Regulation of L-type calcium channel sparklet activity by c-Src and PKC-α

Abstract

The activity of persistent Ca²⁺ sparklets, which are characterized by longer and more frequent channel open events than low-activity sparklets, contributes substantially to steady-state Ca²⁺ entry under physiological conditions. Here, we addressed two questions related to the regulation of Ca²⁺ sparklets by PKC-α and c-Src, both of which increase whole cell Ca_v1.2 current: 1) Does c-Src activation enhance persistent Ca²⁺ sparklet activity? 2) Does PKC-α activate c-Src to produce persistent Ca²⁺ sparklets? With the use of total internal reflection fluorescence microscopy, Ca²⁺ sparklets were recorded from voltage-clamped tsA-201 cells coexpressing wild-type (WT) or mutant Ca_v1.2c (the neuronal isoform of Ca_v1.2) constructs ± active or inactive PKC-α/c-Src. Cells expressing Ca_v1.2c exhibited both low-activity and persistent Ca²⁺ sparklets. Persistent Ca²⁺ sparklet activity was significantly reduced by acute application of the c-Src inhibitor PP2 or coexpression of kinase-dead c-Src. Ca_v1.2c constructs mutated at one of two COOH-terminal residues (Y²¹²²F and Y²¹³⁹F) were used to test the effect of blocking putative phosphorylation sites for c-Src. Expression of Y²¹²²F but not Y²¹³⁹F Ca_v1.2c abrogated the potentiating effect of c-Src on Ca²⁺ sparklet activity. We could not detect a significant change in persistent Ca²⁺ sparklet activity or density in cells coexpressing Ca_v1.2c + PKC-α, regardless of whether WT or Y²¹²²F Ca_v1.2c was used, or after PP2 application, suggesting that PKC-α does not act upstream of c-Src to produce persistent Ca²⁺ sparklets. However, our results indicate that persistent Ca²⁺ sparklet activity is promoted by the action of c-Src on residue Y²¹²² of the Ca_v1.2c COOH terminus.

l-type calcium (ca_v1.2) channels are a primary pathway for Ca²⁺ entry in smooth muscle cells (SMCs) and, therefore, play a key role in regulation of vascular tone (18, 29). Previous studies on Ca_v1.2 channels have revealed the occurrence of a high activity-gating mode characterized by frequent, prolonged channel openings, in addition to a more prevalent gating mode with brief and rare channel openings (13, 23, 24, 39). When recording Ca_v1.2 channel activity using total internal reflection fluorescence (TIRF) microscopy, these low and high activity-gating modes correspond to low activity and persistent Ca²⁺ sparklets, respectively (23, 24, 27, 34).

Persistent Ca²⁺ sparklets account for ∼50% of the steady-state Ca²⁺ entry through Ca_v1.2 channels under physiological conditions, even though they constitute a very small population of the total Ca_v1.2 channels on the plasma membrane (1). Furthermore, an increase in persistent Ca²⁺ sparklet activity has been shown to underlie increased intracellular Ca²⁺ during angiotensin II-induced hypertension and hyperglycemia (27, 28). This evidence points to an important physiological role for the small population of Ca_v1.2 channels that conforms to persistent Ca²⁺ sparklets. In vascular smooth muscle, these events are thought to be highly regulated by the classical isoform of protein kinase C (PKC-α; Refs. 4, 5), and membrane targeting of PKC-α by A-kinase anchoring protein 150 (AKAP150) is considered essential in this process (27).

However, the mechanism of PKC-α action on Ca_v1.2 channels that results in the induction of persistent Ca²⁺ sparklet activity is not clear. Biochemical evidence points to residue S¹⁹²⁸, the canonical PKA phosphorylation site on Ca_v1.2a, (corresponding to S¹⁹⁰¹ on Ca_v1.2c), as a PKC phosphorylation site (36) but whether the phosphorylation of S¹⁹²⁸ by PKC-α translates into an increase in channel function remains unresolved. Electrophysiological and imaging studies demonstrating the regulation of persistent Ca²⁺ sparklet activity by PKC-α have not addressed the question of whether PKC-α phosphorylates the Ca_v1.2 α-subunit directly or acts via another kinase. While c-Src, another highly expressed kinase in SMCs, is known to directly phosphorylate the Ca_v1.2 channel and potentiate whole cell Ca_v1.2 current (2, 12, 14–17, 35, 36), its role in producing persistent Ca²⁺ sparklet activity through Ca_v1.2 channels has not been explored. Regulation of persistent Ca²⁺ sparklet activity by c-Src may or may not be related to PKC-α as several isoforms of PKC have been shown to activate c-Src (3–5, 11). On the basis of this evidence, we hypothesized that c-Src regulates persistent Ca²⁺ sparklet activity and that at least part of the effect of PKC-α on persistent sparklet activity is mediated through c-Src.

MATERIALS AND METHODS

Culture and transfection of tsA-201 cells.

