Convergent regulation of Ca_V1.2 channels by direct phosphorylation and by the small GTPase RAD in the cardiac fight-or-flight response

Significance

The L-type calcium currents conducted by the cardiac Ca_V1.2 calcium channel initiate excitation–contraction coupling and serve as a key regulator of heart rate, rhythm, and force of contraction at rest and in the fight-or-flight response. Our results show that the distal carboxyl-terminal domain of Ca_V1.2 and the small guanosine triphosphatase (GTPase) Ras associated with diabetes (RAD) potently inhibit the activity of Ca_V1.2 channels and further that phosphorylation of the Ca_V1.2 channel in its proximal carboxyl-terminal domain and phosphorylation of RAD act synergistically to increase channel activity in response to β-adrenergic activation of cyclic adenosine monophosphate (cAMP)-dependent protein kinase. Thus, β-adrenergic activation merges two key, convergent intracellular signaling pathways to upregulate Ca_V1.2 channels in the fight-or-flight response.

Abstract

The L-type calcium currents conducted by the cardiac Ca_V1.2 calcium channel initiate excitation–contraction coupling and serve as a key regulator of heart rate, rhythm, and force of contraction. Ca_V1.2 is regulated by β-adrenergic/protein kinase A (PKA)-mediated protein phosphorylation, proteolytic processing, and autoinhibition by its carboxyl-terminal domain (CT). The small guanosine triphosphatase (GTPase) RAD (Ras associated with diabetes) has emerged as a potent inhibitor of Ca_V1.2, and accumulating evidence suggests a key role for RAD in mediating β-adrenergic/PKA upregulation of channel activity. However, the relative roles of direct phosphorylation of Ca_V1.2 channels and phosphorylation of RAD in channel regulation remain uncertain. Here, we investigated the hypothesis that these two mechanisms converge to regulate Ca_V1.2 channels. Both RAD and the proteolytically processed distal CT (dCT) strongly reduced Ca_V1.2 activity. PKA phosphorylation of RAD and phosphorylation of Ser-1700 in the proximal CT (pCT) synergistically reversed this inhibition and increased Ca_V1.2 currents. Our findings reveal that the proteolytically processed form of Ca_V1.2 undergoes convergent regulation by direct phosphorylation of the CT and by phosphorylation of RAD. These parallel regulatory pathways provide a flexible mechanism for upregulation of the activity of Ca_V1.2 channels in the fight-or-flight response.

L-type calcium currents conducted by the Ca_V1.2 calcium channel initiate excitation–contraction coupling and serve as a key regulator of heart rate, rhythm, and force of contraction (1, 2). Ca_V1.2 activity is regulated by cardiac β-adrenergic signaling through cyclic adenosine monophosphate (cAMP) and cAMP-dependent protein kinase A (PKA), and upregulation of the Ca_V1.2 channel has a critical role in mediating increased cardiac output during the fight-or-flight response (3, 4). Understanding the molecular mechanisms linking the β-adrenergic signaling cascade to Ca_V1.2 activity promises to shed light on the pathophysiology of chronic heart failure, supraventricular arrhythmias, and other cardiac disorders associated with autonomic dysfunction. However, the molecular mechanisms through which β-adrenergic regulation, increased cAMP, and PKA phosphorylation enhance the activity of Ca_V1.2 channels have remained elusive (5–8).

Multiple isoforms of Ca_V1.2 channels have been identified in the heart (5–8). The carboxyl-terminal domain (CT) of the pore-forming α₁ subunit mediates interactions between Ca_V1.2 channels that regulate channel activity (9). Deletion of the distal carboxyl terminus (dCT) increases Ca_V1.2 activity (10), and up to 80% of the pore-forming α₁ subunits of Ca_V1.2 expressed in the heart are proteolytically processed at position 1800 (Ca_V1.2Δ1800) (5–8, 11, 12). The dCT associates noncovalently with the channel, reducing calcium conductance by the Ca_V1.2Δ1800-dCT complex relative to the truncated Ca_V1.2Δ1800 or the full-length channel (Ca_V1.2-FL) (12, 13). Deletion of the dCT in mice causes complete loss of PKA regulation of Ca_V1.2 channels (14). PKA phosphorylation of Ser1700 in the Ca_V1.2 carboxyl-terminal domain upregulates Ca_V1.2 channel activity in transfected cells in vitro (15), and Ser1700 is phosphorylated in response to β-adrenergic stimulation of the heart in vivo (16). Alanine substitutions at phosphorylation sites in the proximal carboxyl-terminal domain (pCT) of Ca_V1.2, Ser1700Ala (S1700A) and Ser1700Ala/Thr1704Ala (STAA), reduce β-adrenergic upregulation of Ca_V1.2 current in dissociated ventricular myocytes by approximately two-thirds in genetically modified mice, without significant reduction in expression or localization of the Ca_V1.2 protein (17, 18). The physiological significance of these PKA regulatory sites on Ca_V1.2 was highlighted by further studies revealing the development of lethal heart failure in these mutant mice as they age beyond 200 d (19). However, in a different genetic background, mutation of these phosphoregulatory sites caused loss of expression (20). Moreover, the partial loss of regulation in mice with mutations at Ser1700 and Thr1704 indicated that other regulatory mechanisms are also important (6, 7).

