inhibition is significantly enhanced by the phosphomimetic mutationT79D. Activation of hMR1 by ACh triggers Gq-PLC–mediated hydrolysis of PIP2. The T79C mutant was used instead of the WT to eliminate potential phosphorylation of WT CaM at T79. NS309, a positive SK channel modulator, or a 0 Ca2+ solution (no activity) was used to ensure that the currents recorded were indeed due to activation of SK channels by Ca2+. (b) T79D significantly increases the off-rate, obtained from fitting the time course of reduction of the current amplitudes after application of ACh. (c) T79D promotes inhibition of the SK2 channel activity by ACh-induced PIP2 hydrolysis. The remaining current due to ACh-induced inhibition is reduced from 70.3 ± 5.2% (n = 5, T79C) to 25.1 ± 2.84% (n = 4, T79D; P< 0.001). (d) T79D facilitates inhibition of SK2 by Ci-VSP–triggered dephosphorylation of PIP2. Activation of Ci-VSP by membrane depolarization (prepulse) dephosphorylates PIP2 and inhibits the SK2 channel activity. This inhibitory effect is significantly increased by the T79D mutation, and the remaining current after membrane depolarization is reduced from 88.7 ± 1.6% (n = 4, no Ci-VSP) to 68.6 ± 4.7% (n = 10, T79C + Ci-VSP) and 23.1 ± 5.0% (n = 5, T79D + Ci-VSP). Data in b–d represent mean values ± s.e.m., and the numbers of experiments are shown in the parentheses of the bar graph.

Article Snippet: SK channel modulator NS309 was from Tocris.

Techniques: Phospho-proteomics, Activation Assay, Inhibition, Mutagenesis, Activity Assay, De-Phosphorylation Assay, Membrane

Journal: Cells

Article Title: Altered Ca 2+ Homeostasis in Red Blood Cells of Polycythemia Vera Patients Following Disturbed Organelle Sorting during Terminal Erythropoiesis

doi: 10.3390/cells11010049

Figure Lengend Snippet: PV RBCs show increased Gárdos channel activity. ( A ) CCCP method analysis of PV and CT RBC membrane potential changes upon 100 µM NS309 addition. At the end of the experiment, cells were lysed with 3M NaCl 1% Triton X lysis solution to obtain the zero membrane potential (pHi = pHo) for absolute calculation of membrane potential. CT (mean—black line; SD—grey) and PV (mean—red line; SD—pink) RBCs. Data are displayed as mean with 95% confidence interval; CT ( n = 6) and PV ( n = 8). ( B ) Cell volume assay on Gárdos activity; 0.05% RBCs suspension was prepared in PBS with a final concertation of 0.2% BSA, 1 mM CaCl 2 , and 100 µM NS309. RBC size was measured using CASY before and 2.5, 5, 7.5, and 10 min after 100 μM NS309 addition. Mean with SD, n = 6, Mann–Whitney test. ( C – E ) Patch-clamp analysis. Upon cell catch external solution was added to the wells followed by 10 µM NS3623, 10 µM NS309, 5 µM TRAM-34, and 30 µM GdCl 3 . Currents were measured at room temperature applying −100 to +80 mV ramp voltage protocol for 300 ms, at a holding potential of −30 mV. The cell response was measured in pA at +80 mV. Statistical analysis of the currents at +80 mV in NS309 and TRAM-34 responding cells in ( C ) CT and PV RBCs ( n = 17; n = 48, respectively), ( D ) BaF3 EpoR JAK2 WT ( n = 20) and BaF3 EpoR JAK2 V617F ( n = 48), and ( E ) HEL ( n = 61) and HEL cells treated with 0.3 µM ruxolitinib for 24 h ( n = 63). Cell was considered responsive if it displayed at least a 20% current change. The left panel represents the cell current upon the addition of NS3623 (baseline), NS309, and TRAM-34. The central panel displays NS309-induced current increase, while the right panels represent TRAM-34-induced current decrease. The data are presented as median and box plots (25–75%) with whiskers (10–90%). Mann–Whitney test or Wilcoxon test, * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns—not significant.

Article Snippet: Three minutes after RBC addition, 100 µM of NS309 was added (a compound which shifts Ca 2+ sensitivity of Gárdos of one order of magnitude [ ] (Alomone labs, Jerusalem, Israel) followed by RBC lysis 8 min after RBC addition using 3M NaCl 1% Triton solution (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Activity Assay, Lysis, MANN-WHITNEY, Patch Clamp