The cells were maintained at 37°C in a 5% CO₂ incubator in DMEM supplemented with 10% FBS and 1% penicillin and streptomycin solution. tsA-201 cells between passages 6 and 25 were plated on autoclaved glass coverslips in 35-mm Petri dishes, 24–48 h before transfection. These cells are derived from a human embryonic kidney cell line and immortalized by expression of the T antigen; they do not express any endogenous voltage-gated calcium channels. Cells were transfected with Ca_v1.2 channel subunits [wild-type (WT)/mutant α_1C, β_1b, and α₂δ] along with either green fluorescent protein (GFP) or protein kinase constructs tagged with GFP, using jetPEI (Polyplus transfection). In all protocols, 840 ng of α1c cDNA and 200 ng each of α2-δ and β1b were used for transfection in a 35-mm dish with ∼70% cell confluency. When appropriate, 200 ng c-Src-GFP, PKC-α, and/or GFP cDNA were added to the transfection mixture. Successfully transfected cells were identified on the basis of GFP fluorescence. To confirm that cotransfection of a kinase cDNA with Ca_v1.2c cDNA does not alter the expression of Ca_v1.2c, we compared current densities in cells expressing Ca_v1.2c alone with those of cells coexpressing Ca_v1.2c and c-Src. Both groups were found to have similar current densities [WT Ca_v1.2c peak calcium current (I_Ca) = 8.99 ± 2.18 pA/pF (n = 18); and WT Ca_v1.2c + c-Src peak I_Ca = 10.29 ± 2.66 pA/pF (n = 16)] indicating that cotransfection with a kinase cDNA does not significantly impact expression levels of Ca_v1.2c.

Rat neuronal WT α_1C (accession no. P22002), β_1b, and α₂δ DNA, subcloned into pcDNA3.1 vectors, were gifts from Dr. G. W. Zamponi. Site-directed mutagenesis was carried out in the laboratory of Zamponi (12) to obtain Y²¹²²F and Y²¹³⁹F mutations of the rat neuronal α_1C-subunit. PKC-α tagged with GFP was a gift from Dr. J. Exton. Kinase-dead (kd) c-Src and c-Src tagged with GFP were gifts from Dr. A. P. Braun and Dr. G. E. Davis, respectively.

Electrophysiology.

tsA-201 cells were voltage clamped at −70 mV in the whole cell configuration using an EPC10 (HEKA, Bellmore, NY) or Axopatch 200B (Axon Instruments, Union City, CA) patch-clamp amplifier controlled by Patchmaster (HEKA) or pClamp (Axon Instruments) software, respectively. The cells were perfused with a solution composed of the following (in mM): 140 N-methyl-d-glucamine, 5 CsCl, 1 MgCl₂, 10 glucose, 10 HEPES, and 2 CaCl₂. After gigaseal formation, the bath solution was switched to a solution containing 20 mM CaCl₂ and 120 N-methyl-d-glucamine without any change in the concentrations of the other constituents. The patch pipette solution consisted of the following (in mM): 87 Cs-Asp, 20 CsCl, 1 MgCl₂, 5 MgATP, 10 HEPES, 10 EGTA, and 0.2 Rhod-2. The pH of bath and patch pipette solutions were adjusted to 7.4 using HCl and 7.2 using CsOH, respectively. Patch pipettes filled with pipette solution exhibited resistances between 3 and 6 MΩ. All experiments were performed at room temperature (23, 24).

TIRF microscopy.

Voltage-clamped tsA-201 cells were imaged using a through-the-lens TIRF system coupled to an IX-71 Olympus microscope that was integrated with a high-speed Andor iXON EMCCD camera (Belfast, Ireland). The microscope was equipped with an Olympus PlanApo (×60, 1.45 NA) oil immersion objective and appropriate filter sets to separate excitation (491 nm for GFP and 563 nm for Rhod-2) and emission wavelengths. Images were acquired at a frequency >100 Hz using TILL imaging software (TILL Photonics, Rochester, NY), which also controlled the laser intensity and angle of laser incidence on the objective.

The acquired images were imported into a custom analysis program written in Interactive Data Language (IDL; ITTVIS, Boulder, CO) to perform Ca²⁺ sparklet detection using the amplitude threshold criteria (23, 24). Briefly, a Ca²⁺ fluorescence transient qualified as a sparklet if the average fluorescence amplitude of the 3 × 3 grid of the adjoining pixels (centered at the pixel with the highest fluorescence amplitude) was equal to or greater than the mean basal fluorescence plus 2.5 times its standard deviation.

Mean Ca²⁺ sparklet activity and Ca²⁺ sparklet density analysis.

To test our hypothesis, we compared two parameters across different test groups: mean Ca²⁺ sparklet activity (nP_s) and mean persistent Ca²⁺ sparklet density.

