Fig 1: SK2 phosphomimetic mutation S465D alleviates voltage-dependent inhibition of SK channels by [Ca2+] A, representative traces of [Ca2+]i transients and I SK at baseline and after incubation with ISO (100 nmol L–1 for 3 min) in VMs expressing the rSK2-S465D mutant and dnPLB. B, pooled mean ± SD I–V and peak [Ca2+]/V relationships for (A), n = 7–8, N = 6. I SK: * P = 0.01 (-25 mV), P = 0.02 (-15 mV), P = 0.03 (-5 mV), P = 0.03 (5 mV), P = 0.02 (15 mV), P = 0.008 (25 mV), P = 0.006 (35 mV), P = 0.02 (45 mV, dnPLB), P = 0.008 (55 mV) vs. baseline. ** P = 0.02 (45 mV), P = 0.03 (55 mV) vs. dnPLB. [Ca2+]: * P = 0.01 (-25 mV), P = 0.04 (-25 mV, dnPLB), P = 0.004 (-15 mV), P = 1.4 × 10-4 (-5 mV), P = 0.03 (-5 mV, dnPLB), P = 1.4 × 10-4 (5 mV), P = 0.04 (5 mV, dnPLB), P = 1.7 × 10-4 (15 mV), P = 2.2 × 10-5 (25 mV), P = 0.02 (25 mV, dnPLB), P = 1.5 × 10-5 (35 mV), P = 0.03 (35 mV, dnPLB), P = 6.4 × 10-5 (45 mV), P = 0.001 (55 mV) vs. baseline. ** P = 0.047 (5 mV), P = 0.02 (15 mV), P = 0.01 (25 mV), P = 0.006 (35 mV), P = 0.006 (45 mV), P = 0.03 (55 mV) vs. dnPLB, one-way ANOVA with a Bonferroni post hoc test. C, plot of EC50 values for rSK2 WT and the rSK2-S465D recorded under basal conditions and in the presence of the ß-adrenergic agonist ISO (100 nmol L–1). Mean ± SD of data indicated by line. P = 1 for all comparisons, n = 5-12, N = 4–5, one-way ANOVA.
Fig 2: PLA reveals low level colocalization of SK2-RyR2 complexes with CSQ in Sham and TAB rat VMs A, PLA of SK2 and RyR2 and colocalization with CSQ. Left: each red fluorescent dot represents a site of interaction of proteins that are within 40 nm proximity. Centre: cells were probed with anti-CSQ antibody. Right: merged image showing colocalization of SK2 protein pairs with RyR2. B, Manders coefficients M1 and M2 reveals a low level of colocalization of SK2-RyR2 with CSQ. No significant differences were observed between groups. Student's t test: Sham, n = 8; TAB, n = 15; N = 3 per group. C, SK2, RyR2, Cav1.2a1c and CSQ primary antibodies without PLA protein pairing.
Fig 3: BN-PAGE reveals a physical interaction of SK2 and the a1c subunit of LTCC in Sham and TAB rat VMs Samples for BN-PAGE were prepared from the membrane fraction of freshly isolated Sham and TAB rat VMs. SK2 and the a1c subunit of LTCC can be detected at the same band, indicative of a physical interaction between these two proteins. NKA was used as a loading control. N = 3 per group.
Fig 4: Enhanced phosphorylation of SK2-S465 in TAB rats VMs is PKA-dependent A, representative western blot of SK2 and phosphorylated S465 (left) and plot of p-S465 band optical density (OD) normalized to SK2 levels for Sham and TAB rats (right). Each lane represents a sample from a separate rat. * P = 0.005, paired Student's t test. B, representative western blot of SK2 and phosphorylated S465 (top) and plots of p-S465 band OD normalized to SK2 levels for Sham and TAB rats under baseline conditions, or treated with ISO (* P = 0.01, paired Student's t test), KN93 or H89 (* P = 0.04, one-way ANOVA with a Bonferroni post hoc test) (bottom).
Fig 5: PLA reveals low level colocalization of SK2-Cav1.2a1c complexes with RyR2 in Sham and TAB rat VMs A, PLA of SK2 and Cav1.2a1c, and colocalization with RyR2. Left: each red fluorescent dot represents a site of interaction of proteins that are within 40 nm proximity. Centre: cells were probed with anti-RyR2 antibody. Right: merged image showing colocalization of SK2 protein pairs with RyR2. B, PLA and RyR2 secondary antibodies only, as a technical control. C, quantification of the number of PLA puncta per µm2 in Sham and TAB VMs. No significant differences were observed between groups, Student's t test, Sham n = 29, TAB n = 28, N = 4 per group. D, Manders coefficients M1 and M2 reveals a low level of colocalization of SK2-Cav1.2a1c with RyR2. No significant differences were observed between groups Student's t test: Sham, n = 31; TAB, n = 26; N = 4 per group.
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