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(Circulation Research. 2004;94:1242.)
© 2004 American Heart Association, Inc.

Cellular Biology

Leukemia Inhibitory Factor Activates Cardiac L-Type Ca²⁺ Channels via Phosphorylation of Serine 1829 in the Rabbit Ca_v1.2 Subunit

Eiichi Takahashi, Keiichi Fukuda, Shunichiro Miyoshi, Mitsushige Murata, Takahiro Kato, Makoto Ita, Tsutomu Tanabe, Satoshi Ogawa

From the Institute for Advanced Cardiac Therapeutics (E.T., K.F., S.M.), Cardiopulmonary Division, Department of Internal Medicine (M.M., T.K., S.O.), and Pharmacia-Keio Research Laboratories (M.I.), Shinanomachi Research Park, Keio University School of Medicine, and Department of Pharmacology and Neurobiology (T.T.), Graduate School of Medicine, Tokyo Medical and Dental University, Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Tokyo, Japan.

Correspondence to Keiichi Fukuda, MD, PhD, Institute for Advanced Cardiac Therapeutics, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail kfukuda{at}sc.itc.keio.ac.jp

	Abstract

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We have previously reported that leukemia inhibitory factor (LIF) gradually increased cardiac L-type Ca²⁺ channel current (I_CaL), which peaked at 15 minutes in both adult and neonatal rat cardiomyocytes, and this increase was blocked by the mitogen-activated protein kinase kinase inhibitor PD98059. This study investigated the molecular basis of LIF-induced augmentation of I_CaL in rodent cardiomyocytes. LIF induced phosphorylation of a serine residue in the _1c subunit (Ca_v1.2) of L-type Ca²⁺ channels in cultured rat cardiomyocytes, and this phosphorylation was inhibited by PD98059. When constructs encoding either a wild-type or a carboxyl-terminal–truncated rabbit Ca_v1.2 subunit were transfected into HEK293 cells, LIF induced phosphorylation of the resultant wild-type protein but not the mutant protein. Cotransfection of constitutively active mitogen-activated protein kinase kinase also resulted in phosphorylation of the Ca_v1.2 subunit in the absence of LIF stimulation. In in-gel kinase assays, extracellular signal–regulated kinase phosphorylated a glutathione S-transferase fusion protein of the carboxyl-terminal region of Ca_v1.2 (residues 1700 through 1923), which contains the consensus sequence Pro-Leu-Ser-Pro. A point mutation within this consensus sequence, which results in a substitution of alanine for serine at residue 1829 (S1829A), was sufficient to abolish the LIF-induced phosphorylation. LIF increased I_CaL in HEK cells transfected with wild-type Ca_v1.2 but not with the mutated version. These results provide direct evidence that LIF phosphorylates the serine residue at position 1829 of the Ca_v1.2 subunit via the actions of extracellular signal–regulated kinase and that this phosphorylation increases I_CaL in cardiomyocytes.

Key Words: cardiomyocytes • extracellular signal–regulated kinase • leukemia inhibitory factor • L-type Ca²⁺ channels • phosphorylation

	Introduction

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The cardiac L-type Ca²⁺ channel is the predominant ion channel within the heart, with each cardiomyocyte expressing 30 000 copies.¹ Cardiac L-type Ca²⁺ channels play essential roles in cardiac excitability, in coupling excitation to contraction, and in arrhythmogenesis. The channel is composed of four subunits, ₁, ₂, ß₂, and , and some channel functions are regulated by phosphorylation of the _1c subunit (Ca_v1.2).² Protein kinase A (PKA) has been shown to phosphorylate the serine residue at the carboxyl end of Ca_v1.2,^3,4 and protein kinase C (PKC) may phosphorylate Ca_v1.2 at the amino terminal.⁵ These findings suggest that phosphorylation of the intracellular domain of the cardiac L-type Ca²⁺ channel may be the critical mechanism for modulating its current.

