Fig 2: Stim1 knockout diminishes physiological BK and TRPM4 channel activity.(A) Representative traces of spontaneous transient outward currents (STOCs) in cerebral artery smooth muscle cells (SMCs) from control and Stim1-smKO mice, recorded by perforated patch-clamp electrophysiology over a range of membrane potentials (-60 to 0 mV). (B, C) Summary data showing STOC frequency (B) and amplitude (C) (control, n = 13 cells from four animals; Stim1-smKO, n = 17 cells from five mice; *p<0.05, two-way ANOVA). (D) Representative traces of paxilline (1 µM)-sensitive BK currents in cerebral artery SMCs from control and Stim1-smKO mice, recorded by patch-clamping in conventional whole-cell mode during a series of command voltage steps (-100 to +100 mV). (E) Summary data for whole-cell BK currents (control, n = 6 cells from three mice; Stim1-smKO, n = 7 cells from three mice; two-way ANOVA). (F) Representative traces of TRPM4 currents in cerebral artery SMCs from control and Stim1-smKO mice voltage-clamped at –70 mV, recorded using perforated patch-clamp electrophysiology. TRPM4 currents were evoked as transient inward cation currents (TICCs) by application of negative pressure (–20 mmHg) through the patch pipette and were blocked by bath application of 9-phenanthrol (9-phen; 30 µM). (G) Summary data showing TICC activity as TRPM4 channel open probability (NPo) and (H) TICC amplitude in control (n = 12 cells from five mice) and Stim1-smKO (n = 15 cells from five mice) mice (*p<0.05, unpaired t-test). (I) Representative conventional whole-cell patch-clamp recordings of 9-phenanthrol–sensitive TRPM4 currents in cerebral artery SMCs from control and Stim1-smKO mice. Currents were activated by free Ca2+ (200 µM), included in the patch pipette solution, and were recorded using a ramp protocol from –100 to 100 mV from a holding potential of –60 mV. (J) Summary of whole-cell TRPM4 current density at +100 mV (control, n = 5 cells from three mice; Stim1-smKO, n = 5 cells from three mice, unpaired t-test). ns, not significant. Figure 7—source data 1.Individual data points and analysis summaries for datasets shown in Figure 7.

Fig 3: STIM1 colocalizes with BK and TRPM4.(A) Total internal reflection fluorescence (TIRF)-mode superresolution localization maps of freshly isolated vascular smooth muscle cells (VSMCs) from control mice immunolabeled for BK (red) and STIM1 (green). Colocalized BK and STIM1 clusters were identified by object-based analysis (OBA) and mapped (cyan). Scale bar: 3 µm. Panels to the right show enlarged areas of the original superresolution maps indicated by the white boxes. Arrows show examples of colocalizing clusters. Scale bar: 500 nm. (B) TIRF-mode superresolution localization maps of freshly isolated VSMCs from control mice immunolabeled for TRPM4 (cyan) and STIM1 (magenta). Colocalized TRPM4 and STIM1 clusters were identified by OBA and mapped (yellow). Scale bar: 2 µm. Panels to the right show enlarged areas of the original superresolution maps indicated by the white boxes. Arrows show examples of colocalizing clusters. Scale bar: 500 nm. (C) Colocalization frequency of BK and STIM1 clusters in imaged cells compared to colocalization frequency of BK and STIM1 clusters in randomized maps generated from respective cells. n = 11 cells from four mice (*p<0.05, paired t-test). (D) Colocalization frequency of TRPM4 and STIM1 clusters in imaged cells compared to colocalization frequency of TRPM4 and STIM1 clusters in randomized maps generated from respective imaged cells (n = 11 cells from four mice; *p<0.05, paired t-test). Figure 5—source data 1.Individual data points and analysis summaries for datasets shown in Figure 5.