Proteolytic cleavage and PKA phosphorylation of α_1C subunit are not required for adrenergic regulation of Ca_V1.2 in the heart

Significance

Calcium influx through the cardiac voltage-dependent L-type calcium channel (Ca_V1.2) increases during “fight or flight” through activation of the β-adrenergic and protein kinase A (PKA) signaling pathway. None of the previously identified sites in the α_1C subunit, each painstakingly and singly investigated, were shown to be required for adrenergic modulation of Ca_V1.2. Our approach allowed an unprecedented and massive increase in throughput, because we mutated many potential PKA phosphorylation sites throughout α_1C. By creating transgenic mice expressing either α_1C with alanines substituted for all conserved consensus PKA phosphorylation sites or, separately, α_1C without C-terminal proteolytic cleavage, our paradigm-shifting results show that acute β-adrenergic regulation of Ca_V1.2 does not require phosphorylation of any conserved Ser/Thr of α_1C or the proteolytic cleavage of the C terminus of α_1C.

Abstract

Calcium influx through the voltage-dependent L-type calcium channel (Ca_V1.2) rapidly increases in the heart during “fight or flight” through activation of the β-adrenergic and protein kinase A (PKA) signaling pathway. The precise molecular mechanisms of β-adrenergic activation of cardiac Ca_V1.2, however, are incompletely known, but are presumed to require phosphorylation of residues in α_1C and C-terminal proteolytic cleavage of the α_1C subunit. We generated transgenic mice expressing an α_1C with alanine substitutions of all conserved serine or threonine, which is predicted to be a potential PKA phosphorylation site by at least one prediction tool, while sparing the residues previously shown to be phosphorylated but shown individually not to be required for β-adrenergic regulation of Ca_V1.2 current (17-mutant). A second line included these 17 putative sites plus the five previously identified phosphoregulatory sites (22-mutant), thus allowing us to query whether regulation requires their contribution in combination. We determined that acute β-adrenergic regulation does not require any combination of potential PKA phosphorylation sites conserved in human, guinea pig, rabbit, rat, and mouse α_1C subunits. We separately generated transgenic mice with inducible expression of proteolytic-resistant α_1C. Prevention of C-terminal cleavage did not alter β-adrenergic stimulation of Ca_V1.2 in the heart. These studies definitively rule out a role for all conserved consensus PKA phosphorylation sites in α_1C in β-adrenergic stimulation of Ca_V1.2, and show that phosphoregulatory sites on α_1C are not redundant and do not each fractionally contribute to the net stimulatory effect of β-adrenergic stimulation. Further, proteolytic cleavage of α_1C is not required for β-adrenergic stimulation of Ca_V1.2.

Ca²⁺ influx through the cardiac voltage-dependent L-type calcium channel (Ca_v1.2) initiates excitation–contraction coupling. As part of the “fight-or-flight” response, β-adrenergic agonists signaling through the protein kinase A (PKA) pathway rapidly enhance Ca²⁺ current by increasing the mean open time (mode 2 gating) and open probability of Ca_V1.2 channels (1). The molecular mechanisms of this fundamental regulatory process remain unknown despite decades of investigation, although it is well-established that cAMP-PKA–mediated phosphorylation is a fundamental event (2–4). PKA phosphorylation of Ser¹⁹²⁸ in the cardiac α_1C subunit was initially proposed to be required for adrenergic modulation (5), but this was ruled out by expression, using adenovirus or knock-in strategies, of an α_1C subunit with substitution of Ser¹⁹²⁸ by Ala (6, 7). Ser¹⁵¹² and Ser¹⁵⁷⁰ in α_1C, which are essential for modulation of Ca_V1.2 by Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), are also not required for adrenergic stimulation (8). Ser¹⁷⁰⁰ was then proposed as the primary and essential phosphorylation site responsible for PKA modulation of Ca_V1.2, based on experiments in tsA-201 cells (9). Phosphorylation of Thr¹⁷⁰⁴ by casein kinase II was also proposed to modulate basal Ca_V1.2 function. We recently showed, however, that Ser¹⁷⁰⁰ and Thr¹⁷⁰⁴ are not required for β-adrenergic stimulation of Ca_V1.2 in cardiomyocytes (10). PKA targets in the Ca_V1.2 auxiliary β₂ subunits Ser⁴⁵⁹, Ser⁴⁷⁸, and Ser⁴⁷⁹ (11) are also nonessential (6, 8, 12). Thus, Ca_V1.2 residues previously identified as PKA targets by standard biochemical methodologies, including mass spectrometry and site-directed mutagenesis, are not essential for the rapid β-adrenergic stimulation of Ca_V1.2 in the heart.

