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. 2018 Sep 15;596(20):4863–4877. doi: 10.1113/JP276923

Differential targeting and signalling of voltage‐gated T‐type Cav3.2 and L‐type Cav1.2 channels to ryanodine receptors in mesenteric arteries

Gang Fan 1, Mario Kaßmann 1,2, Ahmed M Hashad 3, Donald G Welsh 4, Maik Gollasch 1,2,5,✉
PMCID: PMC6187032  PMID: 30146760

Abstract

Key points

  • In arterial smooth muscle, Ca2+ sparks are elementary Ca2+‐release events generated by ryanodine receptors (RyRs) to cause vasodilatation by opening maxi Ca2+‐sensitive K+ (BKCa) channels.

  • This study elucidated the contribution of T‐type Cav3.2 channels in caveolae and their functional interaction with L‐type Cav1.2 channels to trigger Ca2+ sparks in vascular smooth muscle cells (VSMCs).

  • Our data demonstrate that L‐type Cav1.2 channels provide the predominant Ca2+ pathway for the generation of Ca2+ sparks in murine arterial VSMCs.

  • T‐type Cav3.2 channels represent an additional source for generation of VSMC Ca2+ sparks. They are located in pit structures of caveolae to provide locally restricted, tight coupling between T‐type Cav3.2 channels and RyRs to ignite Ca2+ sparks.

Abstract

Recent data suggest that T‐type Cav3.2 channels in arterial vascular smooth muscle cells (VSMCs) and pits structure of caveolae could contribute to elementary Ca2+ signalling (Ca2+ sparks) via ryanodine receptors (RyRs) to cause vasodilatation. While plausible, their precise involvement in igniting Ca2+ sparks remains largely unexplored. The goal of this study was to elucidate the contribution of caveolar Cav3.2 channels and their functional interaction with Cav1.2 channels to trigger Ca2+ sparks in VSMCs from mesenteric, tibial and cerebral arteries. We used tamoxifen‐inducible smooth muscle‐specific Cav1.2−/− (SMAKO) mice and laser scanning confocal microscopy to assess Ca2+ spark generation in VSMCs. Ni2+, Cd2+ and methyl‐β‐cyclodextrin were used to inhibit Cav3.2 channels, Cav1.2 channels and caveolae, respectively. Ni2+ (50 μmol L−1) and methyl‐β‐cyclodextrin (10 mmol L−1) decreased Ca2+ spark frequency by ∼20–30% in mesenteric VSMCs in a non‐additive manner, but failed to inhibit Ca2+ sparks in tibial and cerebral artery VSMCs. Cd2+ (200 μmol L−1) suppressed Ca2+ sparks in mesenteric arteries by ∼70–80%. A similar suppression of Ca2+ sparks was seen in mesenteric artery VSMCs of SMAKO mice. The remaining Ca2+ sparks were fully abolished by Ni2+ or methyl‐β‐cyclodextrin. Our data demonstrate that Ca2+ influx through CaV1.2 channels is the primary means of triggering Ca2+ sparks in murine arterial VSMCs. CaV3.2 channels, localized to caveolae and tightly coupled to RyR, provide an additional Ca2+ source for Ca2+ spark generation in mesenteric, but not tibial and cerebral, arteries.

Keywords: Ryanodine receptors, Calcium sparks, T‐type calcium channels, L‐type calcium channels, BKCa channels, Blood pressure

Key points

  • In arterial smooth muscle, Ca2+ sparks are elementary Ca2+‐release events generated by ryanodine receptors (RyRs) to cause vasodilatation by opening maxi Ca2+‐sensitive K+ (BKCa) channels.

  • This study elucidated the contribution of T‐type Cav3.2 channels in caveolae and their functional interaction with L‐type Cav1.2 channels to trigger Ca2+ sparks in vascular smooth muscle cells (VSMCs).

  • Our data demonstrate that L‐type Cav1.2 channels provide the predominant Ca2+ pathway for the generation of Ca2+ sparks in murine arterial VSMCs.

  • T‐type Cav3.2 channels represent an additional source for generation of VSMC Ca2+ sparks. They are located in pit structures of caveolae to provide locally restricted, tight coupling between T‐type Cav3.2 channels and RyRs to ignite Ca2+ sparks.

Introduction

Ca2+ sparks are elementary Ca2+‐release events generated by a single Ca2+‐release unit (CRU) composed of a cluster of ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) (Nelson et al. 1995; Jaggar et al. 1998; Wang et al. 2004). Unlike Ca2+ influx via voltage‐gated Ca2+ channels (VDCCs), Ca2+ release from the SR in the form of Ca2+ sparks paradoxically causes vasodilatation (Nelson et al. 1995; Knot et al. 1998). There are two reasons for this counterintuitive effect of Ca2+ sparks in arterial vascular smooth muscle cells (VSMCs). First, a single spark produces a remarkably high (10–100 μmol L−1) local (∼1% of the cell volume) increase in [Ca2+]i (Perez et al. 1999, 2001), but increases global [Ca2+]i by less than 2 nmol L−1 (Nelson et al. 1995; Jaggar et al. 2000). Second, Ca2+ sparks occur in close proximity to the cell membrane, where every Ca2+ spark activates numerous large‐conductance Ca2+‐sensitive K+ (BKCa) channels, causing K+ efflux (Nelson et al. 1995; Perez et al. 1999; Brenner et al. 2000; Pluger et al. 2000; Sausbier et al. 2005). The resultant ‘spontaneous transient outward currents’ (STOCs) hyperpolarize VSMCs, thereby decreasing Ca2+ entry through L‐type Cav1.2 channels via a deactivation process. The net result of Ca2+ spark–BKCa channel coupling is decreased global [Ca2+]i in VSMCs resulting in vasodilatation (Nelson et al. 1995; Gollasch et al. 1998 b; Brenner et al. 2000; Pluger et al. 2000; Sausbier et al. 2005; Filosa et al. 2006).

