Adrenergic Signaling Controls RGK-Dependent Trafficking of Cardiac Voltage-Gated L-Type Ca2+ Channels Through PKD1

Rationale: The Rad-Gem/Kir-related family (RGKs) consists of small GTP-binding proteins that strongly inhibit the activity of voltage-gated calcium channels. Among RGKs, Rem1 is strongly and specifically expressed in cardiac tissue. However, the physiological role and regulation of RGKs, and Rem1 in particular, are largely unknown. Objective: To determine if Rem1 function is physiologically regulated by adrenergic signaling and thus impacts voltage-gated L-type calcium channel (VLCC) activity in the heart. Methods and Results: We found that activation of protein kinase D1, a protein kinase downstream of &agr;1-adrenergic signaling, leads to direct phosphorylation of Rem1 at Ser18. This results in an increase of the channel activity and plasma membrane expression observed by using a combination of electrophysiology, live cell confocal microscopy, and immunohistochemistry in heterologous expression system and neonatal cardiomyocytes. In addition, we show that stimulation of &agr;1-adrenergic receptor-protein kinase D1-Rem1 signaling increases transverse-tubule VLCC expression that results in increased L-type Ca2+ current density in adult ventricular myocytes. Conclusion: The &agr;1-adrenergic stimulation releases Rem1 inhibition of VLCCs through direct phosphorylation of Rem1 at Ser18 by protein kinase D1, resulting in an increase of the channel activity and transverse-tubule expression. Our results uncover a novel molecular regulatory mechanism of VLCC trafficking and function in the heart and provide the first demonstration of physiological regulation of RGK function.

T he voltage-gated calcium channels play a crucial role in regulating cellular excitability. 1 In cardiac muscle, Ca 2ϩ influx through voltage-gated L-type calcium channels (VLCCs) regulates cardiac rhythm and controls muscle contractility by triggering Ca 2ϩ release from the sarcoplasmic reticulum via excitation-contraction coupling. [2][3][4] The alteration of VLCC density or function at the plasma membrane is a key regulator of Ca 2ϩ -dependent cell signaling, gene expression, and cell growth associated with a variety of cardiac diseases, including heart failure, ischemic heart dysfunction, and cardiac arrhythmias, demonstrating a central role for posttranslational modification of VLCC function in cardiac disease. [5][6][7][8][9] The VLCCs in the heart are multisubunit transmembrane proteins (pore-forming ␣ 1 -subunits, also named Cav1.2, and auxiliary subunits ␣ 2␦ and ␤ 2a ) that open in response to membrane depolarization. 1,4 VLCCs are particularly localized at the sarcolemmal membrane structure called the transverse tubular (T-tubule) system. 10 T-tubules occur at the Z-line and at the end of each sarcomere, and they also show complex networks of branching tubules with both transverse and longitudinal elements, 10 which have crucial roles for the regulation of cardiac muscle contractility and rhythm. 3,4,11,12 Although acute modulation of VLCCs by neurotransmitters such as adrenergic stimulation has been extensively studied, 1,4,[11][12][13][14] surprisingly little is known about the molecular mechanisms underlying dynamic physiological and pathophysiological regulation of VLCC membrane expression by intracellular signal transduction.
Emerging evidence suggests that members of the RGK family (Rem1, Rem2, Rad, and Gem/Kir) strongly inhibit VLCC trafficking and activity when overexpressed in heterologous expression systems or native cells, including heart, skeletal muscle, and brain. 15 In particular, Rem1, a member of the RGK family, is abundantly expressed in the heart. 16 However, in native cells including cardiomyocytes, the physiological role of RGK proteins and their regulation by intracellular signaling are largely unknown.
Here we show that adrenergic stimulation releases Rem1 inhibition of VLCC. The release of Rem1-mediated VLCC inhibition in adult cardiomyocytes dramatically increases both T-tubule VLCC membrane expression and Ca 2ϩ current density. We further show that adrenergic stimulation release of Rem1 inhibition of VLCC results from activation of protein kinase D1 (PKD1), 17,18 a protein kinase downstream of ␣ 1 -adrenoceptor (␣ 1 -AR) signaling, which phosphorylates Rem1 at serine 18. Our results indicate that Rem1 phosphorylation at serine 18 results in increased the Ca 2ϩ current through VLCCs (I Ca ) because of increased VLCC plasma membrane expression. These findings uncover a novel molecular mechanism that modulates VLCC trafficking and function and provide the first demonstration of physiological regulation of RGK function.