The failure to identify a single site as essential for the acute β-adrenergic modulation of Ca_V1.2 led us to propose several possible explanations for these findings: (i) that the phosphorylation of any one of several α_1C residues can induce the adrenergic stimulation of Ca_V1.2 current (redundancy), (ii) that each phosphorylated residue contributes a small fraction of the total effect, and/or (iii) that the critical PKA phosphorylation sites in α_1C have not yet been identified. To test these hypotheses, we replaced with Ala all consensus intracellular PKA phosphorylation sites in α_1C that are conserved in the rabbit, human, guinea pig, mouse, and rat and determined whether elimination of PKA phosphorylation sites in α_1C blocked Ca_V1.2’s responsiveness to adrenergic modulation in cardiomyocytes.

In addition to phosphorylation by PKA, proteolytic cleavage of the α_1C C terminus, occurring in greater than 80% of cardiac Ca_V1.2 channels, has been posited to play an essential role in adrenergic regulation of Ca_V1.2. Specifically, cleavage is proposed to set the basal Ca_V1.2 activity, which is then augmented by adrenergic stimulation (5, 9, 13–18). The functional relevance of proteolytic cleavage of α_1C, however, has not been demonstrated in cardiomyocytes. Indirect evidence, consisting of mass spectrometric analysis of skeletal muscle α_1S proteolytic peptides and sequence alignments of α_1S and α_1C, was the basis for initially proposing Ala¹⁸⁰⁰ in the context of a ¹⁷⁹⁸NNAN motif as the α_1C proteolytic site (13). Although deletion of the ¹⁷⁹⁸NNAN motif did not alter the proteolytic cleavage of α_1C (10), cleavage likely occurs in this general region based upon the observed molecular weight of the truncated α_1C (Fig. 1A). Moreover, the persistence of α_1C cleavage after deletion of the ¹⁷⁹⁸NNAN motif could result from the presence of a similar motif, ¹⁷⁹⁴NANI¹⁷⁹⁷, which is still present when NNAN is deleted. Although the protease responsible for cleavage of α_1C is not known, it was speculated to be calpain-like (13, 15, 17, 19), and a conserved motif rich in Pro, Glu, Ser, and Thr (PEST), which can serve as substrate recognition sites for calpains (20), is just N-terminal to Ala¹⁸⁰⁰. We hypothesized that deletion of these motifs may abrogate C-terminal proteolytic cleavage and enable us to explore the role of proteolytic cleavage in fostering β-adrenergic regulation of Ca_V1.2. Thus, we created two transgenic mouse lines with deletion of either the PEST sequence ¹⁷⁶⁹DTESP alone (ΔPEST) or the PEST sequence and ¹⁷⁹⁴NANINNANN¹⁸⁰² (ΔPEST/PRO) to test the role of proteolytic cleavage of α_1C in regulating Ca_V1.2 function in cardiomyocytes.

Fig. 1.

Putative PKA phosphorylation sites and proteolytic cleavage site in Ca_V1.2 α_1C. (A) Schematic of rabbit cardiac α_1C subunit topology. The putative proteolytic cleavage region is identified; the residues of deleted ¹⁷⁹⁴NANINNANN are shown. The PEST sequence, ¹⁷⁶²DTESPS, is also shown. Solid red circles represent some of the putative PKA phosphorylation sites. (B) Putative PKA phosphorylation sites in rabbit α_1C. Residues in red are predicted phosphorylation sites and were mutated to Ala in the 17-mutant or 22-mutant transgenic mice. Ser and Thr, shown in blue, although not predicted to be phosphorylated residues, were mutated to Ala. Conserved indicates conserved in the human, guinea pig, rabbit, rat, and mouse. Predicted indicates predicted by at least one of the prediction tools, protein kinase A phosphorylation sites using the simplified kinase binding model (pKaPS), Disorder-Enhanced Phosphorylation Sites Predictor (DISPHOS), GPS, NetPhos, and Scansite. The borderline indicates within the “twilight” zone of pKaPS (31). (C) Schematic representation of the binary transgene system. The αMHC-rtTA is the standard cardiac-specific reverse tetracycline-controlled transactivator system. The αMHC_MOD construct is a modified αMHC promoter containing the tet operon (tetO) for regulated expression of FLAG-tagged DHP-resistant (DHP*) α_1C.

Materials and Methods

Reagents.

Nisoldipine and Rp-8-Br-cAMPS were purchased from Santa Cruz Biotechnology. All other chemicals were acquired from Sigma.

Animals.

The α_1C transgenic constructs were generated by fusing rabbit Cacna1c cDNA (accession no. X15539) to the modified murine α-myosin heavy chain (MHC), tetracycline-inducible promoter (“responder” line) vector (a gift of Jeffrey Robbins and Jeffrey Molkentin, University of Cincinnati, Cincinnati) (21, 22). The α_1C subunit was engineered to be dihydropyridine (DHP)-insensitive with the substitutions T1066Y and Q1070M (23, 24). These mice were bred with cardiac-specific (αMHC) doxycycline-regulated, codon-optimized reverse transcriptional transactivator (rtTA) mice obtained via Mutant Mouse Resource and Research Centers (MMRRC) (25) to generate double-transgenic mice. To induce expression, animals received 0.2 g/kg of doxycycline-impregnated food (catalog no. S3888; Bio Serv) for 1 d. The Institutional Animal Care and Use Committee at Columbia University approved all animal experiments.