Voltage‐gated L‐type Cav1.2 channels are presumably the predominant pathway by which extracellular Ca2+ triggers Ca2+ sparks in VSMCs. This perspective grew from studies that inhibited Cav1.2 channels, pharmacologically or genetically (tamoxifen‐inducible smooth muscle‐specific Cav1.2‐specific knockout (SMAKO) mice), and found a substantial decrease in Ca2+ spark frequency (Essin et al. 2007). Local or tight coupling between the Cav1.2 channels and RyRs is not required to initiate Ca2+ sparks. Instead, Cav1.2 channels influenced Ca2+ spark production through their ability to set global cytosolic [Ca2+] and consequently the SR refilling rate. Intriguingly, Ca2+ sparks were not completely eliminated following Cav1.2 channel ablation (Copello et al. 2007; Essin et al. 2007), findings which suggest the presence of an additional Ca2+ influx pathway in Ca2+ spark generation. That pathway could include T‐type CaV3.2 channels, integral membrane proteins selectively blocked by micromolar Ni2+, a divalent ion with limited impact on CaV1.2/CaV3.1 channels when applied at appropriate concentrations (Harraz et al. 2014, 2015). Intriguingly, structural work has further noted that T‐type Cav3.2 channels are located in, or close to, caveolae (Harraz et al. 2014), findings which imply that this arrangement is important for Ca2+ spark ignition (Lohn et al. 2000). Whether T‐type Cav3.2 channels are indeed present in caveolae and contribute to elementary Ca2+ signalling remains uncertain. It is this key question along with the channel's functional interrelationship to CaV1.2 channels that we will explore in this investigation.

Methods

Ethical approval

All animal protocols were approved by the local animal care committee (LAGeSo, Berlin, Germany) and the animal welfare officers of the Max Delbrück Center for Molecular Medicine (MDC) (No. X 9011/16). The study is also conform to the principles and regulations of The Journal of Physiology as described by Grundy (2015). There are no ethical concerns.

Animal procedures

The generation and usage of mice deficient in the smooth muscle Cav1.2 Ca2+ channel (SMAKO, smooth muscle α1c‐subunit Ca2+ channel knockout) have been described previously (Moosmang et al. 2003). Briefly, a conditional lox P‐flanked allele (L2) of the Cav1.2 gene (i.e. exons 14 and 15) was generated by homologous recombination in R1 embryonic stem cells (Seisenberger et al. 2000). In addition, mice carried a knock‐in allele (SM‐CreER T2 (ki); Kuhbandner et al. 2000) that expresses the tamoxifen‐dependent Cre recombinase, CreER T2, from the endogenous SM22 α gene locus, which is selectively expressed in smooth muscle of adult mice. Thus, tamoxifen treatment results in conversion of the lox P‐flanked Cav1.2 allele (L2) into a Cav1.2 knockout allele (L1) specifically in smooth muscle cells (Moosmang et al. 2003; Essin et al. 2007). Mice were maintained at the breeding facility of the MDC in individually ventilated cages under standardized conditions that included a 12 h dark–light cycle and free access to standard chow (0.25% sodium; SSNIFF Spezialitäten, Soest, Germany) and drinking water. At an age of 2–3 months, male SMAKO mice (Cav1.2 flox/flox; SM22α‐CreT2 or Cav1.2 flox/flox; SM22α‐CreT2/T2) and corresponding control mice (Cav1.2 +/+; SM22α‐CreT2/T2, Cav1.2 +/+; SM22α‐CreT2, Cav1.2 +/+, Cav1.2 flox/+ or Cav1.2 flox/flox) were i.p. injected with tamoxifen (30 μg g−1 body weight day−1) for five consecutive days. After 2–4 days, mice were deeply anaesthetized by inhalation of isoflurane until cessation of breathing, then killed by cervical dislocation, and the brain, mesentery and tibial arteries removed. Experiments were performed on the same day with arteries from litter‐matched control and SMAKO mice.