Leukemia inhibitory factor (LIF) is a member of the interleukin-6 family and has a potent hypertrophic effect on cardiomyocytes.⁶ We and others have demonstrated that JAK/STAT,^6,7 mitogen-activated protein kinase,⁸ phosphatidylinositol 3 kinase,⁹ and calmodulin-dependent kinase¹⁰ lie downstream of gp130 in cardiomyocytes and that these pathways play important roles in mediating cardiac hypertrophy. While investigating the molecular mechanism of LIF-induced cardiac hypertrophy, we found that this cytokine increases the L-type Ca²⁺ current (I_CaL) in cardiomyocytes.¹¹ Although the molecular mechanisms by which LIF stimulates I_CaL remains unknown, we have shown it to be independent of PKA and PKC and have shown that the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059 specifically inhibits the LIF-induced amplification of I_CaL. We also identified two extracellular signal–regulated kinase (ERK) consensus sequences (Pro-X-Ser and Thr-Pro) at the carboxyl end of Ca_v1.2 and showed that they are conserved among different species (human, rat, mouse, and rabbit). Based on these findings, we investigated whether LIF can induce phosphorylation of Ca_v1.2 via the ERK1/2 pathway and whether this phosphorylation increases I_Ca,L in cardiomyocytes.

	Materials and Methods

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Materials
Anticardiac L-type Ca²⁺ channel _1c subunit (Ca_v1.2), anti–phospho-ERK1/2, and anti-ERK2 antibodies were purchased from Alomone Labs (Jerusalem, Israel), New England Biolabs (Beverly, Mass), and Santa Cruz Biotechnology (Santa Cruz, Calif), respectively. Angiotensin II and recombinant rat LIF were purchased from Sigma (St Louis, Mo) and Genzyme (Cambridge, Mass), respectively.

Cell Culture
Primary cultures of 1-day-old neonatal and adult Wistar rat cardiomyocytes were prepared as described previously.^6,11 HEK293 and rat aortic smooth muscle cells (P7-10) were cultured as described previously.^12–14

Preparation of Cell Lysates and Immunoprecipitation
Cells were harvested in a lysis buffer containing 10 mmol/L HEPES, pH 7.4, 50 mmol/L sodium pyrophosphate, 50 mmol/L NaF, 50 mmol/L NaCl, 5 mmol/L EDTA, 5 mmol/L EGTA, 100 µmol/L Na₃VO₄, 0.5 mmol/L PMSF, 10 µg/mL leupeptin, and 0.1% Triton X-100.^12,14 The cells were thawed on ice, scraped, sonicated, and centrifuged at 14 000g at 4°C for 30 minutes. Lysates were immunoprecipitated using anti-Ca_v1.2 polyclonal antibody (Alomone) at 4°C for 12 , followed by protein G-Sepharose (Sigma) for 1 hour. Immunoprecipitates were used immediately or stored at –80°C. Western blot analysis was performed as described previously,¹² and the blots were detected by ECL (Amersham Biosciences).

Preparation of Glutathione S-Transferase Fusion Proteins
Three constructs encoding different regions of the C-terminal cytoplasmic domain of rabbit Ca_v1.2¹⁵ (residues 1472 through 2171) were prepared by polymerase chain reaction (PCR). The first spanned residues 1472 through 1699, the second spanned residues 1700 through 1923 (containing the ERK1/2 consensus sequence Pro-Leu-Ser-Pro), and the third spanned residues 1924 through 2171 (containing the ERK1/2 consensus sequence Pro-Ala-Thr-Pro). The orientation and reading frames of all constructs were confirmed by sequencing. The primers used to amplify each region were designed so that PCR products contained a 5' BamHI restriction site and a 3' EcoRI restriction site. Each PCR product was cloned into pGEX-3X. After transformation with glutathione S-transferase (GST)-Ca_v1.2 constructs, cultures of Escherichia coli (BL21) were grown to the sub-log phase, and fusion proteins were induced by exposure to 0.1 mmol/L isopropyl-ß-D-thiogalactopyranoside (Sigma) for 3 hours. Cells were collected, sonicated, and centrifuged, and supernatants were incubated with glutathione-agarose beads (Sigma) overnight at 4°C. Bound fusion proteins were washed extensively and then eluted with 20 mmol/L reduced glutathione, 100 mmol/L Tris-HCl (pH 7.4), and 100 mmol/L NaCl. Protein concentrations were estimated by Coomassie blue staining of the SDS-PAGE separated proteins.¹³