In addition to direct phosphorylation of the CT of Ca_V1.2 α₁ subunits, members of the RGK small guanosine triphosphatase (GTPase) family (Rem, Rad, Rem2, and Gem/Kir) are well-established inhibitors of Ca_V1.1, Ca_V1.2, and Ca_V1.3 in cardiac myocytes and neurons (21). RAD binds to the α₁ and β subunits of Ca_V1.2 and inhibits channel activity (21–23). Although global RAD deletion leads to cardiac hypertrophy in mice, cardiac-specific RAD knockout mice have increased basal I_Ca, elevated ejection fraction, and no indication of concomitant pathological remodeling (24). Recent work has shown that RAD inhibition of Ca_V1.2 can be reversed by PKA phosphorylation in an in vitro expression system in ventricular myocytes, and this effect requires interactions between RAD and the Ca_V1.2 β subunit, which binds to the Ca_V1.2 α₁ subunit via its intracellular domain I–II linker (23). In addition, RAD can reconstitute β-adrenergic/PKA regulation of Ca_V1.2 channels expressed in Xenopus oocytes (25). Together, these studies establish RAD as a key regulator of Ca_V1.2 channels in response to β-adrenergic stimulation and PKA phosphorylation.

Although proteolytically processed Ca_V1.2Δ1800 is a major isoform of the channel in the heart, previous studies of Ca_V1.2 regulation by RAD did not evaluate how regulation of the autoinhibited complex of Ca_V1.2Δ1800 with its noncovalently bound dCT intersects with regulation by the RAD GTPase. In this study, using heterologous coexpression of Ca_V1.2Δ1800, the dCT, and RAD in cultured human embryonic kidney cells, we found that RAD and the dCT both inhibit Ca_V1.2Δ1800 to a similar extent, that simultaneous inhibition can be reversed by PKA stimulation with forskolin, and that this reversal of inhibition is blocked by mutation of PKA phosphorylation sites at Ser1700 and Thr1704 in the pCT and by loss of RAD phosphorylation at its PKA regulatory sites. Our results provide evidence for convergent pathways of regulation of Ca_V1.2 directly by phosphorylation of Ser1700 and Thr1704 in the pCT and indirectly by phosphorylation of RAD, and they further show that the dCT and the RAD GTPase can reconstitute PKA stimulation of the Ca_V1.2 channel in transfected cells in vitro as coregulators.

Results

In order to control the expression of Ca_V1.2 channels, their auxiliary subunits, and their regulatory proteins, we used an in vitro expression system that is well described in previous work (15). Human embryonic kidney tsA-201 cells were transfected with complementary DNAs encoding Ca_V1.2Δ1800 or Ca_V1.2-FL, the auxiliary β_2b and α₂δ subunits, the dCT, and RAD. Ba²⁺ currents were measured, and current–voltage relationships were determined in whole-cell voltage-clamp experiments during repeated depolarizations in 10-mV intervals from a resting membrane potential of −80 mV. In order to assess channel activity, we measured Ba²⁺ tail currents and divided by number of gating charges calculated by integrating the gating current at the reversal potential. This ratio determines the coupling efficiency of pore opening to gating charge movement (Materials and Methods (12, 15)). No systematic changes in the peak amplitude or time course of the Ba²⁺ current or in the gating charge per unit of cell capacitance were observed (Fig. 1 A–D and SI Appendix, Tables S1–S3). Changes in V_1/2 in the presence of RAD (SI Appendix, Fig. S1) were compensated by comparing current amplitudes recorded at the voltage that generated the peak inward current for each construct.