Immunoblots and Immunofluorescence.

Cardiac lysates from 8- to 12-wk-old doxycycline-fed transgenic mice were prepared as described (10). Proteins were size-fractionated, transferred to nitrocellulose membranes, and probed with anti-FLAG antibody (Sigma), anti-α_1C (10), and antitubulin (Santa Cruz Biotechnology) antibodies. Detection was performed with a CCD camera (Carestream Imaging), and ImageQuant software was used for quantification. Isolated cardiomyocytes were fixed for 15 min in 4% paraformaldehyde, and indirect immunofluorescence performed using a 1:200 rabbit anti-FLAG antibody and 1:200 FITC-labeled goat–anti-rabbit antibody (Sigma). Images were acquired using a confocal microscope.

Cellular Electrophysiology.

Membrane currents were measured by the whole-cell patch-clamp method using a MultiClamp 700B amplifier and pCLAMP10 software (Molecular Devices) as described elsewhere (10). The pipette solution contained 40 mM CsCl, 90 mM Cs gluconate, 10 mM 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), 1 mM MgCl₂, 4 mM Mg-ATP, 2 mM CaCl₂, and 10 mm Hepes, adjusted to pH 7.2 with CsOH. After the isolated cardiomyocytes were adequately buffered with 10 mM BAPTA in the internal solution, the isolated cardiomyocytes (26) were superfused with 140 mM tetraethylammonium-Cl, 1.8 mM CaCl₂, 1 mM MgCl₂, 10 mM glucose, and 10 mM Hepes, adjusted to pH 7.4 with CsOH. Pipette series resistances were usually <1 MΩ after 60% compensation. Leak currents and capacitance transients were subtracted by a P/4 protocol. To measure Ca²⁺ peak currents, the cell membrane potential was held at −50 mV and stepped to +10 mV for 350 ms every 10 s. To evaluate the current–voltage (I-V) relationship for Ca²⁺ currents, the same protocol was repeated with steps between −40 mV and +60 mV in 10-mV increments. All experiments were performed at room temperature: 22 ± 1 °C. For I-V curves, we used a Boltzmann distribution: I(V) = G_max * (V − E_rev)/[1 + exp(V_mid − V)/k)], where G_max is maximal conductance, E_rev is reversal potential, V_mid is the midpoint, and k is the slope factor.

Fractional Shortening.

Freshly isolated myocytes were superfused with Tyrode’s solution containing 1.0 mM CaCl₂ and 300 nM nisoldipine. Myocytes were field-stimulated at 1 Hz. Fractional shortening of sarcomere length was measured using the SarcLen module of Ionoptix.

Statistical Analysis.

Results are presented as mean ± SEM. For multiple group comparisons, one-way ANOVA, followed by Tukey’s post hoc test, was performed. For comparisons between two groups, an unpaired Student’s t test was used. Statistical analyses were performed using Prism 6 (Graphpad Software). Differences were considered statistically significant at values of P < 0.05.

Results

Generation of Inducible, Cardiac-Specific PKA Phosphorylation Site Mutant Mice.

The optimal PKA phosphorylation motif is a tetrapeptide with Arg at the second and third positions (termed -2 and -3) before the phosphorylated Ser or Thr and a large hydrophobic residue immediately thereafter (R-R-X-S/T-Φ) (27–29). The positions between -4 and -1 have a strong preference for Arg and, to a lesser extent, for His or Lys (28, 30). We identified all potential intracellular PKA phosphorylation sites (Fig. 1B) in rabbit α_1C using both manual sequence analysis and several web-based PKA phosphorylation prediction tools, including prediction of protein kinase A phosphorylation sites using the simplified kinase binding model (pKaPS) (31), Disorder-Enhanced Phosphorylation Sites Predictor (DISPHOS) (32), GPS (33), NetPhos (34), and Scansite (35). Each phosphorylation site was mutated to Ala if the Ser or Thr in rabbit α_1C was conserved in the rat, mouse, guinea pig, or human. For those conserved sites, we also mutated additional Ser and Thr within several amino acid residues C-terminal to the Arg or Lys to ensure that we fully tested each phosphoregulatory site (residues labeled blue in Fig. 1B). If the site was not conserved among all species, and there was no alternative putative consensus PKA phosphorylation site nearby, we excluded the site from consideration. We also excluded those sites predicted to be extracellular or within the plasma membrane.