Isolation of arterial vascular smooth muscle cells

Arterial VSMCs from mesenteric, tibial, middle and posterior cerebral arteries were isolated as previously described (Gollasch et al. 1998 a; Pluger et al. 2000). Briefly, arteries were removed and quickly transferred to cold (4°C) oxygenated (95% O2–5% CO2) physiological salt solution (PSS) of the following composition (mM): 119 NaCl, 4.7 KCl, 1.2 KH2PO4, 25 NaHCO3, 1.2 MgSO4, 1.6 CaCl2 and 11.1 glucose. The arteries were cleaned, cut into pieces and placed into a Ca2+‐free Hanks’ solution (mM): 55 NaCl, 80 sodium glutamate, 5.6 KCl, 2 MgCl2, 1 mg mL−1 bovine serum albumin (BSA; Sigma‐Aldrich, Taufkirchen, Germany), 10 glucose and 10 HEPES (pH 7.4 with NaOH) containing 0.5 mg mL−1 papain (Sigma‐Aldrich) and 1.0 mg mL−1 DTT for 37 min at 37°C. The segments were then placed in Hanks’ solution containing 1 mg mL−1 collagenase (Sigma‐Aldrich, type F and H, ratio 30% and 70%) and 0.1 mM CaCl2 for 17 min at 37°C. Following several washes in Ca2+‐free Hanks’ solution (containing 1 mg mL−1 BSA), single cells were dispersed from artery segments by gentle triturating. Cells were then stored in the same solution at 4°C. In some experiments, Ca2+ imaging was performed on isolated mesenteric artery segments. The arteries were removed and quickly transferred to cold (4°C) oxygenated (95% O2–5% CO2) PSS. The arteries were cleaned, cut into pieces, and placed into a Ca2+‐free Hanks’ solution containing 0.5 mg mL−1 papain (Sigma‐Aldrich) and 1.0 mg mL−1 DTT for 37 min at 37°C. Following several washes in Ca2+‐free Hanks’ solution (containing 1 mg mL−1 BSA), segments were dispersed from arteries by gentle trituration. Arterial segments were then stored in the same solution at 4°C.

Ca2+ spark measurements

Ca2+ sparks were measured as previously described (Essin et al. 2007). Isolated VSMCs or artery segments were placed onto glass coverslips and incubated with the Ca2+ indicators fluo‐4 AM (10 μmol L−1) and pluronic acid (0.005%, w/v) for 60 min at room temperature in Ca2+‐free Hanks’ solution (Lohn et al. 2000; Pluger et al. 2000). After loading, cells were washed with bath solution for 10 min at room temperature. Isolated cells and intact arterial segments were imaged in a bath solution containing (mM): 134 NaCl, 6 KCl, 1 MgCl2, 2 CaCl2, 10 glucose and 10 HEPES (pH 7.4, NaOH). Images were recorded using a Nipkow disc‐based UltraView LCI confocal scanner (Perkin Elmer, Waltham, MA, USA) linked to a fast digital camera (Hamamatsu Photonics Model C4742‐95‐12ERG, 1344 × 1024 active pixel resolution, 6.45 μm square pixels, Hamamatsu Photonics Co., Hamamatsu, Japan). The confocal system was mounted on an inverted Diaphot microscope with a ×40 oil‐immersion objective (NA 1.3, Nikon Inc., Melville, NY, USA). Images were obtained by illumination with an argon laser at 488 nm, and recording all emitted light above 515 nm. Ca2+ spark analyses were performed off‐line using the UltraView Imaging Suite software (Perkin Elmer). The entire area of each image was analysed to detect Ca2+ sparks. Ca2+ sparks were defined as local fractional fluorescence increase (F/F 0) above the noise level of 1.5. The frequency was calculated as the number of detected sparks divided by the total scan time.

Electrophysiology

Potassium currents were measured in the whole‐cell perforated‐patch mode of the patch‐clamp technique (Gollasch et al. 1996; Essin et al. 2007). Patch pipettes (resistance, 1.5–3.5 MΩ) were filled with a solution containing (in mM): 110 potassium aspartate, 30 KCl, 10 NaCl, 1 MgCl2 and 0.05 EGTA (pH 7.2). The patch pipette solution was supplemented with 200 μg mL−1 amphotericin B, dissolved in dimethyl sulfoxide (DMSO), to measure K+ currents in the whole‐cell perforated‐patch mode. The external bath solution contained (in mM): 134 NaCl, 6 KCl, 1 MgCl2, 2 CaCl2, 10 glucose and 10 HEPES (pH 7.4); holding potential was −60 mV. Whole cell currents were recorded using an Axopatch 200B amplifier (Axon Instruments/Molecular Devices, Sunnyvale, CA, USA) or an EPC 7 amplifier (List, Darmstadt, Germany) at room temperature. Data were digitized at 5 kHz, using a Digidata 1440A digitizer (Axon CNS, Molecular Devices) and pCLAMP software versions 10.1 and 10.2. STOC analysis was performed off‐line using IGOR Pro (WaveMetrics, Lake Oswego, OR, USA) and Microsoft Excel software. A STOC was identified as a signal with at least three times the BKCa single channel current amplitude.

Materials

Fluo‐4‐AM was purchased from Invitrogen (Darmstadt, Germany). All salts and other drugs were obtained from Sigma‐Aldrich (Munich, Deisenhofen or Schnelldorf, Germany) or Merck (Darmstadt, Germany). In cases where DMSO was used as a solvent, the maximal DMSO concentration after application did not exceed 0.5%.

Statistics

Data are presented as means ± SEM. Statistically significant differences in mean values were determined by Student's unpaired t test or one‐way analysis of variance (ANOVA). P‐values < 0.05 were considered statistically significant; n represents the number of cells.

