α1C S1928 Phosphorylation of CaV1.2 Channel Controls Vascular Reactivity and Blood Pressure

Abstract

Background: Increased vascular Ca_V1.2 channel function causes enhanced arterial tone during hypertension. This is mediated by elevations in angiotensin II/protein kinase C signaling. Yet, the mechanisms underlying these changes are unclear. We hypothesize that α1_C phosphorylation at serine 1928 (S1928) is a key event mediating increased Ca_V1.2 channel function and vascular reactivity during angiotensin II signaling and hypertension.

Methods and results: The hypothesis was examined in freshly isolated mesenteric arteries and arterial myocytes from control and angiotensin II-infused mice. Specific techniques include superresolution imaging, proximity ligation assay, patch-clamp electrophysiology, Ca²⁺ imaging, pressure myography, laser speckle imaging, and blood pressure telemetry. Hierarchical "nested" and appropriate parametric or nonparametric t test and ANOVAs were used to assess statistical differences. We found that angiotensin II redistributed the Ca_V1.2 pore-forming α1_C subunit into larger clusters. This was correlated with elevated Ca_V1.2 channel activity and cooperativity, global intracellular Ca²⁺ and contraction of arterial myocytes, enhanced myogenic tone, and altered blood flow in wild-type mice. These angiotensin II-induced changes were prevented/ameliorated in cells/arteries from S1928 mutated to alanine knockin mice, which contain a negative modulation of the α1_C S1928 phosphorylation site. In angiotensin II-induced hypertension, increased α1_C clustering, Ca_V1.2 activity and cooperativity, myogenic tone, and blood pressure in wild-type cells/tissue/mice were averted/reduced in S1928 mutated to alanine samples.

Conclusions: Results suggest an essential role for α1_C S1928 phosphorylation in regulating channel distribution, activity and gating modality, and vascular function during angiotensin II signaling and hypertension. Phosphorylation of this single vascular α1_C amino acid could be a risk factor for hypertension that may be targeted for therapeutic intervention.

Keywords: cardiovascular; clustering; cooperativity; diabetes; hypertension.

Figure 1. pS1928 controls Ca_V1.2 biophysical properties upon Ang II exposure.

A, Representative nifedipine‐sensitive Ba²⁺ currents (I _Ba) elicited by a single voltage step from −70 mV to +10 mV recorded from WT and SA arterial myocytes under control conditions  and after exposure to 100 nmol/L Ang II. The right‐side panel shows summary scatter plots (WT: n=9 cells from 4 mice; SA ct: n=6 cells from 3 mice). Significance was assessed with nested 2‐way ANOVA with Bonferroni post hoc test (P=0.0473). B, Exemplary single‐channel traces obtained in cell‐attached mode using a single voltage step from −80 mV to −30 mV recorded from WT and S1928A arterial myocytes under control conditions and after exposure to 100 nmol/L Ang II. Ca²⁺ was used as the charge carrier to visualize Ca²⁺‐dependent Ca_V1.2 cooperative events. Sections of the traces in the boxes are magnified below for a detailed appreciation of openings and cooperative events. The baseline level is highlighted by a (c) and unitary levels by (o_n). Summary data of (C) channel activity (nPo), (D) channel availability, (E) coupling frequency, and (F) coupling strength (κ) (WT ct: n=13 cells from 7 mice; WT Ang II: n=15 cells from 6 mice; SA ct: n=15 cells from 5 mice; SA Ang II: n=13 cells from 5 mice). Significance was assessed with nested 2‐way ANOVA with Bonferroni post hoc test (P=1.9×10⁻⁹ for 1C; P=0.0615 for D—significant change at P<0.1 and unpaired t test between WT ct and WT Ang II with P=0.0199 (t in graph); P=0.0016 for E; P=0.0132 for F). Data are mean±SEM. P values for relevant comparisons within each panel and supplemental material. Ang II indicates angiotensin II; ct, control conditions; nPO, number of channels X open probability; pS1928, serine 1928 phosphorylation; SA, serine 1928 mutated to alanine; and WT, wild type.

Figure 2. Ang II increased α1_C clustering requires pS1928.