Based on the list, we generated two transgenic mice with inducible cardiomyocyte-specific expression of an N-terminal 3× FLAG-epitope–tagged DHP-resistant α_1C with Ala substitutions of all conserved Ser or Thr, either not including (“17-mutant”) or including (“22-mutant”) residues previously shown to be not required for β-adrenergic stimulation of Ca_V1.2 (rabbit Ser¹⁵¹⁷, Ser¹⁵⁷⁵, Ser¹⁷⁰⁰, Thr¹⁷⁰⁴, and Ser¹⁹²⁸) (Fig. 1 B and C). Several pseudo-wild-type (pWT), 17-mutant, and 22-mutant founder transgenic lines were originally created, and lines demonstrating doxycycline-induced α_1C expression after crossing with the αMHC-rtTA mice were expanded and used for this study. Because the results were consistent within each mouse line, the data were pooled for gender and founders.

Functional Studies of PKA Phosphorylation Site Mutant Mice.

As in prior studies, we used a concentration of 300 nM nisoldipine, which blocked >98% of heterologously expressed WT Ca_V1.2 current in tsA-201 cells, but only blocked 34.6% of DHP-insensitive α_1C (10). In cardiomyocytes isolated from nontransgenic mice, 300 nM nisoldipine inhibited 92.4 ± 1.6% of peak current in the absence of β-adrenergic stimulation (10) and 94.0 ± 1.2% of peak current after 200 nM isoproterenol (Fig. S1). Thus, when recording from cardiomyocytes expressing DHP-resistant transgenic α_1C subunits (discussed below), these data demonstrate that we have effectively blocked the endogenous channels and isolated the transgenic currents, both in the absence and presence of isoproterenol.

Fig. S1.

Nisoldipine inhibits basal and β-adrenergic–stimulated endogenous Ca_V1.2 current equivalently. (A) Diary plot of Ca²⁺ current (I_Ca) amplitude at +10 mV in picoamperes per picofarad (pA/pF) before, after 200 nM isoproterenol, and after 300 nM nisoldipine in presence of 200 nM isoproterenol from nontransgenic mouse. Data are representative of seven similar experiments. (B) Bar graph of fraction current remaining after nisoldipine in cells without isoproterenol (ISO) superfusion (n = 12) and cells with isoproterenol superfusion (n = 7). Mean ± SEM (P = 0.75 by unpaired t test).

Transgenic mice were fed doxycycline-impregnated food overnight to induce the expression of the DHP-resistant transgenic α_1C subunits. Nisoldipine (300 nM) inhibited 51.7 ± 5.8% of peak current in cardiomyocytes isolated from doxycycline-fed pWT α_1C transgenic mice, 33.5 ± 3.9% of peak current in doxycycline-fed 17-mutant α_1C transgenic mice, and 69.5 ± 1.6% of peak current in doxycycline-fed 22-mutant α_1C mice (Fig. 2 A–C). The midpoint potentials, derived from the Boltzmann function, for steady-state activation of the transgenic, nisoldipine-resistant 17-mutant and 22-mutant channels demonstrated a nonsignificant small leftward shift compared with pWT channels, and the slope factors for 17-mutant and 22-mutant transgenic channels were not different from pWT channels (Fig. S2 E and F). Our interpretation of these results is that under basal conditions, the activation of the transgenic 17-mutant and 22-mutant Ca_V1.2 channels is similar to endogenous Ca_V1.2 channels.

Fig. 2.

β-Adrenergic stimulation of Ca_V1.2 current does not require conserved PKA phosphorylation sites in α_1C. (A–C) Exemplar whole-cell Ca²⁺ currents (I_Ca) recorded from freshly dissociated cardiomyocytes of pWT, 17-mutant, and 22-mutant α_1C transgenic mice. Pulses ranged from −70 mV to +10 mV before (black traces) and 3 min after (red traces) 300 nM nisoldipine and 3 min after 200 nM isoproterenol in the presence of nisoldipine (blue traces). (D–F) I-V relationships of pWT, 17-mutant, and 22-mutant α_1C before and after 200 nM isoproterenol, in the presence of 300 nM nisoldipine. (Insets) Series of whole-cell Ca_V1.2 currents recorded from a series of pulses between −40 mV and +60 mV from a holding potential of −50 mV in the absence of 200 nM isoproterenol (red traces) and 3 min after isoproterenol (blue traces). The calibration was 5 picoamperes per picofarad (pA/pF) for 50 ms. Data are representative of pWT (n = 14), 17-mutant (n = 12), and 22-mutant (n = 10) I-V curves. The Boltzmann midpoint (ΔV_mid) induced by isoproterenol: pWT (−2.4 ± 0.5, P < 0.001 by paired t test, n = 12) 17-mutant (−4.5 ± 0.5, P < 0.0001 by paired t test, n = 12), and 22-mutant (−2.8 ± 0.3, P < 0.0001 by paired t test). (G) Combined bar and column scatter plot depicting the fold increase in peak current caused by isoproterenol. Bar graphs are mean ± SEM (n = 18 cardiomyocytes from eight pWT α_1C mice, n = 18 cardiomyocytes from four 17-mutant α_1C mice, and n = 44 cardiomyocytes from eight 22-mutant α_1C mice; P = 0.21 by one-way ANOVA).