In Vivo Phosphate Labeling and Phospho-Amino Acid Analysis
Cells were metabolically labeled with ³²P-orthophosphate (250 µCi/mL) using previously published protocols.¹⁴ Before immunoprecipitation, the samples were equilibrated to contain an equal amount of protein. The Ca_v1.2 subunit from cardiac L-type Ca²⁺ channels was immunoprecipitated and separated by 5% SDS-PAGE. The immunoprecipitates for the phospho-amino acid analysis were incubated with 6N HCl at 106°C for 60 minutes, and the radioactive phospho-amino acids obtained were then applied to a thin-layer chromatography plate. The amino acids were separated by electrophoresis (1000 V for 30 minutes; 10% acetic acid, 1% pyridine in water, pH 3.3).¹⁶ The plates were exposed to radiograph film, and the location of the nonradioactive and radioactive phospho-amino acids was detected by ninhydrin reaction and by radiograph film, respectively.

Construction of Mutant Plasmids and Cell Transfection
A deletion mutant of Ca_v1.2 was prepared by truncation of the C-terminal amino acids (from 1812 through 2171). A second Ca_v1.2 mutant was prepared with a serine to alanine substitution at position 1829. For transient transfections, HEK293 cells at 50% to 60% confluence were seeded onto 100-mm dishes 24 hours before transfection and then cotransfected with 1.0 µg of Ca_v1.2 plasmid, 1.0 µg of ß_2b plasmid, and 1.0 µg of enhanced green fluorescent protein (eGFP) plasmid using an Effecten kit (Qiagen). Successfully transfected cells were then easily identified by their fluorescence. In some experiments, the cells were simultaneously cotransfected with 0.5 µg of either a plasmid containing constitutively active MEK or empty vector. After incubation for 24 hours with the DNA-lipid complexes, the serum-containing medium was changed, and the next day the cells were serum deprived by incubation with 1% calf serum for 24 hours and then labeled with ³²P-orthophosphate.

Perforated Patch-Clamp Recording
Perforated patch-clamp recording was performed in GFP-positive HEK293 cells transfected with constructs encoding wild-type or mutated Ca_v1.2 and rabbit ß_2b subunits¹⁷ for functional analysis of L-type Ca²⁺ channels using slight modifications to previously described protocols.^18,19 The pipette solution contained 170 mmol/L CsCl, 1.1 mmol/L MgCl₂, 2 mmol/L BaCl₂, and 5 mmol/L HEPES (adjusted to pH 7.0 with CsOH). Gramicidin was dissolved in DMSO (100 mg/mL) immediately before the experiment and diluted in the pipette solution to a final concentration of 100 µg/mL (giving a final DMSO concentration of 0.1%). The extracellular solution was Tyrode’s solution, which contained 2 mmol/L BaCl₂, 140 mmol/L NaCl, 4 mmol/L CsCl, 0.5 mmol/L MgCl₂, 5 mmol/L HEPES, and 55 mmol/L glucose (pH 7.4). Cells were mounted on a thermo-controlled bath set at 34°C to 34.5°C, and Tyrode’s solution containing 1000 U/mL LIF was then bath applied. In this system, the solution in the bath was completely changed 5 to 8 minutes after switching the superfusate.