Fig. 1.

RAD inhibits Ca_V1.2Δ1800 and Ca_v1.2-FL. Current/voltage (I/V) curves of Ca_V1.2Δ1800 coexpressed with (red) and without (black) RAD. (A and B) Ca_V1.2-FL. (C and D) Ca_V1.2Δ1800 + dCT. (E) Ca_V1.2Δ1800 − dCT. Representative current recordings at sequential 10-mV depolarizations for full-length (B) and the truncated forms (D) with RAD (red traces) and without RAD (black traces). (F) Peak current amplitudes observed during depolarized potentials between −10 mV and +10 mV. Statistical significance determined via ANOVA and Tukey honest significant difference (HSD) (FL, n = 9; FL + RAD, n = 7; Δ1800, n = 16; Δ1800 + RAD, n = 12; Δ1800 + dCT, n = 16; Δ1800 + dCT + RAD, n = 12; *P < 0.05).

RAD Inhibits Ca_V1.2Δ1800 in the Presence or Absence of the dCT.

Cotransfection of Ca_V1.2-FL with RAD caused 75% inhibition of Ba²⁺ current (−RAD: −8.2 ± 1.2 nA/pC, n = 9; +RAD: −2.1 ± 0.5 nA/pC, n = 7; P < 0.001; Fig. 1 A, B, and F). In cells transfected with Ca_V1.2Δ1800 and RAD, we also observed depressed Ba²⁺ current in current–voltage (I/V) relationships, which retained approximately half the current amplitude observed for cells without RAD (−RAD: −10.1 ± 0.8 nA/pC, n = 16; +RAD: −4.7 ± 0.8 nA/pC, n = 11; P = <0.001; Fig. 1 E and F). When RAD was cotransfected with Ca_V1.2Δ1800 plus the dCT, no additional suppression of peak current was observed in the presence of RAD (+dCT/−RAD: −5.9 ± 0.7 nA/pC, n = 16; +dCT/+RAD: −5.1 ± 0.5 nA/pC, n = 12; P = 0.90), revealing the lack of “additive inhibition” of the autoinhibited Ca_V1.2Δ1800 + dCT complex by RAD (Fig. 1 C and F).These results suggest that the dCT and RAD may serve as convergent coinhibitors that stabilize the same, or similar, inhibited state(s) of Ca_V1.2.

Forskolin Reverses RAD Inhibition of Ca_V1.2.

When the adenylate-cyclase agonist forskolin (FSK) was added to the extracellular solution, Ca_V1.2-FL coexpressed with RAD was significantly stimulated by FSK (−FSK: −2.1 ± 0.5 nA/pC, n = 7; +FSK: −5.8 ± 0.5 nA/pC, n = 7; P = 0.044; Fig. 2 A and D). In addition, increased Ba²⁺ current in the presence of FSK was observed for both Ca_V1.2Δ1800 (−FSK/+RAD: −4.7 ± 0.8 nA/pC, n = 11; +FSK/+RAD: −8.8 ± 0.9 nA/pC, n = 8; P = 0.041) and Ca_V1.2Δ1800 + dCT (−FSK/+RAD: 5.1 ± 0.5 nA/pC, n = 12; +FSK/+RAD: −9.2 ± 1.2 nA/pC, n = 10; P = 0.011) in the presence of RAD (Fig. 2 B–D). By contrast, in the absence of RAD coexpression, FSK did not significantly stimulate Ca_V1.2Δ1800 (−FSK: −10.1 ± 0.8 nA/pC, n = 16; +FSK: −9.5 ± 1.5 nA/pC, n = 6; P = 0.99) or Ca_V1.2Δ1800 + DCT (−FSK: −5.9 ± 0.7 nA/pC, n = 16; +FSK: −6.3 ± 1.1 nA/pC, n = 10; P = 0.99; SI Appendix, Table S1).

Fig. 2.