Fig. S2.

I-V relationships for transgenic Ca_V1.2 channels and endogenous Ca_V1.2 channels are not different. Normalized I-V relationships of Ca²⁺ currents (I_Ca) recorded from pWT α_1C (A), 17-mutant α_1C (B), 22-mutant α_1C (C), and ΔPEST/PRO α_1C (D). Whole-cell Ca_V1.2 currents were recorded from a series of pulses between −40 mV and +60 mV from a holding potential of −70 mV before and 3 min after superfusion of 300 nM nisoldipine (red trace). I-V curve of endogenous Ca_V1.2 (green trace) was derived by subtracting nisoldipine-resistant current from the total Ca_V1.2 current. Mean ± SEM (pWT, n = 10; 17-mutant, n = 7; 22-mutant, n = 8; ΔPEST/PRO, n = 4). (E) Table of Boltzmann function, midpoint (V_mid), and slope for endogenous Ca²⁺ channels and nisoldipine-resistant mutant transgenic Ca²⁺ channels [P = not significant by ANOVA (P = 0.30); endogenous channels, n = 11; pWT, n = 8; 17-mutant, n = 12; 22-mutant, n = 10; ΔPRO/PEST, n = 11]. (F) Normalized I-V relationship of nisoldipine-resistant current of pWT, 17-mutant, 22-mutant, and ΔPEST/PRO. Mean ± SEM.

In the cardiomyocytes isolated from pWT α_1C transgenic mice, isoproterenol increased the nisoldipine-insensitive peak current by a mean of 1.7 ± 0.1-fold (Fig. 2 D and G). In the cardiomyocytes isolated from the 17-mutant and 22-mutant transgenic mice, isoproterenol increased nisoldipine-resistant peak Ca_V1.2 current by a mean of 1.7 ± 0.1-fold and 1.9 ± 0.1-fold, respectively, which is nearly identical to the isoproterenol-induced augmentation of nisoldipine-resistant current in pWT α_1C transgenic cardiomyocytes (Fig. 2 E–G). We observed a hyperpolarizing shift in the I-V curves for currents from both transgenic mutants, similar to the shift observed for pWT (Fig. 2 D–F). Also, similar to the pWT α_1C transgenic mice, the magnitude of the isoproterenol-induced increase in the nisoldipine-resistant Ca²⁺ current was inversely correlated with the basal total Ca_V1.2 current (Fig. S3). The slopes and intercepts of the linear regression lines describing the relationship of total basal current density and response to isoproterenol were not statistically different. This inverse relationship has also been observed for endogenous Ca²⁺ channels in the guinea pig and rat (12).

Fig. S3.

Isoproterenol-induced fold increase in Ca²⁺ current stratified by total basal Ca²⁺ current in picoamperes per picofarad (pA/pF). Lines were fitted by linear regression.

Although we had eliminated the predicted PKA phosphorylation sites in α_1C, the isoproterenol-induced increased current in the cardiomyocytes isolated from the 17-mutant and 22-mutant α_1C transgenic mice was due to PKA, and not due to CaMKII, because inclusion of 10 mM BAPTA in the pipette solution should strongly and rapidly buffer [Ca²⁺], thereby inhibiting CaMKII activity. Moreover, in the 22-mutant mice, we ablated with Ala substitutions the known CaMKII phosphorylation sites in α_1C (36). Taken together, these results demonstrate that the adrenergic regulation of Ca_V1.2 in the heart is dependent upon neither any combination of the five previously identified phosphoregulatory sites in α_1C nor any of the additional 17 consensus PKA phosphorylation sites in α_1C that are conserved among five species.

We considered the possibility that our results were confounded by basal phosphorylation even though the isolated cells are maintained in a physiological solution without catecholamines. To address whether the Ca_V1.2 channels have basal PKA phosphorylation and whether this phosphorylation leads to increased Ca²⁺ currents, we used a cell-permeable cAMP-PKA inhibitor (Rp-8-Br-cAMPS), which functions by occupying cAMP binding sites, thereby preventing dissociation and activation of the PKA holoenzyme (37). The inhibitor can rapidly reverse isoproterenol-mediated up-regulation of Ca_V1.2 of nontransgenic Ca_V1.2 channels by 96.5 ± 12.8% within 1 min (Fig. S4), implying that the cAMP-PKA inhibitor effectively reverses any PKA-mediated increase of basal Ca_V1.2 current. These results also provide additional evidence that the effect of isoproterenol is mediated via PKA, and not CaMKII. In cardiomyocytes isolated from both pWT and the 22-mutant α_1C transgenic mice, the cAMP-PKA inhibitor had no significant inhibitory effect on nisoldipine-resistant basal Ca_V1.2 currents at 1 or 2 min (Fig. 3 A–D). These data indicate that neither endogenous nor transgenic Ca_V1.2 channels are basally activated by PKA in freshly isolated cardiomyocytes.