A pipette resistance of 3 to 4 M was used. Isolated GFP-positive HEK293 cells were selected for the experiment. The Ba²⁺ current was initiated using a 15-ms test pulse from –20 to +10 mV with 10-mV increments and was followed by a 200-ms conditioning prepulse to –50 mV from the holding potential of –80 mV to allow the peak inward current at 0 mV for the test potential to be generated. The series resistance during recording was stable at 20 to 25 M. Because of the geometry and small size of the HEK293 cells, the series resistance might not represent cell access resistance. To confirm good voltage clamping, we determined the voltage of the test pulse that generated the peak I_CaL and the time to reach the peak current (<3 ms) from the onset of the test pulse. The I_CaL was then elicited every 30 seconds by a train step pulse protocol for 0 mV with the same conditioning pulse. The stability of amplitude of I_CaL and the holding current was examined for a few minutes before the administration of LIF. The current-voltage relationship was measured immediately before the train pulse protocol and after the LIF-induced current change was stabilized using the test potential from –40 to +10 mV under the same conditions.

Statistical Analysis
Values are reported as mean±SD. The significance of differences between mean values was determined by ANOVA. Statistical comparisons between the control group and the experimental group were made using Bonferroni’s tests. Differences were considered statistically significant for values of P<0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
LIF Phosphorylates the Ca_v1.2 Subunit of Neonatal Rat L-Type Ca²⁺ Channels at a Specific Serine Residue
Figure 1 depicts the 2D structure of Ca_v1.2 and of the ß_2b subunit of L-type Ca²⁺ channels. Ca_v1.2 contains two consensus sequences at the C-terminal end that are ERK-specific phosphorylation sites.¹⁹ The Pro-Ala-Thr-Pro sequence is conserved in all species examined to date (human, residues 1998 through 2001; rabbit, residues 1986 through 1989),^20,21 and the Pro-Leu-Ser-Pro sequence is also conserved in both humans (residues 1797 through 1800) and rabbits (residues 1827 through 1830).

View larger version (30K): [in this window] [in a new window]   Figure 1. Structure of the L-type Ca2+ channel. Cav1.2 and ß2b subunits are shown. Cav1.2 has a 24-transmembrane domain and a long carboxyl terminal. The carboxyl terminal has various phosphorylation sites, including 2 ERK1/2 consensus sites (Pro-X-Ser/Thr-Pro). These 2 sites are conserved in humans, mice, and rabbits.

To investigate whether LIF phosphorylates Ca_v1.2 via the MEK/ERK pathway, primary cultured rat cardiomyocytes were metabolically labeled with ³²P orthophosphate and stimulated with LIF (1000 U/mL, 15 minutes). Subsequent phosphorylation of Ca_v1.2 was detected by immunoprecipitation. LIF strongly induced phosphorylation of Ca_v1.2, 4.2±1.6-fold greater than the control, and this was inhibited by PD98059 (Figure 2). Phospho-amino acid analysis confirmed that LIF only phosphorylated the serine residues in Ca_v1.2 (Figure 3).

View larger version (30K): [in this window] [in a new window]   Figure 2. LIF phosphorylated the rat L-type Ca2+ channel subunit, Cav1.2. A, In vivo 32P-orthophosphate labeling was performed in LIF-stimulated cardiomyocytes in the presence and absence of PD98059. LIF induced 4.2-fold phosphorylation of Cav1.2, and it was inhibited by PD98059. Bottom, Results of the immunoprecipitation Western blot analysis to demonstrate that each sample contained an equal amount of Cav1.2. Results were the mean of 5 separate experiments, in which each experiment showed similar results. ns indicates not significant vs control [LIF(–), PD98059(–)], *P<0.05 vs control and LIF(+), PD98059(+).

View larger version (44K): [in this window] [in a new window]   Figure 3. Phospho-amino acid analysis by thin-layer chromatography. Phosphorylated Cav1.2 was prepared from the gel. Left, Position of the control phospho-amino acid detected by the ninhydrin reaction. Right, LIF-induced phosphorylation of Cav1.2 was at a serine residue. A representative autoradiogram from 3 independent experiments is shown.