Materials and Methods
An expanded Methods section is available in the Online Data Supplement (http://circres.ahajournals.org/).

Plasmid, Antibodies, and Reagents
All plasmids, antibodies, and reagents used for the experiments are shown in the Online Data Supplement. Antiphospho-Rem1 (Ser18) was generated with a synthetic phosphopeptide corresponding to mouse Rem1 residues 12 to 24.

Cell Culture, Transfection, and Infection
HEK293T cells and Hela cells were transfected with plasmids and used for experiments 24 hours after transfection. Neonatal and adult rat ventricular myocytes were isolated, cultured, and infected with recombinant adenoviruses as previously described. 19 -21 In Vitro Kinase Assays Glutathione S-transferase fusion protein expression plasmids for full-length wild-type (WT-Rem1) and Rem1 mutants were generated and used for in vitro kinase assays for PKD1. 22

Biochemistry
Whole-cell lysates were used for Western blot and immunoprecipitation analyses. 14,23 The expression level of Cav1.2 in the plasma membrane was determined by a cell-surface protein biotinylation assay. 24

Confocal Microscopy
Plasma membrane localization of Cav1.2 was quantified by line scan intensity measurements and reported as membrane/cytosol ratio (M/C ratio). 25 Fast fourier transform power spectra were used for quantification of T-tubular VLCC localization in adult cardiomyocytes. 26

Electrophysiology
Whole-cell patch-clamp experiments were conducted to measure I Ca at room temperature (Ϸ22°C) using extracellular solution containing 10 or 1 mmol/L Ca 2ϩ in HEK293T cells 27 and cardiomyocytes, 14 respectively.

Data and Statistical Analyses
All results are shown as meanϮSE. The number of the cells used for each analysis is shown in parentheses in the graphs. Unpaired Student t tests were performed when comparing two data sets. For multiple comparisons, a one-way ANOVA followed by post hoc Tukey test were performed. Statistical significance was set as PϽ0.05.