FSK increases Ca_V1.2Δ1800 and Ca_v1.2 activity in the presence of RAD. I/V curves of FSK- (blue) and non-FSK (red)–treated cells. (A) Ca_V1.2-FL and RAD. (B) Ca_V1.2Δ1800 + dCT and RAD. (C) Ca_V1.2Δ1800 and RAD. (D) Peak current amplitudes observed during depolarized potentials between −10 mV and +10 mV of cells coexpressing Ca_V1.2Δ1800 and RAD with and without the dCT, in the presence (blue) and absence (red) of FSK. Statistical significance determined via ANOVA and Tukey HSD (Δ1800, n = 16 cells; Δ1800 + FSK, n = 8; Δ1800 + dCT, n = 16; Δ1800 + dCT + FSK, n = 10; Δ1800 + RAD, n = 12; Δ1800 + RAD + FSK, n = 8; Δ1800 + dCT + RAD, n = 12; Δ1800 + dCT + RAD + FSK, n = 10; *P < 0.05).

RAD Inhibition and FSK Stimulation in the Presence of pCT Phosphoregulatory Mutations.

In order to test the effects of phosphoregulatory mutations of Ser1700 and Thr1704 in the pCT (Ser1700A/Thr1704A, STAA) on RAD inhibition, we cotransfected Ca_V1.2 phosphoregulatory mutants with or without RAD (Fig. 3). Cells transfected with Ca_V1.2Δ1800/STAA showed comparable inhibition in the presence of RAD as those expressing wild type (WT) (−RAD: 10.1 ± 1.3 nA/pC, n = 12; +RAD: −3.8 ± 0.5 nA/pC, n = 12; P < 0.001) (Fig. 3 A and C). However, in the presence of the dCT, Ca_V1.2Δ1800/STAA current was substantially reduced in the absence of RAD (Fig. 3 B and C, black) and coexpression of RAD gave only modest further inhibition observed at stimulus voltages between −40 mV and −10 mV (Fig. 3 B and C, red) (I_Ba,peak: −RAD: −6.7 ± 0.3 nA/pC, n = 11; +RAD: −4.6 ± 0.5 nA/pC, n = 12; P = 0.026).

Fig. 3.

RAD inhibition of Ca_V1.2Δ1800 in presence of CT phosphoregulatory mutations. I/V curves of Ca_V1.2Δ1800/STAA with (red) and without (black) RAD coexpression. (A) Ca_V1.2Δ1800/STAA without the dCT. (B) Ca_V1.2Δ1800/STAA + dCT. (C) Peak current amplitudes observed during depolarized potentials between −10 mV and +10 mV. (D) Schematic showing S1700A and T1704A mutation sites, RAD, and dCT inhibition. Statistical significance with ANOVA and Tukey HSD (Δ1800/STAA, n = 12 cells; Δ1800/STAA + dCT, n = 11; Δ1800/STAA + RAD, n = 10; Δ1800/STAA + dCT + RAD, n = 12; *P < 0.05).

To probe the significance of these phosphorylation sites for PKA regulation of Ca_V1.2Δ1800, we tested the effects of FSK stimulation on our Ca_V1.2 mutants (Fig. 4). As observed for WT Ca_V1.2Δ1800, cells expressing mutant Ca_V1.2Δ1800/STAA and RAD without the dCT had a moderate increase in current amplitude in the presence of FSK (Fig. 4B) (−FSK/+RAD: −3.8 ± 0.5 nA/pC, n = 12; +FSK: −9.5 ± 1.3 nA/pC, n = 10; P = 0.002) . In contrast, in the presence of both the dCT and RAD, Ca_V1.2Δ1800/STAA did not conduct increased peak current in the presence of FSK (−FSK: −4.6 ± 0.5 nA/pC, n = 12; +FSK: −4.8 ± 0.7 nA/pC, n = 12; P = 0.96) (Fig. 4 A and D). Similarly, Ca_V1.2Δ1800 with the single S1700A pCT mutation also did not respond to FSK (−FSK: −3.1 ± 0.4 nA/pC, n = 8; +FSK: −3.2 ± 1.0 nA/pC, n = 6; P = 0.41) in the presence of coexpressed dCT and RAD (Fig. 4 C and D), indicating the importance of this PKA site. These results indicate that phosphorylation of Ser1700 is required for β-adrenergic/PKA regulation of the Ca_V1.2 channel when it is inhibited by both RAD and the dCT.

Fig. 4.