A, Exemplary superresolution total internal reflection fluorescence images of WT and SA arterial myocytes labeled for α1_C with 2 magnified areas under control conditions and after exposure to 100 nmol/L Ang II (scale bars=5 μm→5 μm→200 nm). B, Plot of the calculated pair‐correlation function (g(r)) of α1_C clusters in WT and SA arterial myocytes under control conditions and after 100 nmol/L Ang II exposure. Scatter plots of (C) α1_C cluster radius (nm), (D) α1_C cluster density (number of clusters/μm²) and (E) estimated α1_C proteins per cluster (N^cluster) in WT and SA arterial myocytes under control conditions and after 100 nmol/Ls Ang II exposure (WT ct: n=27 cells from 7 mice; WT Ang II: n=34 cells from 7 mice; SA ct: n=38 cells from 5 mice; SA Ang II: n=50 cells from 7 mice). Significance was assessed with nested 2‐way ANOVA with Bonferroni post hoc test (P=0.0268 for C; P=0.0676 for interaction and P=0.0108 for D; P=0.0342 for E). F, Cartoon of the modified PLA approach. Representative maximal projection images of PLA puncta for α1_C‐α1_C interactions in WT (G) and SA (H) male arterial myocytes under control conditions, after Ang II treatment or Ang II+cal C. The fifth image is a representative negative control image for PLA in which only 1 anti‐α1_C‐tagged antibody was added to WT ct arterial myocytes. Dotted lines outline the cells. Scale bars=10 μm. I, Scatter plot of PLA α1_C‐α1_C puncta per cell area (μm²) in WT ct, WT Ang II, WT+cal C, WT Ang II+cal C, SA ct, and SA Ang II arterial myocytes (WT ct: n=23 cells from 5 mice; WT Ang II: n=31 cells from 5 mice; WT+cal C: n=40 cells from 6 mice; WT Ang II+cal C: n=31 cells from 5 mice; SA ct: n=46 cells from 5 mice; SA Ang II: n=46 cells from 5 mice). WT arterial myocytes treated with only 1 of the PLA probes were used as negative control (neg ct; n=4 cells from 2 mice). Significance was assessed with nested 1‐way ANOVA (1W) for WT comparisons or nested 2‐way ANOVA (2W) for WT/SA comparisons with Bonferroni post hoc test (P=4.1×10⁻⁶ for WT comparisons; P=1.3×10⁻⁵ for WT/SA comparisons). Data are mean±SEM. P values for relevant comparisons within each panel and supplemental material. Ang II indicates angiotensin II; cal C, calphostin C; ct, control conditions; PLA, proximity ligation assay; pS1928, serine 1928 phosphorylation; SA, serine 1928 mutated to alanine; and WT, wild type.

Figure 3. pS1928 mediates altered arterial myocyte excitation‐contraction coupling upon Ang II.

A, Simulated Ang II‐mediated effects on arterial myocyte V _M and [Ca²⁺] _i . To simulate the gradual effects of Ang II over time, full modification of Ang II‐dependent model parameters is reached after 180 seconds (s) from the initial administration. The mathematical model was parameterized using a 100% increase in Ca²⁺ currents, 60% reduction in K⁺ currents and 75% increase in TRP currents upon Ang II exposure in WT cells, and 60% reduction in K⁺ currents and 75% increase in TRP currents with no change in Ca²⁺ currents upon Ang II exposure in SA cells. The changes in Ca²⁺, K⁺, and TRP currents are consistent with prior data and results here., , , , , B, Representative traces of perforated whole‐cell recordings from WT and SA mesenteric arterial myocytes in current‐clamp mode with a gap‐free protocol and (C) summary data of V _M under control conditions and after exposure to 100 nmol/L Ang II and 60 mmol/L KCl (WT n=9 cells from 3 mice; SA n=9 cells from 3 mice). HK⁺=60 mmol/L K⁺. Significance was assessed with nested 2‐way ANOVA with Bonferroni post hoc test (P=0.3882 in interaction and P=7.9×10⁻¹³ in treatment). D, Exemplary normalized pseudocolored confocal images at different time points and resulting fluorescence (E) and cell length (F) traces of WT and S1928A mesenteric arterial myocytes loaded with the fluorescent Ca²⁺ indicator fluo‐4 AM. Summary data of (E) peak [Ca²⁺] _i and (F) cell length of WT and SA arterial myocytes after exposure to 100 nmol/L Ang II. In some experiments, WT cells were pre‐treated with 100 nmol/L nif or 100 nmol/L calphostin C (cal C) before application of 100 nmol/L Ang II (WT Ang II: n=4 mice; WT Ang II+nif: n=5 mice; WT Ang II+cal C: n=5 mice; SA Ang II: n=5 mice). Significance was assessed with 1‐way ANOVA with Bonferroni post hoc for comparisons between WT conditions (P=1.4×10⁻⁷ for 3E and P=7.5×10⁻⁸ for 3F). For comparisons between WT and SA conditions, unpaired t test was used. Data are mean±SEM. P values for relevant comparisons within each panel and supplemental material. Ang II indicates angiotensin II; [Ca²⁺] _i , intracellular Ca²⁺ concentration; nif, nifedipine; pS1928, serine 1928 phosphorylation; SA, serine 1928 mutated to alanine; TRP, transient receptor potential; V _M, membrane potential; and WT, wild type.