Fig. S4.

Diary plot of Ca²⁺ current (I_Ca) amplitude at +10 mV in picoamperes per picofarad (pA/pF) before isoproterenol, after 200 nM isoproterenol, and after 200 nM isoproterenol + 30 μM Rp-8Br-cAMPS in cardiomyocytes isolated from nontransgenic (NTG) mice. Data are representative of 10 similar experiments. The isoproterenol-induced increase in Ca²⁺ current was inhibited by 96% (ΔRp/ΔIso) within 1 min of exposure to Rp-8Br-cAMPS, in the continued presence of isoproterenol.

Fig. 3.

Basal Ca²⁺ current in isolated cardiomyocytes is not reduced by cAMP-PKA inhibitor. (A) Diary plot of Ca²⁺ current (I_Ca) amplitude at +10 mV picoamperes per picofarad (pA/pF) of cardiomyocytes isolated from pWT α_1C transgenic mice. The current amplitude after inhibition of endogenous Ca²⁺ current by nisoldipine was set as the baseline. Data are representative of 14 cardiomyocytes for which the Ca²⁺ current was nominally inhibited by 2.8% at 1 min and 4.6% at 2 min after Rp-8Br-cAMPS exposure in the presence of nisoldipine. (B) Bar graph of normalized nisoldipine-resistant Ca²⁺ current before and after Rp-8Br-cAMPS. Mean ± SEM (n = 14 cardiomyocytes; P = 0.2 by repeated measures one-way ANOVA). (C) Diary plot of current amplitude at +10 mV (pA/pF) of cardiomyocytes isolated from 22-mutant α_1C transgenic mice. The current amplitude after inhibition of endogenous Ca²⁺ current by nisoldipine was set as the baseline. Data are representative of eight cardiomyocytes, for which the Ca²⁺ current was nominally inhibited by 2.2% at 1 min and 2.2% at 2 min after Rp-8Br-cAMPS exposure in presence of nisoldipine. (D) Bar graph of the normalized nisoldipine-resistant current (pA/pF) before and after Rp-8Br-cAMPS. Mean ± SEM (n = 8 cardiomyocytes; P = 0.48 by repeated measures one-way ANOVA).

Proteolytic Processing of α_1C.

In cardiomyocytes isolated from nontransgenic mice, native α_1C is detected as a full-length ∼240-kDa band and a cleaved ∼210-kDa band, using an anti-α_1C antibody created against an internal epitope within the intracellular loop of domains II and III (10). To investigate the role of channel cleavage, we created two transgenic mice. In one, we deleted the PEST sequence (ΔPEST) alone. In the second, we deleted both the PEST sequence and ¹⁷⁹⁴NANINNANN¹⁸⁰² (ΔPEST/PRO) (Fig. 4A). Hearts were resected, washed of blood, and immediately frozen. The ratio of cleaved to full-length ΔPEST transgenic α_1C was 48.7 ± 3.0%, which is not significantly different from the 53.9 ± 7.1% cleavage of the pWT α_1C (mean ± SEM, P = not significant by Tukey’s multiple comparison test; Fig. 4 B and C). In contrast, the ratio of cleaved to full-length ΔPEST/PRO was 2.4 ± 0.8% (mean ± SEM, P < 0.0001 by ANOVA/Tukey’s multiple comparison text compared with both pWT and ΔPEST; Fig. 4 B and C), indicating that proteolytic cleavage requires the presence of ¹⁷⁹⁴NANINNANN¹⁸⁰².

Fig. 4.

Identification of C-terminal proteolytic cleavage of α_1C. (A) Sequence of PEST and proteolytic cleavage site of rabbit α_1C. (B) Anti-FLAG antibody (Ab; Upper), anti-α_1C antibody (Middle), and antitubulin antibody (Lower) immunoblots showing FLAG epitope-tagged α_1C expression in isolated cardiomyocytes from nontransgenic (NTG), pWT α_1C, ΔPEST α_1C, and ΔPEST/PRO α_1C transgenic mice. (C) Bar graph of densitometries of cleaved α_1C band divided by truncated + full-length α_1C bands (n = 10 pWT α_1C mice, n = 6 ΔPEST α_1C mice, and n = 10 ΔPEST/PRO α_1C mice; P < 0.0001 by ANOVA, ****P < 0.0001 by Tukey’s multiple comparison test). (D) Percent shortening of sarcomere length after incubation and superfusion of 300 nM nisoldipine-containing solution. Cardiomyocytes were field-stimulated at 1 Hz (P < 0.0001 by ANOVA and Tukey’s multiple comparison test). (E) Immunostaining of ΔPEST/PRO cardiomyocytes. Anti-FLAG antibody, FITC-conjugated secondary antibody, and nuclear labeling with Hoechst stain are shown. Images were obtained with a confocal microscope at 40× magnification. (Scale bar: 10 μm.)