In previous Ca²⁺ sparklet studies (23–27), Ca²⁺ sparklet activity was determined using nP_s analysis (where n = no. of quantal levels and P_s = probability of occurrence of Ca²⁺ sparklet), which is analogous to open probability (nP_o) analysis for single-channel data. We followed a similar approach in this study with one difference. Instead of converting fluorescence units to Ca²⁺ concentration, we computed ΔF_total at each time point in Ca²⁺ sparklet traces. First, the total fluorescence intensity, F_total, for each time point was calculated by summing the Rhod-2 fluorescence over a predefined region from the raw image stacks acquired over a fixed length of time. The area of the predefined region was greater than the entire fluorescence signal produced by the Ca²⁺ sparklet. Subsequently, F_total of the basal fluorescence value was subtracted from F_total observed during channel opening to determine ΔF_total value (1). The quantal ΔF_total value (2,500 arbitrary units), q, was calculated by plotting an all-points histogram of several Ca²⁺ sparklet traces and then fitting the histogram with a multicomponent Gaussian function. Following that, nP_s analysis was performed on Ca²⁺ sparklet traces using “threshold detection analysis” with no duration constraints and a q value of 2,500 arbitrary units in pClamp 9.0. For the purpose of quantal value comparison with previous Ca²⁺ sparklet studies, we converted the fluorescence from some of our Ca²⁺ sparklet traces to Ca²⁺ concentration and obtained a Ca²⁺ sparklet quantal value of 34.4 nM (data not shown); this quantal value was similar to that obtained in other Ca²⁺ sparklet studies (23–25).

Persistent Ca²⁺ sparklet density in each cell was determined by dividing the number of persistent Ca²⁺ sparklet sites by the area of the cell visible in its TIRF image.

Data from cells that were transfected with Ca_v1.2c but did not exhibit measurable I_Ca upon depolarization were not included in the analyses. For mean nP_s calculation, we did not consider cells in which sparklet activity could not be detected even if they expressed measurable I_Ca because it was not possible to calculate an accurate nP_s value in such cases. However, those cells were accounted for in persistent Ca²⁺ sparklet density analysis since any cell that did not exhibit persistent Ca²⁺ sparklets could be assigned a sparklet density value of 0. In addition, we found that 9–10% of our traces fell into the nonstationary category (e.g., Fig. 4A). This was due to the limitation of our image acquisition software where some of the images were acquired during the occurrence of a sparklet event. Inclusion of such traces would lead to an inaccurate estimate (on the lower side) of nP_s. However, every nonstationary trace in this study could be very clearly classified as a persistent or low sparklet activity group except for two traces. Putting those data in either sparklet activity group did not alter our conclusions.

Fig. 4.

Effect of PP2 (10 μM) on Ca²⁺ sparklets in tsA-201 cells coexpressing WT Ca_v1.2c + c-Src (n = 5). A: representative traces of persistent and low activity Ca²⁺ sparklets before and after application of PP2 (10 μM). B: bar plot of means ± SE persistent Ca²⁺ sparklet density before and after PP2 application (N = 5). Bar plots of mean nP_s ± SE of persistent (C; N = 5) and low activity (D; N = 6) Ca²⁺ sparklet sites before and after PP2 application. Error bars represent SE values; n, number of cells; N, number of sparklet sites; *P < 0.05.

Application of drugs.

Xestospongin C (90 μM) was included in the patch-pipette solution to block inositol 1,4,5-trisphosphate receptors. Tetracaine (50 μM) was added to the bath solution to inhibit ryanodine receptors. For some experiments, PP2 (10 μM) was included in the bath solution to inhibit c-Src activity.

Statistical analysis.

Previous analyses of Ca²⁺ sparklet density and Ca²⁺ sparklet activity by two of our laboratories (1, 23–28) have shown that while sparklet density follows a normal distribution, sparklet activity does not if it is not divided into low and persistent sparklet sites. Parametric statistical analyses are appropriate for normally distributed data and nonparametric analyses for nonnormally distributed data. Here, the data as presented followed a normal distribution and thus parametric analyses were used. A one-tail unpaired t-test was used to compare the Ca²⁺ sparklet activities and persistent Ca²⁺ sparklet densities of the control and test groups. A one-tail paired t-test was used to evaluate the effect of PP2 on Ca²⁺ sparklets. P < 0.05 was considered to be statistically significant.

Chemical reagents.

Xestospongin C and PP2 were purchased from Calbiochem (San Diego, CA). Rhod-2 was procured from Invitrogen (San Diego, CA). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO).

RESULTS

Ca²⁺ fluorescence events in tsA-201 cells.

TIRF imaging of cells expressing WT Ca_v1.2c channels confirmed the presence of Ca²⁺ sparklets (Fig. 1). Based on their nP_s values, Ca²⁺ sparklets were characterized as either persistent (nP_s ≥ 0.2) or low (nP_s < 0.2) activity events. We also recorded Ca²⁺ fluorescence events similar to Ca²⁺ sparklets from cells expressing GFP alone in the absence of measurable I_Ca upon depolarization (Fig. 2A). Since we had no objective reasons for excluding such anomalous events, they were analyzed in the same manner as Ca²⁺ sparklets. Importantly, the mean number of anomalous event sites per cell in cells expressing GFP alone (n = 9 cells) was significantly lower than the mean number of Ca²⁺ sparklet sites per cell observed after expression of Ca_v1.2c channels (+GFP; n = 10 cells; P < 0.05; Fig. 2B). Persistent Ca²⁺ sparklets were not observed in absence of Ca_v1.2c expression (Fig. 2, C and D), and the activity of all anomalous events, measured as nP_s, was <0.2, similar to low activity Ca²⁺ sparklets (Fig. 2E). These results indicated that Ca_v1.2c channels were responsible for all of the persistent Ca²⁺ sparklet activity under the recording conditions used in our experiments and the occurrence and inclusion of anomalous Ca²⁺ events would not influence the persistent Ca²⁺ sparklet data analysis in further protocols.