Stimulation of Ca_V1.2Δ1800 in presence of CT phosphoregulatory mutations and RAD. I/V curves of Ca_V1.2Δ1800 with phosphoregulatory mutations with and without FSK. (A) Ca_V1.2Δ1800/STAA with dCT and RAD coexpression with and without FSK stimulation as indicated. (B) Ca_V1.2Δ1800/STAA with RAD without dCT coexpression with and without FSK stimulation as indicated. (C) Ca_V1.2Δ1800/S1700A with dCT and RAD coexpression with and without FSK stimulation. (D) Peak current amplitudes observed in cells expressing Ca_V1.2Δ1800/STAA and S1700A with and without FSK stimulation. Statistical significance determined via ANOVA and Tukey HSD (Δ1800/STAA + dCT + RAD, n = 12 cells; Δ1800/STAA + dCT + RAD + FSK, n = 12; Δ1800/STAA + RAD, n = 12; Δ1800/STAA + RAD + FSK, n = 10; Δ1800/S1700A + dCT + RAD, n = 8; Δ1800/S1700A + dCT + RAD + FSK, n = 6; *P < 0.05).

FSK Stimulation Is Suppressed by RAD Phosphoregulatory Mutations.

Coexpression of a nonactivatable RAD with its four PKA sites mutated to alanine (Ser25Ala, Ser38Ala, Ser272Ala, and Ser300Ala) (RAD/4M) with Ca_V1.2Δ1800 and the dCT resulted in significantly greater inhibition of Ca_V1.2 channels than with WT RAD, suggesting that basal phosphorylation of these sites partially reduces the inhibitory effects of WT RAD in the absence of activation of PKA (compare red and purple symbols in Fig. 4A). Coexpression of Ca_V1.2Δ1800 plus the dCT and RAD/4M also resulted in a channel complex with no increased activity in the presence of FSK (Fig. 5 A and B; no FSK: −4.1 ± 0.9 pA/pC, n = 9; +FSK: −4.3 ± 1.0 pA/pC, n = 6; P = 0.89). As S1700A and STAA mutations also eliminated channel stimulation, these findings suggest that phosphorylation of both RAD and the pCT is necessary for maximal upregulation by PKA of the Ca_V1.2Δ1800 + dCT form of the channel.

Fig. 5.

Stimulation of Ca_V1.2 Δ1800 in presence of RAD with phosphoregulatory mutations. I/V curves of Ca_V1.2Δ1800 + dCT coexpressed with RAD with phosphoregulatory mutations (RAD/4M). (A) Ca_V1.2Δ1800 with dCT + RAD with and without FSK (red and cyan markers, respectively) and Ca_V1.2Δ1800 with dCT + RAD/4M with and without FSK (purple and orange markers, respectively). (B) Peak current amplitudes observed in cells expressing Ca_V1.2Δ1800 + dCT with WT RAD and RAD/4M with and without FSK stimulation. Statistical significance determined via ANOVA and Tukey HSD (Δ1800 + dCT + RAD, n = 12 cells; Δ1800 + dCT + RAD + FSK, n = 10; Δ1800 + dCT + RAD_4M, n = 9; Δ1800 + dCT + RAD_4M, n = 6; *P < 0.05).

Discussion

The cardiac fight-or-flight response is an important determinant of overall cardiovascular system function and is an essential therapeutic target for cardiac disorders ranging from chronic heart failure to tachyarrhythmias to postmyocardial infarction therapy (26). Although a major effort has been made to elucidate the cardiac β-adrenergic signaling pathway, the detailed mechanism linking stimulation of β₁-adrenergic receptors to increased activity of Ca_V1.2 and upregulation of the excitation–contraction coupling apparatus remains elusive. The CT mediates interactions between Ca_V1.2 channels that regulate channel activity (9), and a critical role has been demonstrated for direct phosphorylation of the CT in mediating FSK or isoproterenol-induced upregulation of channel activity (15). Studies in mutant mice found complete loss of PKA regulation with deletion of the dCT (14), and partial (approximately two-thirds) loss of the β-adrenergic response in the absence of direct phosphorylation of Ca_V1.2 at known sites in the pCT (17, 18). The small GTPase RAD is a potent inhibitor of Ca_V1.2 channels, and recent work implicates reversal of this inhibition by PKA phosphorylation of RAD as a key element in upregulation of Ca_V1.2 channel activity in response to β-adrenergic stimulation (23–25). In this work, we have addressed the hypothesis that direct phosphorylation of the CT by PKA and indirect regulation via RAD phosphorylation act in a convergent manner to increase the activity of Ca_V1.2 channels in response to PKA phosphorylation. Our results show that inhibition of Ca_V1.2 channel activity by RAD and the dCT, followed by relief of this inhibition by phosphorylation of RAD and the pCT, can give rise to robust PKA stimulation of Ca_V1.2Δ1800, an important cardiac form of the channel. Notably, we did not find a requirement for expression of an A-kinase anchoring protein (AKAP) in contrast to previous studies that required precise expression ratios of AKAP15 or another AKAP to reconstitute PKA regulation in a similar heterologous expression system (15, 27). We speculate that RAD may serve as an AKAP itself or bind an AKAP to bring PKA to the signaling complex.