Results

Contribution of T‐type Cav3.2 channels to Ca2+ sparks

We first studied the contribution of Cav3.2 channels to Ca2+ spark generation in VSMCs using 50 μM Ni2+ as a selective blocker (Harraz et al. 2015). Ca2+ sparks were measured in Fluo‐4‐AM‐loaded VSMCs in the absence (control) or presence of Ni2+ (Fig. 1). Ni2+ decreased the Ca2+ spark frequency in mesenteric artery VSMCs (0.085 ± 0.01 Hz, control vs. 0.046 ± 0.01 Hz, with Ni2+, P < 0.05, Fig. 1 C). In contrast, Ni2+ failed to decrease Ca2+ spark frequency in tibial or cerebral VSMCs (Fig. 1 D and E). These data suggest that the contribution of T‐type Cav3.2 channels to Ca2+ spark generation varies according to the vascular bed from which VSMCs originate.

Figure 1. The CaV3.2 channel inhibitor Ni2+ (50 μM) decreased Ca2+ spark events in VSMCs from mesentery but not tibial and cerebral arteries.

Figure 1

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A, Ca2+ fluorescence images of a Fluo‐4‐AM‐loaded control (CaV1.2+/+) VSMC and time course of Ca2+ fluorescence changes in the cellular region of interest (ROI; upper panel). Cell boundary is marked with dashed line. B, same as A but in the presence of Ni2+ (50 μM). C, the frequency of Ca2+ sparks was reduced by 50 μM Ni2+ (n = 98 vs. 87 VSMCs, respectively, P < 0.05). D and E, Ni2+ did not change the frequency of Ca2+ sparks in tibial (D, n = 80 vs. 97, respectively, P > 0.05) or cerebral VSMCs (E, n = 86 vs. 96, respectively, P > 0.05). Cells were scanned for a total time of 30 s, images were recorded at a rate of 5 s−1. Cells were isolated from 3 mice in each group; 25–35 cells per mouse were recorded and analysed. * P < 0.05; n.s., not significant.

T‐type Cav3.2 channels are located in caveolae to trigger Ca2+ sparks

Caveolae are plasma membrane pits (diameter, ∼50 nm) rich in cholesterol and sphingolipids (Rodal et al. 1999). We used methyl‐β‐cyclodextrin, a cholesterol‐depleting drug, to unfold caveolae (Fig. 2; Lohn et al. 2000) and to explore the contribution of caveolar vs. non‐caveolar Cav3.2 channels in triggering Ca2+ sparks. Isolated mesenteric artery VSMCs (Fig. 2) were initially exposed to Ni2+ (50 μM) or incubated with methyl‐β‐cyclodextrin (10 mM). Both interventions decreased the Ca2+ spark frequency and the fraction of cells with sparks by ∼30%. Addition of Ni2+ to mesenteric artery VSMCs treated with methyl‐β‐cyclodextrin did not further reduce Ca2+ spark frequency or the percentage of cells firing. Neither Ni2+ nor methyl‐β‐cyclodextrin significantly reduced the amplitudes of the Ca2+ sparks (Fig. 3).

Figure 2. Caveolae pit strcutures unfloded through cholesterol depletion by methyl‐β‐cyclodextrin.

Figure 2

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A, electron microscopy image of a control mesenteric VSMC. B and C, electron microscopy images of mesenteric VSMCs after treatment with methyl‐β‐cyclodextrin (10 mM).

Figure 3. Caveolar disruption reduced Ca2+ sparks in isolated mesenteric artery VSMCs.

Figure 3

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A, Ca2+ fluorescence images of a Fluo‐4‐AM‐loaded control (CaV1.2+/+) mesenteric VSMC and time course of Ca2+ fluorescence changes in the cellular ROI (upper panel). B, same as A but with cells incubated with methyl‐β‐cyclodextrin (10 mmol L−1) to disrupt caveolae. C–E, summary of the results. Ca2+ spark frequency (C), fraction of cells producing Ca2+ sparks (D), and amplitude of Ca2+ sparks (E) in control cells (n = 72), in cells (n = 70) incubated with Ni2+ (50 μM), in cells (n = 74) incubated with methyl‐β‐cyclodextrin (10 mM), and in cells (n = 76) incubated with Ni2+ (50 μM) following methyl‐β‐cyclodextrin (10 mM) treatment. WT, wild‐type. Cells were isolated from 3 mice in each group; 20–35 cells were recorded and analysed from each mouse. * P < 0.05; n.s., not significant.

There are at times confounding issues with examining Ca2+ spark generation in individual cells, and thus we repeated the preceding experiments on intact mesenteric artery segments (Fig. 4). Both Ni2+ and methyl‐β‐cyclodextrin reduced Ca2+ sparks frequency and percentage of cells with sparks by ∼30%; the combination of both drugs had no additional effects. Either drug alone or in combination failed to impact Ca2+ spark amplitude. Cumulatively, these results suggest that caveolar Cav3.2 channels trigger Ca2+ sparks in mesenteric artery VSMCs.

Figure 4. CaV3.2 channel blockade or caveolar disruption reduced Ca2+ sparks in mesenteric artery segments.

