Spatiotemporal Control of Vascular Ca V 1.2 by α1 C S1928 Phosphorylation

Figures

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  Figure 1.. Increased CaV1.2 cooperativity and α1C clustering in cells of diabetic patients. A, Representative cell-attached recordings of arterial myocytes of people with diabetes mellitus (DM)] and without (ND) diabetes during a 2 s depolarizing step from −80 mV to −30 mV. B, Magnified areas from boxed areas in A, highlighting the closed (c) and unitary CaV1.2 opening levels (on). Summary data of (C) number of channels X open probability (nPo), unitary events (D) open-time and (E) closed-time histograms, (F) availability, (G) coupling frequency and (H) coupling strength (ND n=17 cells from 6 human samples; DM n=13 cells from 6 human samples). (I) Representative super-resolution total internal reflection fluorescence (TIRF) images of arterial myocytes labeled for α1C subunits with 2 magnified areas showing cluster size and distribution in arterial myocytes from ND and DM samples (scale bars=10 μm→1 μm→0.2 μm). Amalgamated data for (J) cluster size and (K) cluster density. (L) Representative nearest-neighbor interpolation maps color-coded as a function of the number of neighbors within a 250 nm radius and summary data fitted with Gaussian curves (ND n=29 cells from 6 human samples; DM n=29…

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  Figure 2.. pS1928 controls CaV1.2 cooperative gating upon HG. A, B, Voltage dependency of activation and steady-state inactivation fitted with a Boltzmann model from wild-type (WT) and S1928A arterial myocytes under control/10 mmol/L D-glucose conditions and 20 mmol/L D-glucose (WT: n=9 cells from 4 mice; S1928A: n=8 cells from 4 mice). C, Representative cell-attached recordings from WT and S1928A arterial myocytes during a 2 s depolarizing step from −80 mV to −30 mV upon 10 mmol/L and 20 mmol/L D-glucose. D, Magnified areas from boxed regions in C highlighting the closed (c) and unitary CaV1.2 opening levels (on). Summary data of (E) nPo, (F, G) unitary events open and closed-time histograms for WT and S1928A, (H) availability, (I) coupling frequency and (J) coupling strength (WT n=9 cells from 4 mice; S1928A n=11 cells from 4 mice). Data are mean±SEM. Significance was assessed with extra sum-of-squares F test (A: V50-activation P=0.5601, V50-inactivation P=0.0989; B: WT V50-activation P=0.0013, V50-inactivation P=0.1067, S1928A V50-activation P=0.1273, V50- inactivation P=0.2640; F: P=0.0002 and G: P=0.0521) and nested two-way ANOVA (E: P=0.0007, H: P=0.0345, I: P=0.0314 and J: P=0.0280)…

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  Figure 3.. pS1928 mediates increased α1C clustering upon HG. A, Representative images of proximity ligation assay (PLA) puncta of α1C-α1C interactions from WT and S1928A myocytes upon 10 mmol/L and 20 mmol/L D-glucose. Dashed lines represent cells’ footprints and summary puncta/μm2 (WT 10 D-glu n=32 cells from 5 mice, 20 D-glu n=37 cells from 5 mice; S1928A 10 D-glu n=22 cells from 3 mice, 20 D-glu n=22 cells from 3 mice). B, Representative super-resolution TIRF images of arterial myocytes labeled for α1C with 2 magnified areas showing cluster size and distribution in WT and S1928A arterial myocytes (scale bars=10 μm→1 μm→ 0.2 μm). Amalgamated data for (C) cluster size and (D) cluster density. (E) Representative nearest-neighbor interpolation maps color-coded as a function of the number of neighbors within a 250-nm radius and frequency distribution fitted with Gaussian curves (WT 10 D-glu n=27 cells from 3 mice, 20 D-glu n=27 cells from 3 mice; S1928A 10 D-glu n=20 cells from 3 mice, 20 D-glu n=19 cells from 3 mice). Data are mean±SEM. Significance was assessed with nested 2-way ANOVA with Bonferroni post-hoc test (A: P=0.0281, C: P=8.0×10−13, D: P=0.5568) and with extra sum-of-squares F…

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  Figure 4.. S1700 phosphorylation (pS1700) is not required for HG-induced CaV1.2 cooperative gating. (A) Exemplary whole-cell nifedipine-sensitive IBa traces to a depolarizing step from −70 mV to +10 mV and amalgamated current density from WT and S1700A (n=9 cells from 3 mice). (B) Representative cell-attached recordings from WT and S1700A cerebral arterial myocytes during a 2 s depolarizing step from −80 mV to −30 mV upon 10 mmol/L and 20 mmol/L D-glucose. (C) Magnified areas from boxed regions in B highlighting the closed (c) and unitary CaV1.2 opening levels (on). Summary data of (D) nPo, (E) availability, (F) coupling frequency and (G) coupling strength (n=9 cells from 3 mice). H, Representative diameter recording, and summary percentage myogenic tone from S1700A cerebral arteries upon 10 mmol/L and 20 mmol/L D-glucose (n=6 arteries from 5 mice). Data are mean±SEM. Significance was assessed with nested t-test. P values within each panel and Table S8.

