Fig 2: Comparison of SK2-LI obtained with antibodies directed against the C-terminal and the N-terminal region of the SK2 peptide. Adjacent sections of SK2-LI detected with ANTI-CSK2 (a, c, e, g and i) and Anti-NSK2 (b, d, f, h and j). In hippocampus, SK2-LI was detected in molecular layer and in strata oriens and radiatum of CA1 (a, b). In granule cell layer, SK2-LI could be detected mainly in the plasma membrane (c, d). In pyramidal layer, somata and apical dendrites showed expression of SK2 peptide (e, f). In the amygdala, SK2-LI in basolateral nuclei was detected in cell somas (g, h). In the neocortex, SK2-LI detected with the N-terminal antibody showed only moderate immunoreactivity in cells of layer V (j), while SK2-LI detected with Anti-CSK2 was found as well in layer IV and VI (i). sr stratum radiatum, sp stratum pyramidale, so stratum oriens, slm stratum lacunosum moleculare, ml molecular layer, gl granule cell layer, pl pleomorphic cell layer, BLA basolateral amygdala, IV, V, VI layers of the cortex, Scale bar 200 µm in a, b; 30 µm in c–f; 60 µm in g–j

Fig 3: 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 4: 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 5: SK2 and Rab11 co-immunostaining in hippocampal CA1 region of WT and AS mice at different time points after baseline recording (used as control) and TBS applications with or without FSK treatment. (a) Representative images of SK2 (red) and Rab11 (green) co-immunostaining. Scale bar, 5 µm. (b) Quantitative results (mean ± SEM) of mean fluorescent intensity (MFI) of SK2 in different experimental groups (p = 0.0267, WT Baseline vs WT TBS 60 min, p = 0.0077, WT Baseline vs WT FSK/TBS 15 min, p < 0.001, WT Baseline vs WT FSK/TBS 60 min, p < 0.001, WT baseline vs AS Baseline, p < 0.001, AS Baseline vs AS TBS 15 min, p = 0.0020, AS Baseline vs AS FSK/Baseline, p < 0.001, AS Baseline vs AS FSK/TBS 15 min, p < 0.001, AS Baseline vs AS FSK/TBS 60 min, p < 0.001, AS TBS 60 min vs AS FSK/TBS 60 min, n = 3 slices from 3 mice/group, two-way ANOVA followed by Tukey test). (c) Quantitative results (mean ± SEM) of puncta dually labeled by anti-SK2 and anti-Rab11 antibodies in different experimental groups (p = 0.0361, WT Baseline vs WT TBS 15 min, p = 0.0014, WT Baseline vs WT FSK/TBS 15 min, p = 0.0149, WT Baseline vs WT FSK/TBS 60 min, p < 0.001, WT baseline vs AS Baseline, p = 0.0313, AS Baseline vs AS TBS 15 min, p = 0.0032, AS Baseline vs AS FSK/Baseline, p < 0.001, AS Baseline vs AS FSK/TBS 15 min, p = 0.011, AS Baseline vs AS FSK/TBS 60 min, p = 0.0017, AS TBS 60 min vs AS FSK/TBS 60 min, n = 3 slices from three mice/group, two-way ANOVA followed by Tukey test). See also Supplementary Fig. 1.