Constitutively Active MEK Phosphorylates ERK1/2 and Ca_v1.2
To confirm that the MEK/ERK pathway is involved in the phosphorylation of Ca_v1.2, we cotransfected HEK293 cells with constructs encoding wild-type Ca_v1.2 and ß_2b subunits and constitutively active MEK1 (ca-MEK1)²² or vector alone and measured phosphorylation. Western blot analysis using anti–phospho-ERK antibody revealed that ca-MEK1 phosphorylated ERK1/2. Metabolic labeling with ³²P-orthophosphate showed that ca-MEK1 also induced phosphorylation of wild-type Ca_v1.2. The intensity of the phosphorylation of Ca_v1.2 was 2.6-fold greater than controls (P<0.05, n=3) (Figure 4A).

View larger version (33K): [in this window] [in a new window]   Figure 4. LIF phosphorylated the Cav1.2 subunit of the L-type Ca2+ channel in vitro at the carboxyl terminal via the MEK/ERK pathway. A, Wild-type-Cav1.2 was cotransfected with constitutive active MEK1 (caMEK1) and ß2b subunit, and phosphorylation of Cav1.2 was detected. The caMEK1-transfected cells showed phosphorylation of Cav1.2 in the absence of LIF stimulation, indicating that the MEK1/ERK1 pathway was sufficient to induce phosphorylation. A representative autoradiogram from 4 independent experiments is shown. B, Wild-type (WT) or a deletion mutant (Del) lacking the carboxyl terminal of Cav1.2 was transfected into HEK293 cells with the ß2b subunit, and the cells were stimulated with LIF. The wild-type Cav1.2 was phosphorylated with LIF, but the deletion mutant was unaffected by LIF. Results were the mean of 5 separate experiments, in which each experiment showed similar results. ns indicates not significant vs control [LIF(–), PD98059(–)], *P<0.05 vs control.

LIF Phosphorylates the Carboxyl-Terminal of Ca_v1.2
To determine whether LIF phosphorylates the carboxyl-terminal of Ca_v1.2, we transfected HEK293 cells with either the rabbit wild-type Ca_v1.2 or a Ca_v1.2 carboxyl-terminal deletion mutant, along with the ß_2b subunit. The cells were metabolically labeled with ³²P-orthophosphate and stimulated with LIF. Although the wild-type Ca_v1.2 was phosphorylated 2.9-fold greater than the control (n=5, P<0.05), the deletion mutant was not phosphorylated by LIF at all (n=5, not significant) (Figure 4B).

LIF Phosphorylates the Carboxyl-Terminal of Ca_v1.2 From Amino Acids 1812 through 2171
To identify the phosphorylation site, we prepared deleted GST-fusion proteins of Ca_v1.2 as a substrate and performed in-gel kinase assays. The three deleted fusion proteins contained either no ERK1/2 consensus sequences, the Pro-Leu-Ser-Pro sequence, or the Pro-Ala-Thr-Pro sequence (see also Figure 1). Serum-starved primary cultured rat neonatal cardiomyocytes and rat aortic smooth muscle cells were incubated for 15 minutes with LIF and angiotensin II, respectively, and in-gel kinase assays were performed using myelin basic protein (MBP), GST, or deleted Ca_v1.2 GST-fusion proteins. ERK1/2, which was activated by LIF, phosphorylated the MBP and the protein containing the Pro-Leu-Ser-Pro consensus sequence, and this phosphorylation was blocked by preincubation with PD98059 (Figure 5). These findings indicate that activated ERK1/2 can phosphorylate the carboxyl terminal of Ca_v1.2 between amino acids 1700 and 1923.

View larger version (44K): [in this window] [in a new window]   Figure 5. LIF phosphorylated the Cav1.2 subunit of the L-type Ca2+ channel in vitro at the carboxyl terminal from positions 1700 through 1923 via the MEK/ERK pathway. GST-fusion proteins were prepared for 3 regions of the carboxyl terminal of Cav1.2. Part I, 1472 through 1699; part II, 1700 through 1923; and part III, 1924 through 2171. In-gel phosphorylation of the GST-fusion proteins was assessed by using LIF-stimulated cardiomyocyte lysates (top) or angiotensin II–stimulated RASM cell lysates (bottom). Arrows indicate that 42- and 44-kDa ERK1/2 phosphorylated the substrates (MBP and part II). A representative autoradiogram from 3 independent experiments is shown.