Figure 4. pS1928 sustains arterial constriction upon Ang II.

A, Representative normalized diameter traces of WT and SA male mesenteric arteries pressurized to 60 mm Hg and treated with 100 nmol/L Ang II and 0 Ca²⁺+ 1 μmol/L nifedipine. The peak and plateau tones are highlighted by arrows. Scatter plots of (B) basal tone, (C) Ang II‐induced peak diameter reduction, and (D) plateau tone (n=6 mice per group). Significance was assessed with unpaired t test for all plots. (E) Representative pseudocolored blood flow (ie, flux) images of WT and SA male mesenteric arteries through a laparotomy before and after exposure to Ang II and 0 Ca²⁺ plus a vasodilatory mix (described in the Methods section). (F) Exemplary normalized arterial diameter (upper panel) and normalized blood flux (lower panel) before and after exposure to increasing concentrations of Ang II (from log−10 mol/L to log−5 mol/L) and subsequent application of 0 Ca²⁺ plus a vasodilatory mix. G and H, Summary plots of basal tone and basal flux, respectively (n=6 mice per group). Significance was assessed with unpaired t test. I and J, Exemplary 3‐dimensional graphs highlighting the peak and 5‐minute normalized arterial diameter and blood flux responses, respectively, to the range of Ang II concentrations in mesenteric arteries from WT and SA male mice. In some experiments, mesenteric arteries from WT mice were pretreated with 1 μmol/L nifedipine before the application of the range of Ang II concentrations to examine the role of nifedipine‐sensitive channels in Ang II‐induced alterations in arterial diameter and blood flux in vivo. K and L, Amalgamated data of normalized diameter and blood flux, respectively, in mesenteric arteries from WT, WT+nifedipine, and SA male mice after 5 minutes in response to a range of Ang II concentrations (WT: n=8 mice; WT+nifedipine: n=8 mice; SA: n=7 mice). Significance was assessed with multiple unpaired t test (relevant comparisons between WT and SA data in Figure 4K: P=0.0197 at −9 M Ang II; P=0.0571 at −8 M Ang II; P=0.0004 at −7 M Ang II; P=0.0085 at −6 M Ang II; P=0.1250 at −5 M Ang II, and between WT and SA data in Figure 4L: P=0.9999 at −9 M Ang II; P=0.0817 at −8 M Ang II; P=0.0139 at −7 M Ang II; P=0.0018 at −6 M Ang II; P=0.0088 at −5 M Ang II). Data are mean±SEM. P values for relevant comparisons within each panel and supplemental material. Ang II indicates angiotensin II; nif, nifedipine; pS1928, serine 1928 phosphorylation; SA, serine 1928 mutated to alanine; and WT, wild type.

Figure 5. Increased Ca_V1.2 activity and α1_C clustering during hypertension.