RAD and dCT as Coinhibitors of Ca_V1.2Δ1800.

Previous work in both heterologous and transgenic mouse expression systems has primarily evaluated the regulation of Ca_V1.2-FL by RAD (22, 23, 25), whereas the more-abundant form in the heart is the truncated Ca_V1.2Δ1800, likely in complex with the proteolyzed, autoinhibitory dCT (11, 12). A proposed model for coregulation of Ca_V1.2Δ1800 by RAD, dCT, and PKA phosphorylation is shown in Fig. 6. Our findings indicate that the presence of either RAD or the dCT substantially inhibits Ca_V1.2Δ1800 activity (Fig. 6, Left [I₁] and Right [I₂]), and the presence of both RAD and dCT maximally inhibits Ca_V1.2Δ1800 activity (Fig. 6, I_1 + 2, Bottom). Strikingly, inhibition by the dCT and RAD is not additive, suggesting that these two parallel mechanisms may arrive at the same, or similar, inhibited state(s) (Fig. 6, Bottom). These partially (Fig. 6, Left, Right) or maximally (Fig. 6, Bottom) inhibited states of Ca_V1.2Δ1800 channels are poised for direct β-adrenergic upregulation by phosphorylation of the CT (15, 17, 18) and indirect regulation by phosphorylation of RAD (23, 25).

Fig. 6.

Model of Ca_V1.2Δ1800 coregulation by RAD and dCT. Schematic diagram integrating Ca_V1.2Δ1800 inhibition by RAD, the dCT, and pCT phosphorylation at S1700. A, activated state; I, inhibited state.

Parallel Upregulation of Ca_V1.2Δ1800 by PKA Phosphorylation of the pCT and RAD.

In the presence of dCT and RAD to generate the fully inhibited state (Fig. 6, I_1 + 2, Bottom), activation of PKA regulation by FSK leads to a substantial increase in Ca_V1.2 channel activity (Fig. 2B). This increase in Ca_V1.2 activity requires phosphorylation of the pCT on Ser1700 (Fig. 4 B and C) and phosphorylation of RAD (Fig. 5). These results support the direct pathway (Fig. 6, blue arrow) from the fully inhibited state of Ca_V1.2 (Fig. 6, I_1 + 2, Bottom) to the activated state (A, Fig. 6, Top). Our results also support the direct pathway from the partially inhibited state in the presence of RAD (I₁, Fig. 6, Left) to the activated state (A, Fig. 6, Top). However, we did not detect activation of Ca_V1.2 by the direct pathway from the partially inhibited state in the presence of the dCT (I₂; Fig. 6, Right) to the activated state (A, Fig. 6, Top). FSK activation of PKA signaling may not be sufficient to support this transition without stimulation of the phosphorylation of the CK-II site at Thr1704, which may require a direct CK-II–related stimulus for phosphorylation from the intact β-adrenergic pathway.

Taken together, our findings strongly support the hypothesis that PKA-dependent regulation of Ca_V1.2Δ1800 arises through the convergence of the autoinhibition/direct phosphorylation pathway and the RAD-mediated indirect regulatory pathway. Unexpectedly, we observed that the activity of this channel complex is regulated in a convergent manner through two molecular pathways stimulated by activation of PKA. Loss of PKA phosphorylation at Ca_V1.2 position S1700 blunted PKA-mediated upregulation when both the dCT and RAD were present, but upregulation was preserved when RAD was present but the dCT was absent. We found that loss of phosphorylation of PKA sites on RAD also reduced PKA-dependent upregulation of Ca_V1.2, suggesting that both direct PKA phosphorylation of Ca_V1.2 and PKA phosphorylation of RAD are required for robust PKA-dependent regulation.