Figure 4

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A, Ca2+ fluorescence images of Fluo‐4‐AM‐loaded control (WT, CaV1.2+/+) mesenteric artery segments. B, same as A but in the presence of the CaV3.2 channel blocker Ni2+ (50 μM). C–E, summary of the results. Ca2+ spark frequency (C), fraction of cells producing Ca2+ sparks (D), and amplitude of Ca2+ sparks (E) in control cells (n = 83), in cells (n = 75) incubated with Ni2+ (50 μM), in cells (n = 80) after incubation with methyl‐β‐cyclodextrin (dextrin, 10 mM), and in cells (n = 77) incubated with Ni2+ (50 μM) and methyl‐β‐cyclodextrin (10 mM). WT, wild‐type. Cells were isolated from 3 mice in each group; 20–35 cells were recorded and analysed from each mouse. * P < 0.05; n.s., not significant.

Individual contributions of Cav3.2 and Cav1.2 channels to Ca2+ sparks

We next studied the individual contributions of Cav1.2 and Cav3.2‐mediated Ca2+ sparks. We first used the Cav1.2 blocker Cd2+ (200 μM; Essin et al. 2007) to demonstrate a role of Cav1.2 channels in Ca2+ spark generation. As previously observed (Essin et al. 2007), Cd2+ reduced Ca2+ spark frequency (∼70%) and the percentage of cells firing in mesenteric artery VSMCs; Cd2+ did not affect Ca2+ spark amplitude (Fig. 5; Essin et al. 2007). We next studied the role of Cav3.2 channels in triggering those remaining sparks and found that both Ni2+ and methyl‐β‐cyclodextrin completely abolished Ca2+ spark events in VSMCs treated with Cd2+ (Fig. 5 B and C).

Figure 5. CaV1.2 or CaV3.2 channel blockade reduced Ca2+ sparks in isolated mesenteric artery VSMCs.

Figure 5

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A, Ca2+ fluorescence images of a Fluo‐4‐AM‐loaded mesenteric VSMC (WT, CaV1.2+/+) in the presence of the CaV1.2 channel blocker Cd2+ (200 μM) and time course of Ca2+ fluorescence changes in the cellular ROI indicated (upper panel). B, same as A but after incubation of cells with methyl‐β‐cyclodextrin (dextrin, 10 mM, 90 min at room temperature). C, same as A but in the presence of Ni2+ (50 μM). D and E, summary of the results. Ca2+ spark frequency (D), fraction of cells producing Ca2+ sparks (E), and amplitude of Ca2+ sparks (F) were significantly reduced vs. control when Cd2+ was present alone or combined with methyl‐β‐cyclodextrin treatment or the CaV3.2 channel blocker Ni2+ (50 μM). The two latter interventions also significantly inhibited Ca2+ sparks compared to Cd2+ treatment. n = 98 control cells, n = 59 cells treated with Cd2+, n = 77 cells treated with dextrin and Cd2+, and n = 59 cells treated with Ni2+ and Cd2+. Cells were isolated from 3 mice in each group; 18–35 cells were recorded and analysed from each mouse. * P < 0.05.

We next confirmed our results using a genetic mouse model taking advantage of tamoxifen‐inducible SMAKO mice (Essin et al. 2007). Ca2+ spark frequency and the percentage of cells firing Ca2+ sparks was diminished in mesenteric VSMCs of SMAKO mice. Ni2+ completely abolished the remaining Ca2+ sparks (Fig. 6). Similar results were observed using methyl‐β‐cyclodextrin to deplete caveolae (Fig. 6). Cd2+ had no effect on the frequency and percentage of cells firing Ca2+ sparks (Fig. 6). Note, 2 μM thapsigargin, a potent inhibitor of the sarcoplasmic/endoplasmic reticular Ca2+‐ATPase (SERCA), completely blocked Ca2+ spark generation in VSMCs (n = 75 cells from 3 mice), as previously reported (Nelson et al. 1995). We finally confirmed our results by measuring STOCs in isolated mesenteric artery VSMCs. STOCs were measured in methyl‐β‐cyclodextrin‐ and/or Ni2+‐treated cells at a holding potential of −40 mV, a physiological membrane potential and one that should drive T‐type Ca2+ channel‐mediated Ca2+ sparks, enabling the activation of large‐conductance Ca2+‐activated K+ channels (Harraz et al. 2015). Figure 7 shows that STOC frequencies were reduced by Ni2+ and methyl‐β‐cyclodextrin in a non‐additive manner. In contrast, methyl‐β‐cyclodextrin and Ni2+ had no effect on amplitudes of STOCs. Cumulatively, our data demonstrate that the Cav3.2 channels reside in caveolae and are key regulators of Ca2+ sparks in mesenteric arteries.

Figure 6. Ni2+ and methyl‐β‐cyclodextrin, but not Cd2+, inhibited Ca2+ sparks in mesenteric artery VSMCs from SMAKO mice.