A, Scatter plots of (A) channel activity (nPo), (B) channel availability, (C) coupling frequency and (D) coupling strength (κ) in sham and hypertension WT and SA arterial myocytes (WT sham: n=15 cells from 5 mice; WT hypertension: n=16 cells from 7 mice; SA sham: n=11 cells from 4 mice; SA hypertension: n15 cells from 5 mice). Significance was assessed with nested 2‐way ANOVA with Bonferroni post hoc test (P=0.0054 for A; P=0.0068 for B; P=0.1721 for interaction and P=0.0099 for treatment for C; P=0.4589 for interaction and P=0.0001 for treatment for D). E, Representative super‐ TIRF images of sham and hypertension WT and SA arterial myocytes labeled for α1_C with 2 magnified areas (scale bars = 5 μm → 200 nm). F, Plot of the calculated pair‐correlation function (g(r)) of α1_C clusters in sham and hypertension WT and SA arterial myocytes. Scatter plots of (G) α1_C cluster radius (nm), (H) α1_C cluster density (number of cluster/μm²) and (I) estimated α1_C proteins per cluster (N^cluster) in sham and hypertension WT and SA arterial myocytes (WT sham: n=16 cells from 5 mice; WT hypertension: n=37 cells from 6 mice; SA sham: n=25 cells from 6 mice; SA hypertension: n=26 cells from 6 mice). Significance was assessed with nested 2‐way ANOVA with Bonferroni post hoc test (P=0.0216 for G; P=0.9509 for H; P=0.6589 for interaction and 0.0056 for genotype for I). J, Scatter plot of PLA α1_C‐α1_C puncta per cell area (μm²) in sham and hypertension WT and SA arterial myocytes (WT sham: n=29 cells from 6 mice; WT hypertension: n=50 cells from 7 mice; SA sham: n=28 cells from 5 mice; SA hypertension: n=27 cells from 5 mice). WT sham arterial myocytes treated with only 1 of the PLA probes were used as negative control (neg ct; n=4 cells from 2 mice). Significance was assessed with nested 2way ANOVA with Bonferroni post hoc test (P=0.0252). K, Representative superresolution TIRF images of BPN and BPH arterial myocytes labeled for α1_C with 2 magnified areas (scale bars=5 μm→200 nm). Scatter plots of (L) α1_C cluster radius (nm), (M) α1_C cluster density (number of cluster/μm²), and (I) estimated α1_C proteins per cluster (N^cluster) in BPN and BPH arterial myocytes (BPN: n=47 cells from 5 mice; BPH: n=47 cells from 6 mice). Significance was assessed with nested t test. Data are mean±SEM. P values for relevant comparisons within each panel and supplemental material. BPH indicates blood pressure highp; BPN, blood pressure normal; ct, control conditions; HTN, hypertension; nPO, number of channels X open probability; PLA, proximity ligation assay; SA, serine 1928 mutated to alanine; TIRF, total internal reflection fluorescence; and WT, wild type.

Figure 6. pS1928 regulates Ca_V1.2 function, vascular reactivity, and blood pressure during hypertension.

Representative diameter recordings over a pressure range (from 10 to 100 mm Hg) and plot summary data of percentage myogenic tone obtained using mesenteric arteries from WT (A) and SA (B) sham and hypertension mice (WT sham: n=12 arteries from 5 mice; WT hypertension: n=9 arteries from 5 mice; SA sham: n=8 arteries from 5 mice; S1928A hypertension: n=7 arteries from 5 mice). Significance was assessed with nested t‐test. Plots of systemic (C) and diastolic (D) blood pressure at day 0 (blue circles) and day 7 (red circles) from sham and hypertensive WT and SA mice using radio telemetry (n=5 mice per group). Significance was assessed with 3‐way ANOVA with Bonferroni post hoc test (P=0.0017 for C; P=0.0299 for D). E, Scatter plot of ΔMAP from day 0 to day 7 in sham and hypertensive WT and SA mice (n=5 mice per group). Significance was assessed with 2‐way ANOVA with Bonferroni post hoc test (P=1.4×10⁻⁴). Data are mean±SEM. P values for relevant comparisons within each panel and supplemental material. ΔMAP, change in mean arterial pressure; HTN, hypertension; pS1928, serine 1928 phosphorylation; SA, serine 1928 mutated to alanine; WT, wild type.

Figure 7. Model by which Ang II/PKC signaling promotes α_1C pS1928 to modulate the α1_C/Ca_V1.2 channel spatial and temporal properties leading to changes in global [Ca²⁺] _i , myogenic tone, blood flow, and blood pressure during hypertension.

Image created with Biorender.com. Ang II indicates angiotensin II; [Ca²⁺] _i , intracellular Ca²⁺ concentration; PKC, protein kinase C; pS1928, serine 1928 phosphorylation; and S1928, serine 1928.