␣ 1 -AR Stimulation Attenuates the Inhibitory Effect of Rem1 on VLCC Function and Plasma Membrane Expression
Rem1 is expressed in cardiomyocytes 15 but is not endogenously expressed in HEK293T cells (online Figure I). To explore whether adrenergic signaling can release the inhibitory effects of Rem1 on I Ca , we coexpressed VLCC subunits with Rem1 and ARs (␣ 1 -AR or ␤ 1 -AR) in HEK293T cells and determined the subcellular VLCC localization using confocal microscopy. 26 Cav1.2 (pore-forming ␣-subunit), ␤ 2a , and ␣ 2␦ subunits were cotransfected. Cotransfection of all three subunits resulted in the distinct expression of GFP-tagged Cav1.2 in the surface membrane ( Figure 1A, B, online Figure II). As previously reported, 28 without coexpression of ␤ 2a subunits, Cav1.2 was not expressed at the plasma membrane (online Figure II). In addition, coexpression of ␣ 2␦ subunits increased the surface membrane expression level of Cav1.2-␤ 2a channels.
Rem1 coexpression caused Cav1.2 to be largely retained at the endoplasmic reticulum (ER) ( Figure 1A, B, Figure 2A, B). Remarkably, the inhibitory effect of Rem1 on VLCC surface expression was dramatically attenuated by ␣ 1 -AR stimulation (10 mol/L phenylephrine [Phe] for 2 hours; Figure 1A, B), concomitant with Cav1.2 redistribution from the ER to the plasma membrane (Figure 2A, B). We determined the dose-dependence of 2 hours of Phe treatment on Cav1.2 membrane expression and found that 0.1 mol/L Phe significantly increased channel membrane expression, with a maximal effect at 10 mol/L (online Figure III). The increase in VLCC surface expression by Phe was blocked by the ␣ 1 -AR antagonist prazosin (1 mol/L), confirming that the effect is mediated through ␣ 1 -ARs (M/C ratio of Phe-treatedϭ0.93Ϯ0.29, nϭ13; untreatedϭ0.81Ϯ0.16, nϭ35; Pϭ0.71). Acute ␣ 1 -AR stimulation (30 seconds-15 minutes) did not significantly alter VLCC localization, but VLCCs gradually redistributed to the surface membrane after 1 hour of stimulation (online Figure VI). In the absence of Rem1 expression, VLCC membrane expression was not enhanced by Phe stimulation (online Figure II). Rem1-mediated reduction in Cav1.2 surface expression and relief by ␣ 1 -AR stimulation were also confirmed by a cell-surface protein biotinylation assay 24 (online Figure V). VLCCs function was estimated in whole-cell patch experiments. Consistent with reduction in surface membrane expression, Rem1 expression markedly decreased I Ca as previously reported for Rem1 and other RGKs. 15 The ␣ 1 -AR stimulation (10 mol/L Phe for 2 hours) restored I Ca magnitude to levels comparable to that observed in the absence of Rem1 without altering the voltage dependence of channel activation ( Figure 1D, online Table I). Acute activation of ␣ 1 -AR signaling did not activate I Ca both in the presence and absence of Rem1 in this cell line (online Figure VI). To functionally assess the plasma membrane expression level of the channels under these conditions, we activated channels with the Ca 2ϩ channel agonist Bay K 8644. 21 In control cells, I Ca was significantly increased (online Figure VII) as previously reported 21 by Bay K 8644 treatment. In Rem1transfected cells, I Ca was also significantly increased by Bay K 8644 treatment. However, the average fold increase in I Ca is the same among these three groups and I Ca in Rem1transfected cells still remained at lower levels than those observed in the absence of Rem1 or in the presence of Rem1 after Phe stimulation (online Figure VII). These results indicate that I Ca inhibition by Rem1 under these conditions is mainly attributable to a decrease in VLCC plasma membrane expression, which can be released by ␣ 1 -AR stimulation.

Non-standard Abbreviations and Acronyms
To explore whether ␤ 1 -adrenergic signaling can also release the inhibitory effects of Rem1 on I Ca , we coexpressed VLCC subunits with Rem1 and ␤ 1 -ARs in HEK293Tcells. However, the inhibitory effect of Rem1 on VLCC trafficking was not reversed by ␤ 1 -AR stimulation (100 nmol/L isoproterenol for 2 hours) in these experiments (online Figure VIII). Because ␤ 1 -AR shows agonist-induced internalization during long-term agonist stimulation, not observed with ␣ 1A -AR (online Figure IX), we also used a direct adenylyl cyclase activator (1 mol/L forskolin for 2 hours) to directly activate downstream ␤ 1 -AR signaling. The inhibitory effect of Rem1 on VLCC trafficking also was not reversed by forskolin applications (online Figure VIII), indicating that release of Rem1 inhibition on I Ca is specific to ␣ 1 -AR signaling.