Fig. 1.

Total internal reflection fluorescence (TIRF) images and fluorescence traces from a tsA-201 cell exhibiting low activity (A) and persistent Ca²⁺ sparklets (B). AU, arbitrary units.

Fig. 2.

Whole cell Ca²⁺ current and Ca²⁺ sparklet activity in tsA-201 cells expressing wild-type (WT) Ca_v1.2c + green fluorescent protein (GFP) or GFP alone. A: representative calcium current (I_Ca) traces evoked by a depolarization step from −70 mV to +30 mV in GFP alone and WT Ca_v1.2c expressing tsA-201 cells. Bar plots of means ± SE (B) Ca²⁺ sparklet or anomalous Ca²⁺ fluorescence event sites per cell and (C) persistent Ca²⁺ sparklet densities in cells expressing WT Ca_v1.2c + GFP (n = 10) and GFP alone (n = 9). Bar plots of mean Ca²⁺ sparklet activity (nP_s; where n = no. of quantal levels and P_s = probability of occurrence of Ca²⁺ sparklet) ± SE of persistent (D) and low (E) activity Ca²⁺ sparklets in cells expressing GFP alone (n = 7) and WT Ca_v1.2c + GFP (n = 10). Error bars represent SE values; n, number of cells; *P < 0.05.

c-Src underlies persistent Ca²⁺ sparklets through Ca_v1.2c in tsA-201 cells.

To investigate the role of c-Src in regulation of persistent Ca²⁺ sparklet activity, we employed pharmacological means as well as manipulated the expression of proteins of interest in tsA-201 cells. First, TIRF imaging was performed on tsA-201 cells expressing c-Src alone and WT Ca_v1.2c + c-Src. The latter test group (n = 9 cells) exhibited almost 10-fold higher persistent Ca²⁺ sparklet density than cells expressing only c-Src (n = 9 cells; P < 0.05; Fig. 3A). Similar to nontransfected cells, we also observed anomalous Ca²⁺ events in cells expressing c-Src alone (Fig. 3, B and C). However, all of these anomalous Ca²⁺ events had nP_s <0.2 with the exception of one event (n = 9 cells). These data further corroborated our inference that the occurrence of anomalous Ca²⁺ events would not substantially affect persistent Ca²⁺ sparklet data analysis. Of note, the activity and density of persistent Ca²⁺ sparklets in cells coexpressing WT Ca_v1.2c and c-Src were similar to those observed in cells expressing WT Ca_v1.2c alone (compare Figs. 2 and 3). This observation suggests at least two possible explanations. First, c-Src may not be involved in the production of persistent Ca²⁺ sparklets. Second, endogenous c-Src expression may be sufficient to promote persistent sparklet activity. Indeed, tsA-201 cells have been shown previously to express endogenous c-Src (12).

Fig. 3.

Ca²⁺ sparklet analysis in tsA-201 cells expressing c-Src alone or with WT Ca_v1.2c. A: bar plot of means ± SE persistent Ca²⁺ sparklet density in cells expressing WT Ca_v1.2c + c-Src (n = 9) and c-Src alone (n = 9). Bar plots of mean nP_s ± SE of persistent (B) and low (C) activity Ca²⁺ sparklets in cells expressing WT Ca_v1.2c + c-Src (n = 8) or c-Src alone (n = 6). Error bars represent SE values; n, number of cells; *P < 0.05.

We substantiated the role of c-Src in producing persistent Ca²⁺ sparklet activity through Ca_v1.2c by recording Ca²⁺ sparklets from cells coexpressing WT Ca_v1.2c and c-Src before and after treatment with 10 μM PP2, a membrane-permeable c-Src antagonist (Fig. 4). Application of PP2 resulted in a 2.3- and 6.5-fold decrease in the mean density and activity respectively, of persistent Ca²⁺ sparklets (N = 5 sparklet sites; n = 5 cells; P < 0.05; Fig. 4, A–C) without significantly changing the mean activity of low activity Ca²⁺ sparklet sites (N = 6 sparklet sites; n = 5 cells; P > 0.05; Fig. 4D). To address whether endogenous c-Src was responsible for producing persistent Ca²⁺ sparklets, we tested the effect of PP2 on Ca²⁺ sparklet activity in cells expressing WT Ca_v1.2c alone. Indeed, PP2 application decreased persistent Ca²⁺ sparklet density (Fig. 5A) while reducing Ca²⁺ sparklet activity significantly in cells expressing WT Ca_v1.2c alone (N = 8 sparklet sites; n = 5 cells; P < 0.05; Fig. 5B). Interestingly, PP2 treatment also resulted in a significant reduction in the activity of low activity Ca²⁺ sparklets (N = 13 sparklet sites; n = 5 cells; P < 0.05; Fig. 5C).