Figure 6

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A, Ca2+ fluorescence images of a Fluo‐4‐AM‐loaded SMAKO (smooth muscle‐specific CaV1.2−/−) mesenteric VSMC and time course of Ca2+ fluorescence changes in the cellular ROI indicated (upper panel). B, same as A but in the presence of the CaV1.2 channel blocker Cd2+ (200 μM). C, same as A but after incubation of cells with methyl‐β‐cyclodextrin (10 mM, 90 min at room temperature). D, same as A but in the presence of the CaV3.2 channel blocker Ni2+ (50 μM). E–G, summary of the results. Ca2+ spark frequency (E), fraction of cells producing Ca2+ sparks (F), and amplitude of Ca2+ sparks (G) were completely inhibited after incubation of the cells with methyl‐β‐cyclodextrin or in the presence of Ni2+, but not in Cd2+ (n = 123 non‐treated, control cells; n = 97 cells treated with Cd2+; n = 112 cells treated with methyl‐β‐cyclodextrin; n = 91 cells treated with Ni2+; cells were isolated from 3 mice in each group; 30–45 cells were recorded and analysed from each mouse). n.s., not significant.

Figure 7. Ni2+ and methyl‐β‐cyclodextrin reduced in a non‐additive manner STOCs in mesenteric artery VSMCs.

Figure 7

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A, original recordings of STOCs in mesenteric artery smooth muscle cells before and after application of Ni2+ (50 μM). Holding potential was −40 mV. B, same as A but following incubation of cells with methyl‐β‐cyclodextrin (10 mM). C and D, summary of the results. STOC frequencies (C) and amplitudes of STOCs (D) in control cells (n = 7 cells from 3 mice, 2–3 cells were recorded and analysed from each mouse), and in cells treated with methyl‐β‐cyclodextrin (dextrin, 10 mM) (n = 8 cells from 5 mice, 1–3 cells were recorded and analysed from each mouse). * P < 0.05; n.s., not significant.

Discussion

In this study, we used pharmacological tools and smooth muscle‐specific Cav1.2 channel knockout (SMAKO) mice (Essin et al. 2007) to clearly ascertain the contribution of caveolar T‐type Cav3.2 channels in the triggering of Ca2+ sparks in VSMCs. Findings reveal that L‐type Cav1.2 channels are the dominant pathway mediating Ca2+ influx in VSMCs, SR refilling and consequently Ca2+ spark generation (Essin et al. 2007). They also show the presence of a secondary component in mesenteric arteries driven by T‐type Cav3.2 channels, which are selectively blocked by micromolar Ni2+ (Lee et al. 1999; Harraz et al. 2014, 2015). Findings based on the use of methyl‐β‐cyclodextrin suggest that caveolae are important in local Ca2+ spark generation and Cav3.2 channels, contrary to Cav1.2 channels, are located in caveolae in close apposition to RyRs (Fig. 8).

Figure 8. Proposed model of the role of CaV1.2 and CaV3.2 channels in Ca2+ sparks generation.

Figure 8

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Ca2+ sparks are produced by opening of clustered ryanodine receptors (RyRs) in the SR, which produces a negative‐feedback effect on vasoconstriction. This vasodilatory effect is mediated by activation of large‐conductance Ca2+‐activated K+ (BKCa) channels, which results in hyperpolarization of VSMCs and reduced global cytosolic [Ca2+]. The majority (∼70‐80%) of Ca2+ sparks are triggered by CaV1.2 channels contributing to global cytosolic [Ca2+], which in turn influences luminal SR calcium via SERCA. A minority (∼20–30%) of Ca2+ sparks are generated by local and tight coupling between CaV3.2 channels in caveolae to initiate Ca2+ sparks. CaV3.2 channels are located in pit structures of caveolae. In line with this model, depletion of caveolae or CaV3.2 channel blockade similarly decreased Ca2+ sparks by ∼20%. SERCA, calcium pump; SR, sarcoplasmic reticulum; VSMC, mesenteric artery vascular smooth muscle cell.

Cav1.2 channels play a predominant role in the generation of Ca2+ sparks

Ca2+ sparks have been observed in various contractile tissue (Tsugorka et al. 1995; Cheng et al. 1996; Jaggar et al. 2000), but in arterial VSMCs they paradoxically cause vasodilatation (Nelson et al. 1995; Knot et al. 1998). This is due to the fact that Ca2+ sparks occur in close proximity to the cell membrane, where they activate large‐conductance Ca2+‐sensitive K+ (BKCa) channels (Perez et al. 1999; Brenner et al. 2000; Pluger et al. 2000; Sausbier et al. 2005). The resultant ‘spontaneous transient outward currents’ (STOCs) hyperpolarize VSMCs, an electrical event that decreases Ca2+ entry through L‐type Cav1.2 channels and which induces vasodilatation (Nelson et al. 1995; Gollasch et al. 1998 b; Brenner et al. 2000; Pluger et al. 2000; Sausbier et al. 2005; Filosa et al. 2006). This study provided two further lines of evidence linking L‐type CaV1.2 channels to the generation of Ca2+ sparks. First, deletion of the smooth muscle‐specific Cav1.2 channel gene in (SMAKO) mice decreased Ca2+ spark frequency by 50%, 80% and 75% in tibial, cerebral and mesenteric arteries, respectively (Essin et al. 2007). Second, Cd2+, a selective inhibitor for high voltage‐activated L‐type Cav1.2 channels but not low voltage‐activated T‐type Ca2+ channels (Fox et al. 1987; Akaike et al. 1989; Ryu & Randic, 1990), diminished the frequency of Ca2+ sparks by 75%. These data align with previous results and indicate that Cav1.2 channels act as the primary Ca2+ influx pathway responsible for ∼70–80% of Ca2+ spark events in VSMCs (Essin et al. 2007).