␣ 1 -AR Stimulation Regulates VLCC Function and Plasma Membrane Expression Through PKD1-Mediated Phosphorylation of Rem1 at Ser18
PKD1 is a newly described serine/threonine protein kinase involved in ␣ 1 -AR signaling that plays important roles in the cardiovascular system. 18,19 Ser18 of Rem1 lies within a PKD consensus motif LXRXX(T*/S*) ( Figure 3A) 18,19 conserved across multiple eukaryotic Rem1 species (online Figure X). The amino acid sequence surrounding Ser 290 of Rem1 also is closely related to the PKD consensus motif ( Figure 3A). To examine whether PKD1 could phosphorylate Rem1 at either of these potential sites, we performed in vitro kinase assays using glutathione S-transferasefusion proteins and found that Ser18 (but not Ser290) is a PKD1-specific phosphorylation site in Rem1 ( Figure 3B). We further demonstrated that PKD1 phosphorylates Rem1-Ser18 in situ ( Figure 3C) using a custom-made antibody (online Figure XI). Moreover, PKD1 interacted with Rem1, and this interaction increased when PKD1 was activated (online Figure XII). To test whether ␣ 1 -AR signaling phosphorylates Rem1-Ser18 through PKD1 activation, we cotransfected ␣ 1 -AR and Rem1 into HEK293T cells and stimulated the cells with phenylephrine. In unstimulated cells, a low-level Rem1 phosphorylation was observed, suggesting that PKD signaling has some activity under basal conditions ( Figure 3D). Phosphorylation of Rem1-Ser18 was increased within 30 seconds of Phe stimulation, concomitant with endogenous PKD1 activation, and this effect remained for up to 2 hours ( Figure 3D Similarly, coexpression of a kinase-negative PKD1 mutant reduced the phosphorylation of Rem1 and abolished rescue of I Ca produced by Phe (online Figure XVI). Results from direct cell application of a PKC activator and cAMP analog demonstrated that PKD1 activation and Rem1 phosphorylation occurs downstream of PKC but not cAMP (online Figure VIII). Collectively, these results indicated that PKD1 directly phosphorylates Rem1-Ser18 on ␣ 1 -AR stimulation, promoting VLCC plasma membrane localization and, thus, increasing I Ca .
Rem1-Ser18 is a potential phosphorylation site suggested to be required for the binding RGKs to the scaffolding protein 14-3-3 in vitro, which is thought to regulate subcellular RGK localization. 15 However, the upstream signaling pathway that controls RGK phosphorylation remains unknown. We found that Ser18 phosphorylation promoted Rem1 binding to 14-3-3 (online Figure XVII) and translocation to the nucleus (online Figure XVIII), suggesting that Ser18 phosphorylation increases the ability of 14-3-3 to recruit Rem1, thereby interfering with the ability of Rem1 to associate with VLCC and inhibit VLCC surface membrane trafficking and function. Rem1 in unstimulated cells strongly colocalized with the ER marker, consistent with the role of Rem1 in suppressing VLCC membrane expression.  . The membrane/cytosol ratio (M/C ratio) of fluorescence intensity is shown compared with control cells transfected with VLCC subunits (middle). Current-voltage relationships were obtained from these three groups (right). B, Mutation of the PKD phosphorylation site in Rem1(S18A) attenuated PKD1-induced VLCC expression at the plasma membrane. VLCC subunits and mutant Rem1-S18A were cotransfected with or without PKD-SE. Representative confocal image from a cell cotransfected with VLCC, Rem1-S18A, and PKD-SE shows that GFP-CaV1.2 subunits were primarily localized within the cytosolic region of the cell (left). The M/C ratio of fluorescence intensity compared with control cells (middle). Current-voltage relationships were obtained from these three groups (right). C, Expression of mutant Rem1-S18A blocked phenylephrine (Phe)-induced enhancement of Ca 2ϩ channel expression in the plasma membrane. VLCC subunits, ␣ 1 -AR and mutant Rem1-S18A were cotransfected in HEK293T cells. Cells were treated with or without Phe for 2 hours. Representative confocal image of cells transfected with VLCC subunits, ␣1-AR, and Rem1-S18A shows that GFP-␣ 1C -subunits were localized in the surface membrane after Phe stimulation (left). The M/C ratio of fluorescence intensity is shown compared with control cells (middle). Current-voltage relationships were obtained from these three groups (right).