Fig. 5.

Effect of PP2 (10 μM) on Ca²⁺ sparklets in tsA-201 cells expressing WT Ca_v1.2c alone (n = 5). A: bar plot of means ± SE persistent Ca²⁺ sparklet density before and after PP2 application (N = 5). Bar plots of means nP_s ± SE of persistent (B; N = 8) and low activity (C; N = 13) Ca²⁺ sparklet sites before and after PP2 application. Error bars represent SE values; n, number of cells; N, number of sparklet sites; *P < 0.05.

To further test the involvement of c-Src, we coexpressed WT Ca_v1.2c with kinase dead c-Src (kd c-Src) in tsA-201 cells to competitively inhibit endogenous c-Src (Fig. 6). kd c-Src harbors a single point mutation in the kinase domain of the enzyme resulting in ablation of c-Src kinase activity. Out of six cells cotransfected with WT Ca_v1.2c and kd c-Src, persistent Ca²⁺ sparklet activity was detected in only one cell. Evaluation of persistent Ca²⁺ sparklet density revealed a 3.4-fold reduction in cells transfected with WT Ca_v1.2c and kd c-Src (n = 6 cells) compared with cells coexpressing WT Ca_v1.2c and c-Src (n = 9 cells; P < 0.05; Fig. 6A). The means of persistent Ca²⁺ sparklet activities of the two transfection groups were comparable but could not be statistically compared due to an insufficient number of persistent sparklets in the WT Ca_v1.2c + kd c-Src transfection group (Fig. 6B). As expected, the means for nP_s of low activity Ca²⁺ sparklets in cells coexpressing c-Src or kd c-Src with WT Ca_v1.2c were similar (Fig. 6C). Collectively, these data suggest that c-Src expression promotes persistent Ca²⁺ sparklet activity.

Fig. 6.

Ca²⁺ sparklet analysis in tsA-201 cells expressing WT Ca_v1.2c with c-Src or kd c-Src. A: bar plot of means ± SE persistent Ca²⁺ sparklet density in cells expressing WT Ca_v1.2c + c-Src (n = 9) and WT Ca_v1.2c + kd c-Src (n = 6). Bar plots of mean nP_s ± SE of persistent (B) and low (C) activity Ca²⁺ sparklets in cells expressing WT Ca_v1.2c + c-Src (n = 8) and WT Ca_v1.2c + kd c-Src (n = 3). Error bars represent SE values; n, number of cells; *P < 0.05.

c-Src phosphorylates Ca_v1.2c at residue Y²¹²² to produce persistent Ca²⁺ sparklets.

To elucidate the mechanism by which c-Src enhances Ca_v1.2c activity, we tested the role of two potential phosphorylation sites (Y²¹²² and Y²¹³⁹) on the COOH terminus of rat Ca_v1.2c (2, 12). Coexpression of the Y²¹²²F Ca_v1.2c construct with c-Src (n = 19 cells) resulted in a 3.4-fold decrease in the density of persistent Ca²⁺ sparklets with respect to cells transfected with WT Ca_v1.2c + c-Src (n = 19 cells; P < 0.05; Fig. 7A). No such differences were observed between mean persistent Ca²⁺ sparklet densities of cells expressing Y²¹³⁹F Ca_v1.2c + c-Src and WT Ca_v1.2c + c-Src (P > 0.05; Fig. 7A). Neither of the single point mutations on Ca_v1.2c led to significant changes in the mean activities of persistent or low activity Ca²⁺ sparklets compared with cells expressing WT Ca_v1.2c + c-Src (Fig. 7, B and C). These findings suggest that Y²¹²² on the COOH terminus of Ca_v1.2c is the major phosphorylation site involved in production of persistent Ca²⁺ sparklets by c-Src.

Fig. 7.

Ca²⁺ sparklet analysis in tsA-201 cells coexpressing c-Src with WT, Y²¹²²F or Y²¹³⁹F Ca_v1.2c constructs. A: bar plot of means ± SE persistent Ca²⁺ sparklet density in cells expressing WT Ca_v1.2c + c-Src (n = 19), Y²¹²²F Ca_v1.2c + c-Src (n = 19), and Y²¹³⁹F Ca_v1.2c + c-Src (n = 17). Bar plots of mean nP_s ± SE of persistent (B) and low (C) activity Ca²⁺ sparklets in cells expressing WT Ca_v1.2c + c-Src (n = 17), Y²¹²²F Ca_v1.2c + c-Src (n = 17), and Y²¹³⁹F Ca_v1.2c + c-Src (n = 12). Error bars represent SE values; n, number of cells; *P < 0.05.