Cav3.2 channels can trigger Ca2+ sparks without involvement of Cav1.2 channels

Beside L‐type Cav1.2 channels, voltage‐gated T‐type Ca2+ channels have been detected in VSMCs in numerous vascular beds, including mesenteric, cerebral and coronary arteries (Chen et al. 2003; Braunstein et al. 2009; Abd El‐Rahman et al. 2013). Recent studies indicate that T‐type Cav3.2 channels could provide sufficient Ca2+ influx to activate Ca2+ sparks to cause STOCs affecting the arterial myogenic response (Harraz et al. 2014, 2015). Using genetic tools and the Cav3.2 blocker Ni2+, the authors found that Ca2+ sparks and STOCs have been suppressed by ∼25%. Thus, T‐type Ca2+ channels could contribute to Ca2+ spark events and consequently BKCa channel activation to elicit negative feedback control of arterial tone (Harraz et al. 2015). In order to rule out non‐specific effects of Ni2+ on Cav1.2 channel‐mediated Ca2+ spark generation in mesenteric arteries, we used Cd2+ or the genetic ablation of VSMC Cav1.2 channels (SMAKO mice) before probing Ni2+ effects on Ca2+ sparks. We found that Ni2+ completely abolished Ca2+ sparks after silencing Cav1.2 channels. Our data provide firm evidence that T‐type channels provide sufficient Ca2+ influx to trigger Ca2+ sparks in the absence of L‐type Cav1.2 channels in mouse mesenteric arteries but not in tibial and cerebral arteries. Based on our results, we conclude that T‐type channels seem to be responsible for 20–30% of the Ca2+ spark events in mesenteric arteries, which are resistant to L‐type Cav1.2 channel block. The relative contribution of L‐type vs. T‐type channels in triggering Ca2+ sparks depends on the membrane voltage of the cells (Hashad et al. 2017). Since L‐type channels are high voltage‐activated Ca2+ channels, they cause Ca2+ sparks at relatively high membrane potentials. In contrast, T‐type channels are low voltage‐activated Ca2+ channels, which can trigger Ca2+ sparks only at relatively low membrane potentials (Hashad et al. 2017). Given that we mainly worked with isolated cells, it is possible that the higher contribution of L‐type channels could be due to a more depolarized state of the VSMCs.

Cav3.2 channels are localized in caveolae

Caveolae are cholesterol and sphingolipid enriched membrane microdomains (Anderson, 1998) that form flask‐shaped plasma membrane structures in contractile cells including vascular smooth muscle (Feron et al. 1996; Lo et al. 2016; Keshavarz et al. 2017). A number of transport proteins reside in caveolae, including but not limited to L‐type Ca2+ channels, Na+ channels and the Na+/Ca2+ exchanger (Yarbrough et al. 2002; Brazer et al. 2003; Pani & Singh, 2009; Shaikh et al. 2010). Cholesterol depletion by methyl‐β‐cyclodextrin disrupts these lipid rafts and thus provides a means to assess their functional importance (Rodal et al. 1999; Lohn et al. 2000; Smart & Anderson, 2002). We previously proposed that caveolae are important in local Ca2+ spark generation (arterial smooth muscle cells and atrial cardiac muscle; Lohn et al. 2000) but were unable to define the caveolemmal Ca2+ influx pathways involved. In this regard, we applied methyl‐β‐cyclodextrin to study the putative role of caveolae in Ca2+ spark ignition in VSMCs. We found that methyl‐β‐cyclodextrin reduced the frequency of Ca2+ sparks/STOCs and the percentage of cells firing Ca2+ sparks by ∼20–30% in mesenteric VSMCs. A similar reduction has been observed in mesenteric VSMCs lacking caveolin‐1 (Suzuki et al. 2013), which suggests that the lack of caveolae attenuates generation of Ca2+ sparks and consecutive translation into electrical STOC signals.

Intriguingly, we found that the T‐type Cav3.2 blocker Ni2+ failed to inhibit Ca2+ sparks in VSMCs after methyl‐β‐cyclodextrin treatment, a finding which suggest that T‐type channels reside in caveolae. This interpretation aligns with past ultrastructural findings which used immunogold labelling to highlight that Cav3.2 channels are associated with caveolae structures (Harraz et al. 2014). To confirm the importance of caveolemmal T‐type Cav3.2 channels in triggering Ca2+ sparks, we further analysed the impact of methyl‐β‐cyclodextrin treatment or T‐type Cav3.2 channel blockade by Ni2+ in mesenteric VSMCs without functional L‐type Cav1.2 channels (i.e. blocked by Cd2+ or genetically ablated using SMAKO mice). In all experimental settings, we failed to observe Ca2+ sparks, consistent with T‐type Cav3.2 channels localizing to caveolae and contributing to the generation of Ca2+ sparks. Unfolding caveolae by using methyl‐β‐cyclodextrin to enlarge the diffusional space between the caveolar Ca2+ influx channels and RyRs in CRUs (Lohn et al. 2000), we found that Ca2+ spark events triggered by Cav3.2 channels were completely inhibited. We conclude that a close apposition of Cav3.2 channels in caveolae to RyRs is crucial for T‐type Cav3.2 to generate Ca2+ sparks in mesenteric artery VSMCs. Our results suggest that T‐type Cav3.2 channels in caveolar microdomains exhibit tight and locally restricted control of RyRs in CRUs via local Ca2+ elevations (Fig. 8). This pathway contrasts the role of L‐type Cav1.2 channels, which represent the predominant pathway to mediate Ca2+ influx in VSMCs contributing to global cytosolic [Ca2+], which in turn triggers indirectly Ca2+ sparks via luminal SR calcium load (Essin et al. 2007; Fig. 8). Recent data indicate that caveolin‐1 can also facilitate the molecular interaction between L‐type Cav1.2 and BKCa channels in caveolae. However, the relevance of these findings is still unknown as the signalling pathway has only been described in cultured VSMCs (Suzuki et al. 2013) or in VSMCs of non‐mesenteric origin (Cheng & Jaggar, 2006). Noteworthy, Ni2+ increased STOC frequency in superior epigastric arteries at −30 mV (Mullan et al. 2017), which might contribute to vasoconstriction of this type of blood vessel.