␣ 1 -AR Stimulation Enhances VLCC Expression at the Plasma Membrane Through PKD1-Dependent Phosphorylation of Rem1 at Ser18 in Neonatal Cardiomyocytes
Our results indicate that ␣ 1 -AR stimulation results in PKD1-mediated phosphorylation of Rem1 at Ser18 and a subsequent increase in VLCC surface membrane expression and function after heterologous expression in HEK293T cells. Next, we investigated the role of the proposed ␣ 1 -AR-PKD1-Rem1-VLCC signaling pathway in native cardiomyocytes. We found that Rem1 is expressed in whole-cell lysates of neonatal rat ventricular myocytes and that ␣ 1 -AR stimulation by Phe (30 minutes) promoted PKD1 activation and Rem1 phosphorylation at Ser18 (online Figure XIX). In addition, we determined the subcellular localization of VLCC in neonatal cardiomyocytes before and after ␣ 1 -AR stimulation by Phe using an anti-Cav1.2 antibody ( Figure 5, online Figure XX). In agreement with previous reports, 11,29 Cav1.2 labeling was observed both at the surface membrane and intracellularly under basal conditions (online Figure XX). After ␣ 1 -AR stimulation (10 mol/L Phe for 2 hours), Cav1.2 was preferentially localized at the plasma membrane with additional nuclear punctuate staining ( Figure 5A, online Figure XX), presumably because of the stimulation of endogenous Rem1. The M/C ratio was significantly increased by Phe stimulation (Figure 5A, C, online Figure  XX). To confirm the involvement of PKD1 activity in this effect, myocytes were infected with GFP-tagged kinasenegative PKD and cellular localization of Cav1.2 was determined before and after ␣ 1 -AR stimulation. GFP infection alone did not alter Cav1.2 localization either before or after Phe treatment (compare with Figure 5A, online Figure XX). However, kinase-negative PKDinfected myocytes did not show a significant increase in Cav1.2 plasma membrane expression in response to Phe ( Figure 5B, C). These results demonstrate that ␣ 1 -AR stimulation induces an increase of VLCC surface membrane expression through a PKD1-dependent mechanism in neonatal cardiomyocytes. To confirm the involvement of Rem1 phosphorylation at Ser18 in this process, myctagged WT-Rem1 or Rem1-S18A was overexpressed by adenoviral infection and the subcellular localization of Cav1.2 was assessed before and after ␣ 1 -AR stimulation ( Figure 5D-F). Overexpression of both WT-Rem1 (M/C ratioϭ0.93Ϯ0.15, nϭ9) and Rem1-S18A (M/C ra-tioϭ0.88Ϯ0.09, nϭ10) significantly decreased the M/C ratio compared to control (LacZ-infected cells, M/C ra-tioϭ1.63Ϯ0.24, nϭ11; Pϭ0.03 and 0.01, respectively). In WT-Rem1-expressing cells, Phe stimulation promoted Cav1.2 redistribution to the plasma membrane and significantly reduced the degree of colocalization with Rem1 ( Figure 5D, F, online Figure XXI). However, in Rem1-S18A-infected myocytes, Phe stimulation did not alter Cav1.2 subcellular localization or Rem1-Cav1.2 colocalization ( Figure 5E, F, online Figure XXI). These results indicate that ␣ 1 -AR stimulation increases VLCC membrane expression in neonatal cardiomyocytes through PKD-dependent phosphorylation of Rem1 at Ser18.