Convergent Regulation by RAD and Direct Protein Phosphorylation: Toward a Coherent Model of the Cardiac Fight-or-Flight Response.

Over the past 2 decades, evidence has emerged linking dCT proteolysis, dCT autoinhibition, pCT phosphorylation, β subunit phosphorylation and trafficking, AKAP association, RAD phosphorylation, and several other factors coupled to increased Ca_V1.2 activity following β-adrenergic stimulation (5–8). Although some inconsistencies and questions remain to be resolved, a unified mechanism involving the CT, RAD, and the Ca_Vβ subunit is emerging, with each piece likely contributing aspects of the β-adrenergic response in additive or synergistic interactions that have yet to be fully elucidated. An interesting possibility is that the partially preserved β-adrenergic response to high doses of isoproterenol observed in global S1700A and STAA mice, as well as the preserved β-adrenergic response of the transgenic phosphomutant mouse models, might arise due to cell type–specific adaptive changes in the PKA and RAD pathways. We anticipate that advances in understanding of Ca_V1.2 regulation in the fight-or-flight response will help guide future work in the development of novel inotropic agents, antihypertrophic medications, and next-generation neurohormonal blocking agents for cardiac disorders.

Regulation of Ca_V1.2 Channels in Neurons and Vascular Smooth Muscle.

Ca_V1.2 channels are present in numerous cell types, including neurons and vascular smooth muscle (26). In neurons, upregulation of the activity of Ca_V1.2 channels primes excitatory synapses for long-term potentiation through PKA phosphorylation of Ser1928 in the dCT (28), and phosphorylation of both Ser1700 and Ser1928 in the CT by PKA controls channel mobility in the plasma membrane (29). In vascular smooth muscle, glucose-mediated increases in Ca_V1.2 activity are associated with phosphorylation of Ser1928 and increased vasoconstriction in hyperglycemia (30). We speculate that cell type–specific interactions with RAD and other members of the Rem family of small G proteins, as we have described here, may lead to convergent tissue-specific regulation in these other cell types.

Materials and Methods

Cell Culture and Transfection.

Human embryonic kidney tsA-201 cells were transfected as previously described with pcDNA3.1 plasmids encoding rabbit Ca_V1.2 (full-length, Δ1800 containing residues 1–1,800, and the dCT containing residues 1,801–2,122) (31), rat β₂b subunit (32), the α₂δ subunit (33), and RAD (WT and 4M [containing Ser25Ala, Ser38Ala, Ser272Ala, and Ser300Ala mutations]) (23) in a 1:1:1:0.75:1 molar ratio unless otherwise specified (12, 15). Data were collected across at least three transfections per expression condition, and experiments with and without RAD were performed in parallel using identical transfection conditions.

Electrophysiology.

Forty-eight hours after transfection, cells were immersed in extracellular solution containing 150 mM Tris, 10 mM glucose, 1 mM MgCl₂, and 10 mM BaCl₂ with pH adjusted to 7.4 with MeSO₄. The intracellular solution contained 135 mM CsCl₂, 10 mM ethylene glycol tetra-acetic acid, 1 mM MgCl₂, 4 mM MgATP, and 10 mM Hepes with pH adjusted to 7.3 with CsOH. Voltage was clamped at −80 mV using 3–5 MΩ glass electrodes with 50–70% resistance compensation. Current traces were sampled at 5 kHz after antialias filtering at 2 kHz. A −p/4 leak subtraction protocol was applied to each recording, and current amplitudes during each depolarization were normalized to gating charge measured at the reversal potential. Peak currents were determined as the largest current amplitude observed during any depolarization recorded at 10-mV intervals ranging from −80 mV to + 80 mV (12, 15). For stimulated cell recordings, 10 μM FSK was added to extracellular buffer bathing the cells, followed by a 10-min equilibration period before recording. Pooled results for the voltage dependence of activation (V_1/2), peak Ba²⁺ current, rate of decay of the Ba²⁺ current, and gating charge normalized to cell capacitance are presented in the SI Appendix, Fig. S1 and Tables S1–S3.

Statistical Analysis.

Data are shown as means ± SEM of number of measurements performed. Statistical significance was tested with Student’s t test for pairwise analysis and one-way ANOVA followed by Tukey’s test for comparison of multiple conditions.

Data, Materials, and Software Availability

All data are included in the manuscript and/or SI Appendix.