PKC-α does not induce persistent Ca²⁺ sparklets via c-Src.

To test if PKC-α induces persistent Ca²⁺ sparklet activity by activating endogenous c-Src, we performed Ca²⁺ sparklet experiments on cells coexpressing PKC-α and Y²¹²²F Ca_v1.2c, Y²¹³⁹F Ca_v1.2c, or WT Ca_v1.2c. The mean persistent Ca²⁺ sparklet densities of the Y²¹²²F Ca_v1.2c + PKC-α (n = 12 cells) and Y²¹³⁹ F Ca_v1.2c + PKC-α (n = 8 cells) transfection groups did not show any significant reduction compared with the WT Ca_v1.2c + PKC-α (n = 9 cells) transfection group (Fig. 8A). Similarly, the mean nP_s values of both low and persistent Ca²⁺ sparklets in cells expressing Y²¹²²F Ca_v1.2c + PKC-α (n = 7 cells; P > 0.05) or Y²¹³⁹F Ca_v1.2c + PKC-α (n = 7 cells; P > 0.05) were similar to those in cells expressing WT Ca_v1.2c + PKC-α (n = 9 cells) (Fig. 8, B and C). Furthermore, inhibition of endogenous c-Src by PP2 (10 μM) did not change the activity (N = 4 sparklet sites; n = 3 cells) or the density (N = 3 sparklet sites; n = 3 cells) of PKC-α-induced persistent Ca²⁺ sparklets in cells coexpressing PKC-α and WT Ca_v1.2c (P > 0.05; Fig. 9, A and B). In contrast, PP2 application led to an increase in the activity of low activity Ca²⁺ sparklet sites (N = 9 sparklet sites; n = 7 cells; P < 0.05; Fig. 9C). Overall, these data suggest that PKC-α does not induce persistent Ca²⁺ sparklet activity through c-Src.

Fig. 8.

Ca²⁺ sparklet analysis in tsA-201 cells coexpressing PKC-α with WT, Y²¹²²F or Y²¹³⁹F Ca_v1.2c constructs. A: bar plot of means ± SE persistent Ca²⁺ sparklet density in cells expressing WT Ca_v1.2c + PKC-α (n = 9), Y²¹²²F Ca_v1.2c + PKC-α (n = 12), and Y²¹³⁹F Ca_v1.2c + PKC-α (n = 8). Bar plots of mean nP_s ± SE of persistent (B) and low (C) activity Ca²⁺ sparklets in cells expressing WT Ca_v1.2c + PKC-α (n = 9), Y²¹²²F Ca_v1.2c + PKC-α (n = 7), and Y²¹³⁹F Ca_v1.2c + PKC-α (n = 7). Error bars represent SE values; n, number of cells.

Fig. 9.

Effect of PP2 (10 μM) on Ca²⁺ sparklets in tsA-201 cells coexpressing WT Ca_v1.2c and PKC-α. A: bar plot of means ± SE of persistent Ca²⁺ sparklet density before and after PP2 application (N = 3; n = 3). Bar plots of mean nP_s ± SE of persistent (B; N = 4; n = 3) and low activity (C; N = 9; n = 7) Ca²⁺ sparklet sites before and after PP2 application. Error bars represent SE values; n, number of cells; N, number of sparklet sites; *P < 0.05.

DISCUSSION

The present study is the first to investigate the role of c-Src in the production of persistent Ca²⁺ sparklets. Our findings suggest that c-Src promotes persistent Ca²⁺ sparklets by phosphorylation of Ca_v1.2c on residue Y²¹²². We could not find evidence that PKC-α acts via c-Src to induce persistent Ca²⁺ sparklet activity of Ca_v1.2c. The possibility of parallel actions of c-Src and PKC-α conforms to existing ideas about the complex regulation of Ca_v1.2 channels to fully explain their central roles in the regulation of many physiological functions.

One challenge that arose in the present study was dealing with the presence of anomalous Ca²⁺ events in untransfected tsA-201 cells. The origin of these anomalous events could not be resolved, but they might be related to the endogenous expression of Ca²⁺-permeable transient receptor potential channels in this cell line. Similar events have been observed in Xenopus oocytes expressing N-type Ca²⁺ channels at negative (−60 to −120 mV) holding potentials and have been reported to have similar amplitudes, durations and spatial spread as N-type Ca²⁺ channel fluorescence events (7). Reportedly, the amplitudes of those anomalous events decreased with the application of depolarizing pulses that were used to record events associated with N-type Ca²⁺ channels. We could not use a similar strategy to eliminate anomalous Ca²⁺ events for at least two reasons: 1) Ca_v1.2 channels exhibit a higher open probability with membrane depolarization, which results in increased basal Ca²⁺ fluorescence (24); and 2) the driving force for Ca²⁺ decreases at higher potentials, leading to smaller changes in Ca²⁺ fluorescence upon Ca_v1.2 channel opening that are more difficult to resolve. Due to these limitations, we focused primarily on the analysis of persistent Ca²⁺ sparklet activity. The frequency of anomalous Ca²⁺ events resembling persistent Ca²⁺ sparklets was very low, thus making our results less prone to errors caused by the presence of such events among persistent Ca²⁺ sparklets in cells that expressed Ca_v1.2c alone or with kinases.