␣ 1 -AR Stimulation Enhances T-Tubule VLCC Expression in Adult Ventricular Myocytes Through PKD1-Dependent Phosphorylation of Rem1 at Ser18
We next investigated the role of the ␣ 1 -AR-PKD1-Rem1-VLCC signaling pathway in the heart by measuring Cav1.2 localization and I Ca function in adult rat ventricular myocytes in response to sustained ␣ 1 -AR stimulation. By using the plasma membrane marker wheat germ agglutinin, we confirmed that the cellular morphology, T-tubule structure, and its periodicity (Ϸ1.8 m) 10 were preserved in our cultured myocytes up to 40 hours after infection ( Figure 6A, D, online Figures XXII, XXIII).
We measured the effect of Rem1 and Rem1(S18A) overexpression and ␣ 1 -AR stimulation on Cav1.2 localization and I Ca in adult cardiomyocytes. We used fast fourier transform power spectral analysis of Cav1.2 immunofluorescence to quantify VLCC T-tubular localization. 26 In WT-Rem1-overexpressing myocytes, ␣ 1 -AR stimulation promoted Rem1 phosphorylation, Cav1.2 T-tubule redistribution, and partially recovered I Ca without changes in Cav1.2 protein expression ( Figure 6A-C, online Figure XXV). Rem1 colocalized well with T-tubule Cav1.2 channels before, but not after, Phe stimulation (online Figure XXVI). In Rem1-S18A-overexpressing adult ventricular myocytes, ␣ 1 -AR-mediated regulation of VLCC T-tubule expression and I Ca activation were not observed ( Figure 6D-F). These results indicate that ␣ 1 -AR stimulation increases both VLCC function and T-tubule localization in adult cardiomyocytes through PKDdependent phosphorylation of Rem1 at Ser18. All experiments were measured 40 hours after Rem1 adenovirus infection; the effects of Rem1 overexpression on channel membrane localization were not observed 24 hours after infection with WT-Rem1 adenovirus (online Figure XXV), although cells expressed four-times to five-times more Rem1 compared to endogenous Rem1 expression, consistent with the slow turnover observed for the Cav1.2 protein. 30 Forty hours after infection with WT-Rem1 adenovirus, myocytes expressed eight-times to 10-times more Rem1 compared to endogenous Rem1 (online Figure XXV).
To determine whether PKD1 activation could regulate VLCC without overexpression of Rem1, presumably by regulating endogenous Rem1, we measure the effect of ␣ 1 -AR stimulation (10 mol/L Phe for 2 hours) on VLCC T-tubular distribution and current. Phe treatment increased I Ca in both freshly isolated and cultured adult ventricular myocytes (online Figures XXIII, XXIV). Cultured cardiomyocytes were infected with Lac-Z as control for Rem1infected cells. VLCC T-tubular distribution was increased after Phe application without changing the total Cav1.2 expression levels (online Figures XXIII, XXV). T-tubular redistribution of Cav1.2 induced by ␣ 1 -AR stimulation was abolished by infection with kinase-negative PKD (online Figure XXVI).