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  Figure 5.. pS1928 is necessary for increased arterial myocyte Ca2+ and contractility upon HG. A, Representative traces of perforated whole-cell recordings from WT and S1928A cerebral arterial myocytes in current-clamp mode with a gap-free protocol and summary data of Vm upon 10 mmol/L D-glu, 20 mmol/L D-glue and 60 mmol/L KCl (WT n=10 cells from 3 mice; S1928A n=8 cells from 3 mice). (B) Representative normalized pseudo-colored confocal images at different time points and resulting fluorescence (black) and cell length (red) traces of WT and S1928A cerebral arterial myocytes loaded with the fluorescent Ca2+ indicator fluo-4 AM. Summary data of peak [Ca2+]i and cell length of WT and S1928A arterial myocytes upon 20 mmol/L D-glu or 20 mmol/L L-glu ([Ca2+]i: WT 20 mmol/L D-glu n=17 cells from 9 mice, 20 mmol/L L-glu n=8 cells from 5 mice; S1928A 20 mmol/L D-glu n=12 cells from 5 mice; cell length: WT 20 mmol/L D-glu n=14 cells from 6 mice, 20 mmol/L L-glu n=8 cells from 5 mice; S1928A 20 mmol/L D-glu n=12 cells. From 5 mice). (C) Representative pseudo-colored blood flow images of WT and S1928A cerebral pial arteries through a cranial window exposed to 10 mmol/L D-glu, 20 mmol/L D-glu or 0 Ca2+…

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  Figure 6.. pS1928 is required for increased vascular α1C clustering in diabetes. (A) Representative images of PLA puncta and summary puncta/μm2 for α1C-α1C interactions in sham and streptozotocin (STZ)-treated WT and S1928A arterial myocytes (WT sham n=40 cells from 6 mice; WT STZ n=34 cells from 6 mice; S1928A sham n=32 cells from 5 mice; S1928A STZ n=34 cells from 5 mice). Dashed lines represent the cells’ footprints. (B) Representative super-resolution total internal reflection fluorescence (TIRF) images of arterial myocytes labeled for α1C with 2 magnified areas showing cluster size and distribution in arterial myocytes from sham or STZ-treated WT and S1928A mice (scale bars=10 μm→1 μm→0.2 μm). (C) Amalgamated data for cluster size. (D) Representative nearest-neighbor interpolation map color-coded as a function of the number of neighbors within a 250 nm radius and frequency distribution data fitted to Gaussian curves (WT sham n=34 cells from 6 mice, WT STZ n=29 cells from 5 mice; S1928A sham n=41 cells from 5 mice, S1928A STZ n=34 cells from 5 mice). Data are mean±SEM. Significance was assessed with nested 2-way ANOVA with Bonferroni post-hoc test (A: P=0.0024, C: P=0.0079) and with…

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  Figure 7.. Functional changes in CaV1.2, arterial contractility, and blood flow during diabetes require pS1928. A, Magnified areas of traces in S6B highlighting the closed (c) and unitary CaV1.2 opening levels (on) of representative cell-attached recordings from sham or STZ WT and S1928A arterial myocytes during a 2 s depolarizing step from −80 mV to −30 mV. Summary data of CaV1.2 (B) nPo, (C) availability, (D) coupling frequency, (E) coupling strength and (F, G) unitary events open and closed-time histograms obtained from WT and S1928A cells (WT sham n=19 cells from 7 mice; WT STZ n=10 cells from 5 mice; S1928A sham n=6 cells from 5 mice; S1928A STZ n=8 cells from 5 mice). H, Representative pseudo-colored blood flow images of sham and STZ-treated WT and S1928A cerebral pial arteries through a cranial window exposed to 10 mmol/L D-glu and 0 Ca2+ + vasodilatory mix. Summary data of (I) myogenic tone and (J) flux (WT sham n=9 arteries from 5 mice; WT STZ n=9–10 arteries from 5 mice; S1928A sham n=10–11 arteries from 5 mice; 1928A STZ n=12 arteries from 5 mice). Data are mean±SEM. Significance was assessed with nested 2-way ANOVA with Bonferroni post-hoc test (B: P=0.0009, C: P=0.0318, D:…