Having generated a mutant Ca_V1.2 in cardiomyocytes for which the C terminus was resistant to proteolytic cleavage by the endogenous proteases that act on the endogenous Ca_V1.2, we then asked whether proteolytic cleavage of the C terminus was required for either trafficking or function of cardiac Ca_V1.2. We used two complementary methods to assess appropriate trafficking to the t-tubules. First, we assessed whether the transgenic myocytes contracted normally in response to Ca²⁺ influx through the ΔPEST/PRO Ca_V1.2. Cardiomyocyte contraction requires Ca²⁺ influx via Ca_V1.2 in t-tubules, which triggers Ca²⁺-induced Ca²⁺ release from neighboring ryanodine receptor intracellular Ca²⁺ release channels and subsequent contraction. Superfusion of nisoldipine inhibited the contraction of nontransgenic cardiomyocytes to electric field stimulation at 1 Hz (Fig. 4D), confirming the necessity of Ca_V1.2. In cardiomyocytes isolated from both pWT and ΔPEST/PRO α_1C mutant transgenic mice, field-stimulated contraction persisted in the presence of nisoldipine (Fig. 4D; P < 0.0001 for both pWT and ΔPEST/PRO compared with nontransgenic cardiomyocytes by ANOVA, Tukey’s multiple comparison test, P = not significant between pWT and ΔPEST/PRO). Thus, the nisoldipine-resistant ΔPEST/PRO channels can trigger contraction similar to the pWT channels. Second, we observed the location of ΔPEST/PRO Ca_V1.2 channels by immunocytochemistry. Anti-FLAG antibody immunofluorescence of fixed cardiomyocytes from pWT and ΔPEST/PRO mutant transgenic mice also showed a striated pattern consistent with surface membrane distribution of expressed α_1C subunits and t-tubular localization (Fig. 4E). No anti-FLAG antibody immunofluorescent signal was detected in cardiomyocytes isolated from nontransgenic mice, and no immunofluorescence was detected in either pWT or ΔPEST/PRO transgenic cardiomyocytes when the anti-FLAG antibody was omitted (Fig. 4E and Fig. S5).

Fig. S5.

Immunostaining of pWT and ΔPEST/PRO cardiomyocytes. Incubation was performed with FITC-conjugated secondary antibody and nuclear labeling with Hoechst stain, but without anti-FLAG antibody. Images were obtained with a confocal microscope at a magnification of 40×. (Scale bar: 10 μm.)

With this foundation, we then tested whether the absence of C-terminal cleavage affected the ability of the ΔPEST/PRO Ca_V1.2 channels to respond to isoproterenol. We measured Ca_V1.2 currents in adult cardiomyocytes from ΔPEST/PRO α_1C mutant transgenic mice. Nisoldipine (300 nM) inhibited only 41.9 ± 5.3% of peak current, implying that the transgenic channels trafficked to the membrane and were functional (Fig. 5 and Figs. S2D and S6). The midpoint and slope of the ΔPEST/PRO α_1C channels were not different from the midpoint and slope of pWT channels (Fig. S2 E and F). These findings imply that under basal conditions, the C-terminal proteolytic cleavage of α_1C is not required for expression and basal function of the Ca²⁺ channel. In the cardiomyocytes isolated from ΔPEST/PRO α_1C transgenic mice, isoproterenol increased the nisoldipine-insensitive peak current by a mean of 1.7 ± 0.04-fold (Fig. 5 A–C). Taken together, these results demonstrate that C-terminal proteolytic cleavage of α_1C is not required for β-adrenergic regulation of Ca_V1.2 in the heart.

Fig. 5.

DHP-resistant currents in ΔPEST/PRO transgenic mice. (A) Exemplar whole-cell Ca_V1.2 currents recorded from pulses from −70 mV to +10 mV before (black trace), 3 min after 300 nM nisoldipine (red trace), and 3 min after 200 nM isoproterenol in the presence of nisoldipine (blue trace). (B) Representative I-V relationship of ΔPEST/PRO Ca²⁺ current (I_Ca) without and with 200 nM isoproterenol. (Insets) Series of whole-cell Ca_V1.2 currents recorded from a series of pulses between −40 mV and +60 mV from a holding potential of −70 mV in the absence of isoproterenol (red traces) and 3 min after 200 nM isoproterenol (blue traces). Data are representative of 14 similar I-V curves. The Boltzmann midpoint (ΔV_mid) is induced by isoproterenol: ΔPEST/PRO: −2.4 ± 0.7 (P < 0.01 by paired t test; n = 12). (C) Combined bar and column scatter plot depicting the fold increase in peak current caused by 200 nM isoproterenol. Bar graphs are mean ± SEM. Presented pWT data are the same as in Fig. 2 (n = 18 cardiomyocytes from eight pWT α_1C mice and n = 36 cardiomyocytes from six ΔPEST/PRO α_1C mice; P = 0.92 by unpaired t test).