Discussion
In the present study, we characterize a novel molecular mechanism for the regulation of VLCC cell-surface expression. We show that PKD1 induces an increase in cell-surface VLCC density through phosphorylation of small GTP-binding protein Rem1 in response to ␣ 1 -AR stimulation, leading to a subsequent increase in Ca 2ϩ channel activity (Figure 7). Our study demonstrates that Rem1 is a PKD1 substrate and that a novel ␣ 1 -AR-PKD1-Rem1 signaling pathway dynamically regulates VLCC function in cardiomyocytes. In particular, in adult ventricular myocytes, adrenergic stimulation releases Rem1 inhibition of VLCCs, resulting in an increase in channel activity and expression at T-tubules. T-tubule localization of VLCCs is key to the control cardiac excitability and contractility. 3,4,11,12 Our results uncover a novel molecular regulatory mechanism of VLCC trafficking and function, and provide the first demonstration of physiological regulation of RGK function.
Previous reports proposed that RGK-mediated Ca 2ϩ current suppression in heterologous expression systems results from either a decrease in the number of pore subunits of Ca 2ϩ channels expressed at the plasma membrane [31][32][33][34][35] or an inhibition of surface membrane channel activity. 21,34,36,37 Our data agree with Rem1 decreasing VLCC expression at plasma membrane in both a heterologous expression system and in native cardiomyocytes. Moreover, changes in VLCC membrane localization correlated well with the functional effects observed. Our data suggest that whereas short-term expression of Rem1 may inhibit channel activity without decreasing membrane expression, longer-term Rem1 expression leads to a decrease in channel membrane levels. This is consistent with the slow turnover observed for this channel 30 and suggests that Rem1 may decrease channel insertion into the plasma membrane. Interestingly, the PKD1-Rem1 (S18)-mediated increase in membrane expression of VLCC is observed after only 1 hour of persistent activation of the signaling, suggesting that PKD1-Rem1 signaling can release a VLCC reserve that would help maintain VLCC activity at persistently high adrenergic states. Taken together, our data indicate channel trafficking to be a major contributor to PKD1-Rem1 regulation of VLCC in cardiomyocytes.
Our data would also suggest that the increase in VLCC membrane expression through ␣ 1 -AR-PKD1-Rem1 signaling could contribute to the cytosolic Ca 2 overload when catechol- amine levels are chronically and strongly increased under pathophysiological stress conditions, such as cardiac hypertrophy and heart failure. However, potential limitations of the present study include the alteration in integrity of plasma and intracellular membrane systems such as T-tubules and ER membranes in isolated cardiomyocytes after culture and after 2 hours of Phe treatment. Further investigation would be needed to clarify the detailed mechanism and the role of endogenous expression levels of Rem1 in the regulation of VLCC membrane expression by ␣ 1 -AR-PKD1 signaling in cardiomyocytes and in vivo under the physiological conditions. Among RGKs, both Rem1 and Rad are expressed at significant mRNA and protein levels in the heart 15 and are thought to regulate VLCC function. 21,34,38,39 Although Ser18 is conserved in both proteins, 15 it is not part of a PKD1 substrate motif in Rad. However, further investigation is needed to clarify the involvement of Rad in the regulation of VLCC by ␣ 1 -AR-PKD1 signaling in cardiomyocytes. Changes in ER morphology in Rem1-overexpressing cells were observed (Figures 1, 2), possibly because of the Rem1 regulation of cytoskeleton dynamics, as has been reported for other RGKs. 15 Although morphological remodeling of the ER by Rem1 might contribute to Rem1-mediated inhibition of VLCC trafficking, further studies are needed to more precisely define the underlying mechanism.
In conclusion, we provide the first evidence to our knowledge for a receptor-mediated signaling pathway that can dynamically regulate Rem1 inhibition of VLCCs (Figure 7) in the cardiovascular system. Specifically, we show that ␣ 1 -AR-PKD1-mediated phosphorylation of Rem1-S18 dramatically attenuates Rem1 suppression of VLCC membrane expression and function by promoting the association of Rem1 with 14-3-3 and, consequently, by reducing the colocalization of Rem1 and VLCCs. Because alterations in VLCC T-tubular membrane expression and function are implicated in cardiovascular disease, 7,11,12,26 the PKD1-Rem1-VLCCs regulatory pathway will provide new insight into understanding cardiac excitation-contraction coupling regulation and also will provide new therapeutic perspectives for cardiac hypertrophy, heart failure, and arrhythmias. . Proposed ␣ 1 -adrenoceptor (AR)-protein kinase D1 (PKD1)-Rem1-14 to 3-3 mechanism for the regulation of voltagegated L-type calcium channel (VLCC) membrane expression. 1, Rem1 blocks VLCC membrane expression and inhibits VLCC activity by retaining VLCCs in endoplasmic reticulum (ER). 2, The ␣ 1 -AR stimulation activates PKD1, which then directly phosphorylates Rem1-Ser18. 3, Phosphorylation of Rem1-S18 increases binding to 14-3-3 and induces Rem1 translocation from cytosol to the nucleus. 4, Rem1 nuclear translocation attenuates the inhibitory effect of Rem1 on VLCC expression, releasing the channel from the ER to traffic to the plasma membrane.