Effect of c-Src on persistent Ca²⁺ sparklet activity.

c-Src activation has previously been shown to increase whole cell Ca_v1.2c current in response to insulin-like growth factor-I and α₅β₁ integrin activation (2, 12, 36), and c-Src has been implicated in the basal regulation of Ca_v1.2c current as well (36). In the present study, we tested the involvement of c-Src in the production of persistent Ca²⁺ sparklet activity. Our results suggest that c-Src promotes persistent Ca²⁺ sparklet activity by acting on residue Y²¹²² of the COOH terminus of Ca_v1.2c (Fig. 7). Of the various intracellular regions on rat Ca_v1.2c, only the region from amino acid residues 1,932 to 2,143, containing tyrosine residues at positions 2,122 and 2,139, is known to be phosphorylated by c-Src (2). Furthermore, previous studies have provided evidence for phosphorylation of Y²¹²² by c-Src, resulting in an increase in Ca_v1.2 channel current (2, 12, 36). Interestingly, in the present study, mutation of Y²¹²² on Ca_v1.2c did not completely eliminate persistent Ca²⁺ sparklets (Fig. 7, A and B). There are at least two possible explanations for the residual persistent Ca²⁺ sparklet activity: 1) c-Src phosphorylates another tyrosine residue on Ca_v1.2c, or 2) the presence of another Ca_v1.2c activating mechanism independent of c-Src. Here, we used PP2 treatment and kd c-Src overexpression to selectively inhibit c-Src-induced persistent Ca²⁺ sparklets. These c-Src inhibition strategies have been used successfully in previous studies (2, 12, 36), and both approaches attenuated the occurrence of persistent Ca²⁺ sparklets here (Figs. 4 and 6).

An interesting observation was that PP2 application significantly reduced both persistent Ca²⁺ sparklet activity and density in cells expressing WT Ca_v1.2c and c-Src whereas the Y²¹²²F mutation on Ca_v1.2c or expression of kd c-Src had an effect only on persistent Ca²⁺ sparklet density. Exclusion of cells that did not show any sparklet activity (see materials and methods) based on nP_s analysis may have masked the attenuating effect of the Y²¹²²F mutation or kd c-Src expression on nP_s values. Another plausible explanation could be the presence of a population of persistent Ca²⁺ sparklets regulated by another kinase like PKC in addition to that promoted by c-Src. Such a population of persistent Ca²⁺ sparklets might not be affected by the Y²¹²²F mutation or kd c-Src expression.

The activity of a channel at any given time is determined by the balance of the activity of specific kinases and phosphatases in its immediate vicinity. The balance between the local activities of PKC-α, PP2A, and PP2B has previously been suggested to produce different patterns of Ca_v1.2 activity across the plasma membrane, and these sites with differential activities have been classified as silent, low, and persistent Ca²⁺ sparklet activity sites (23). Preferential phosphorylation of some Ca_v1.2 channels over the others by PKC-α, based on the colocalization of PKC-α with Ca_v1.2, has been suggested to be responsible for the production of localized persistent Ca²⁺ sparklet activity (24). Immunofluorescence and confocal imaging experiments indicate that Ca_v1.2 channels have a diffuse distribution while PKC-α is distributed in clusters near the plasma membrane (24). Whether c-Src has a clustered distribution on the cell membrane under the conditions of our experiments is not known; however, c-Src has been shown in multiple studies to localize preferentially to focal adhesions (36, 20). Based on the observation that both low activity and persistent Ca²⁺ sparklets occur in cells transfected with WT Ca_v1.2c and c-Src, we can speculate that c-Src, like PKC-α, regulates a small population of Ca_v1.2 channels on the plasma membrane. In that case, the activity of a sparklet site may be determined by the activities of nearby c-Src and possibly unidentified phosphatases.

Although the experiments in this study were performed on tsA-201 cells expressing Ca_v1.2c, it is highly likely that c-Src also induces persistent Ca²⁺ sparklet activity in native vascular smooth muscle cells. Most vascular smooth muscle cells have high expression levels of c-Src (30). c-Src activation has been shown to increase Ca_v1.2 current in rat arteriolar, rabbit portal vein, and human colonic smooth muscle cells (5, 12, 14–17, 36). Because Ca_v1.2c and Ca_v1.2b share identical sequences (in rat) around residue Y²¹²², these results are likely to be applicable to rat Ca_v1.2b. Both rabbit and human Ca_v1.2b lack the tyrosine residue corresponding to Y²¹²², suggesting that in those species Ca_v1.2b is phosphorylated by c-Src at other tyrosine residues (17).

Mechanism of PKC-α action on persistent Ca²⁺ sparklet activity.