A limitation of our study is that we silenced all L‐type Cav1.2 channels through pharmacological or genetic tools. Therefore, we cannot discriminate between the contribution of caveolar vs. non‐caveolar L‐type Cav1.2 channels in triggering Ca2+ sparks. However, since (i) Cd2+ did not alter Ca2+ sparks in VSMCs from SMAKO mice and (ii) Ni2+ and methyl‐β‐cyclodextrin treatment showed non‐additive inhibitory effects on Ca2+ sparks and STOCs, it seems unlikely that L‐type Cav1.2 channels reside within caveolae to ignite Ca2+ sparks in mesenteric artery VSMCs.

Cav1.2/Cav3.2–RyR–BKCa pathway in VSMCs

BKCa channel activation is a negative regulator of membrane potential in VSMCs (Yuan et al. 2010; Wang et al. 2012). BKCa channels are activated by Ca2+ sparks, discrete events regulated by Ca2+ influx triggering the cytosolic gate on RyR directly or altering the SR load. The participation of Cav1.2 channels and SERCA pumps in setting SR load and triggering Ca2+ sparks is well established (Maggi et al. 1995; Zaccolo et al. 2002; Venetucci et al. 2012). In this study, we demonstrate that SERCA pump inhibition by thapsigargin completely blocked the generation of Ca2+ sparks triggered by T‐type Cav3.2 channels. Although L‐type Cav1.2 channels act as the primary Ca2+ influx pathway and are responsible for ∼70–80% Ca2+ spark events in mesenteric VSMCs (Essin et al. 2007), T‐type Cav3.2 channels in caveolar microdomains seem to enable L‐type Cav1.2‐resistent Ca2+ spark events (∼20–30%). Unfolding the pit structure of caveolae did block the Ni2+‐sensitive Ca2+ spark events in a thapsigargin‐sensitive manner. As such, we conclude that Cav3.2 channels in the caveolae are responsible for the ignition of Ca2+ sparks from SR CRUs. The tight association between Cav3.2 channels and RyRs seems to be necessary to enabling this biological process (Fig. 6).

In conclusion, our data demonstrate that L‐type CaV1.2 channels provide the predominant Ca2+ pathway for the generation of Ca2+ sparks in murine arterial VSMCs. Furthermore, T‐type CaV3.2 channels represent an additional source for generation of VSMC Ca2+ sparks in mesenteric, but not tibial and cerebral, arteries. We suggest that the T‐type Cav3.2 channels are located in pits structures of caveolae to provide locally restricted, tight coupling between T‐type Ca2+ channels and RyRs to ignite Ca2+ sparks.

Additional information

Competing interests

None declared.

Author contributions

G.F., M.K. and A.M.H. were responsible for the collection and analysis of data. D.G.W. and M.G. were responsible for the conception and design of the experiments. G.F. and M.G. drafted the manuscript. All authors were responsible for interpretation of the data, contributed to the drafting and revised the manuscript critically for important intellectual content. All authors have approved the final version of the manuscript and agree to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.

Funding

M.G. is supported by grants from the Deutsche Forschungsgemeinschaft (DFG), by the DZHK (German Centre for Cardiovascular Research) and by the BMBF (German Ministry of Education and Research). M.K. is supported by the DZHK (German Centre for Cardiovascular Research) and by the BMBF (German Ministry of Education and Research). G.F. is supported by the CSC (China Scholarship Council). A.M.H. is a Vanier scholar (Canadian Institute of Health Research) and was supported by salary studentship from Alberta Innovates Health Solutions (AIHS). D.G.W. is supported by an operating grant from the Canadian Institute of Health Research. He is the Rorabeck Chair in Molecular Neuroscience and Vascular Biology at the University of Western Ontario, Canada. M.G. and D.G.W. are supported by the Deutsche Akademische Austauschdienst (DAAD).

Biography

Gang Fan is a second year doctoral student working under the supervision of M.G. and M.K. at the Experimental and Clinical Research Center (ECRC) of Charité – Universitätsmedizin Berlin. His research work focuses on voltage‐gated calcium channels and ryanodine receptors in smooth muscle cells.

graphic file with name TJP-596-4863-g001.gif

Edited by: Don Bers & Fernando Santana

G. Fan and M. Kaßmann contributed equally to this work.

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