Fig. S6.

(A) Combined bar graph and column scatter plot for total peak current density in picoamperes per picofarad (pA/pF) of cardiomyocytes isolated from pWT and ΔPEST/PRO α_1C transgenic mice. Mean ± SEM (P = 0.32 by unpaired t test). (B) Fraction of nisoldipine-resistant current in pWT and ΔPEST/PRO transgenic cardiomyocytes (P = 0.14 by unpaired t test).

Discussion

Prior studies used biochemical and mass spectrometry to identify single residues in α_1C and β₂ subunits phosphorylated by PKA as candidate targets for acute β-adrenergic regulation of Ca_V1.2 in heart. Our data show that none of those identified sites in α_1C are required for the acute fight-or-flight modulation of Ca_V1.2 by β-adrenergic agonists in the heart. For both the 17-mutant and 22-mutant mouse lines, we found that acute β-adrenergic regulation of Ca_V1.2 was not diminished, implying that Ser or Thr residues in α_1C that are common to the five species and within a consensus PKA phosphorylation site are not the functionally relevant PKA phosphorylation sites for acute modulation of Ca_V1.2 by β-adrenergic agonists. A role for the previously identified PKA phosphorylation sites within the Ca_V1.2 β₂ subunit has been excluded by several studies (6, 8, 12). Our results suggest that the field may have been focusing on the wrong targets.

Most recently, Ser¹⁷⁰⁰ has been proposed as the critical PKA phosphorylation site in α_1C for mediating acute β-adrenergic regulation of Ca_V1.2 in the heart (9, 38). Our studies of Ser¹⁷⁰⁰, in isolation (10) and now in combination with other putative PKA phosphorylation sites, do not support its role as was suggested by a knock-in mouse (38). Advantages of our approach are that expression of the mutant channels is conditional, short-term, and limited, thereby leading to fewer secondary compensatory effects, such as heart failure, which reduces normal β-adrenergic responsiveness. The potential confounders due to compensatory changes with a chronic, knock-in model are also illustrated by the decreased basal Ca_V1.2 current in cardiomyocytes isolated from an S1700A knock-in mouse, which was speculated to be secondary to reduced basal phosphorylation of Ser¹⁷⁰⁰ by PKA (38, 39). Using a potent cAMP-PKA inhibitor that can fully reverse β-adrenergic stimulation of Ca_V1.2 when applied after isoproterenol, we demonstrate that Ca_V1.2 current is not up-regulated by cAMP-PKA–mediated pathways in unstimulated cardiomyocytes isolated from pWT α_1C mice.

Our approach also allowed us to investigate the role of proteolytic cleavage of the α_1C C terminus. Proteolytic cleavage and the subsequent assembly of the cleaved C-terminal fragment with the truncated α_1C have been proposed to play important roles in basal and adrenergic regulation of Ca_V1.2 (9, 17, 18). Truncation of α_1C, either at Gly¹⁷⁹⁶ (immediately N-terminal to the identified protease cleavage site) or Asp¹⁹⁰⁴ caused a marked reduction in the cell surface expression of α_1C and in the Ca_V1.2 current in the heart, but not in vascular smooth muscle, leading to cardiac failure and perinatal death (18, 40). Because the distal C terminus, after cleavage from an endogenous channel, likely remains associated with the cardiac Ca_V1.2 complex (41), expression of α_1C with a truncated distal C terminus does not mimic a physiological state. Here, we identified the proteolytic site of the cardiac Ca²⁺ channels and disrupted it. This strategy enabled us to explore adrenergic regulation with the C terminus of α_1C still present but unable to be cleaved. We found that prevention of C-terminal proteolytic cleavage did not alter the expression, trafficking, basal function, or adrenergic regulation of the Ca_V1.2 in the heart. The functional effects of cleavage of α_1C remain unclear, but our studies demonstrate that proteolytic cleavage of α_1C in the heart is not required for β-adrenergic regulation of Ca_V1.2.

Taken together, our studies rule out a role for conserved PKA phosphorylation sites in α_1C contributing to acute regulation of β-adrenergic stimulation of Ca_V1.2. Moreover, our data show that these key residues are not redundant and do not each contribute combinatorially to a net stimulatory effect. Whether redundancy could exist with the Ca_V1.2 β subunit is not clear at this stage.