PKC-α activation is linked to an increase in persistent Ca²⁺ sparklet activity in freshly isolated cerebral arterial myocytes and in tsA-201 cells expressing WT Ca_v1.2c + PKC-α (23, 24); however, the mechanism by which PKC-α acts on Ca_v1.2 channels is not well understood. In this study, we tested whether c-Src acts downstream of PKC-α in the series of events that lead to increased persistent Ca²⁺ sparklet activity. PKC has been shown to lie upstream of c-Src in signaling pathways controlling actin reorganization (3, 4), podosome formation (11), as well as in the regulation of smooth muscle Ca_v1.2 channels in smooth muscle (5). PKC may activate c-Src directly or indirectly, and c-Src activation mechanisms may also vary among the various PKC isoforms expressed by any particular cell type. If PKC-α acts solely via c-Src to produce persistent Ca²⁺ sparklets, mutation of Y²¹²² should have resulted in at least a partial reduction of persistent Ca²⁺ sparklet activity. However, the data in Fig. 8 do not support such an effect. Furthermore, PP2 application did not significantly change persistent Ca²⁺ sparklet density or activity in cells coexpressing WT Ca_v1.2c + PKC-α (Fig. 9, A and B). These data clearly suggest that PKC-α induces persistent Ca²⁺ sparklet activity in a c-Src independent manner. In the same test groups, the PP2-induced increase in nP_s of low activity Ca²⁺ sparklets was intriguing (Fig. 9C), particularly as PP2 inhibited low activity Ca²⁺ sparklets in cells expressing WT Ca_v1.2c alone (Fig. 5C). This fundamental difference in the effect of PP2 on low activity Ca²⁺ sparklets in the presence or absence of PKC-α may be related to the ∼10-fold difference in the basal nP_s level for low activity Ca²⁺ sparklets. A possible explanation is that PP2 is acting to inhibit c-Src phosphorylation of tyrosine residues in other regions of Ca_v1.2 when PKC is activated.

By what alternative mechanisms could PKC-α induce persistent Ca²⁺ sparklet activity? It is possible that PKC-α directly phosphorylates Ca_v1.2c to produce persistent Ca²⁺ sparklets. The NH₂ terminus of the cardiac Ca_v1.2 isoform has been implicated in both PKC-mediated stimulation and inhibition of Ca_v1.2 (21, 31, 32), but the NH₂ terminus of the (neuronal) Ca_v1.2c isoform lacks the potential serine and threonine PKC phosphorylation sites of the Ca_v1.2a (cardiac) and Ca_v1.2b (smooth muscle) isoforms. The occurrence of persistent Ca²⁺ sparklets in cells expressing WT Ca_v1.2c + PKC-α suggests that phosphorylation of the NH₂ terminus of Ca_v1.2c by PKC-α is not required to produce persistent Ca²⁺ sparklet activity. Site S¹⁹⁰¹ on Ca_v1.2c (S¹⁹²⁸ on Ca_v1.2a and b) is conserved among different Ca_v1.2 channel isoforms in different species and has long been considered the primary PKA phosphorylation site on Ca_v1.2 channels (6, 10, 22, 33). However, some recent studies have challenged the role of S¹⁹²⁸ phosphorylation in PKA-mediated potentiation of Cav1.2 current (8, 9, 19) by suggesting that PKA acts primarily by phosphorylation of a more proximal residue, S¹⁷⁰⁰ to relieve the inhibition by the noncovalently associated distal COOH-terminal inhibitory domain (8, 37). The S¹⁹²⁸ residue of Ca_v1.2 is also implicated as a PKG phosphorylation site using in vitro kinase assays, but PKG phosphorylation of S¹⁹²⁸ does not seem to be involved in PKG-mediated inhibition of Ca_v1.2 current. In addition, biochemical experiments provide evidence for S¹⁹²⁸ phosphorylation by PKC-α (38), but whether this phosphorylation event translates into increased channel function or sparklet activity remains unknown.

In conclusion, our data show that c-Src can enhance the activity of persistent Ca²⁺ sparklets in transfected tsA-201 cells through phosphorylation of Ca_v1.2c at residue Y²¹²². However, cells coexpressing Y²¹²²F Ca_v1.2c and PKC-α continue to show an increase in persistent Ca²⁺ sparklet activity. These findings indicate that PKC-α does not act upstream of c-Src to produce persistent Ca²⁺ sparklets under these conditions.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grants P01 HL-095486, HL-085870, HL-085686, and HL-098200 and American Heart Association Grants 0735251N and 0840094N.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: J.G., L.F.S., and M.J.D. conception and design of research; J.G., M.F.N., P.G., J.-T.C., J.L.M., and L.F.S. performed experiments; J.G. and J.L.M. analyzed data; J.G., M.F.N., P.G., J.-T.C., L.F.S., and M.J.D. interpreted results of experiments; J.G., J.L.M., and M.J.D. prepared figures; J.G. and M.J.D. drafted manuscript; J.G., M.F.N., L.F.S., and M.J.D. edited and revised manuscript; J.G. and M.J.D. approved final version of manuscript.