Ca_v1.2 Is Specifically Phosphorylated by LIF at Serine 1829
To confirm that the LIF-induced phosphorylation of the carboxyl-terminal of the L-type Ca²⁺ channel actually occurred at the ERK target sequence (Pro-Leu-Ser-Pro, 1827 through 1830), we cotransfected HEK293 cells with the ß_2b subunit and constructs encoding either the S1829A mutant Ca_v1.2 or wild-type Ca_v1.2. Cells were then metabolically labeled with ³²P-orthophosphate. After treatment with LIF, the wild-type Ca_v1.2 was phosphorylated 2.9-fold over the control, whereas the mutant Ca_v1.2 remained unphosphorylated (Figure 6), indicating that Ca_v1.2 was specifically phosphorylated at serine 1829.

View larger version (32K): [in this window] [in a new window]   Figure 6. Cav1.2 is specifically phosphorylated by LIF at serine 1829. Wild-type Cav1.2 (WT) or the point mutant Cav1.2 (serine 1829 to alanine substitution, MT) was transfected together with the ß2b subunit into HEK293 cells and metabolically labeled with ortho-32P. Cells were stimulated with LIF for 15 minutes, and the wild-type or mutant Cav1.2 was immunoprecipitated. The increased intensity of the phosphorylation of the Cav1.2 was normalized by the intensity of the phosphorylation in the absence of LIF stimulation in wild-type and mutant cells, respectively. Results are the mean of 3 separate experiments, in which each experiment showed similar results. ns indicates not significant vs control [LIF(–), PD98059(–)], *P<0.05 vs control.

LIF Increased I_CaL in the Wild-Type Ca_v1.2 but not in the S1829A Mutant Ca_v1.2
To confirm that the S1829A point mutation in Ca_v1.2 abolished the response to LIF, we measured the peak inward current in HEK293 cells cotransfected with the ß_2b subunit and either wild-type or mutant Ca_v1.2. In our preliminary conventional whole-cell patch clamp experiment, the peak inward current varied from 0 to 6 nA in wild-type (n=57) and from 0 to 2 nA in mutant (n=30) cells, but the difference was not statistically significant. Little or no inward current (<50 pA) was observed in 10 GFP-positive wild-type cells or in four GFP-positive mutant cells. Although some cells displayed large currents (>1nA; n=2 in wild-type, n=1 in mutant), the current density was generally similar between the two groups. In the perforation patch-clamp experiment, the amplitude of the current was stable during the observation period, suggesting no natural run down. The amplitude of the peak current 5 minutes after exposure to LIF increased in the wild-type Ca_v1.2 cells (38±40%, n=7), whereas there was a small decrease in current in mutant cells (–16±10%, n=7, P<0.05 versus wild-type). A representative time course of the increase in I_CaL is shown in Figure 7. The representative original current trace was shown in online Figures 1 and 2 (available in the online data supplement at http://circres.ahajournals.org). Cells transfected with wild-type showed an increase in the amplitude of the current 5 minutes after exposure to LIF that reached a maximum after 20 minutes. Cells transfected with mutant showed a small decrease immediately after the change of solution that was stable for >30 minutes. Furthermore, extracellular application of 10 µmol/L dB-cAMP or 8Br-cAMP did not increased the I_CaL in wild-type cells, suggesting there is no PKA pathway to phosphorylate the L-type Ca²⁺ channel in HEK293 cells (data not shown). These results indicated that the LIF-induced increase in I_CaL was mediated by the phosphorylation of serine 1829.

View larger version (24K): [in this window] [in a new window]   Figure 7. LIF increased ICaL in the wild-type Cav1.2 but not in the S1829A mutant Cav1.2. The function of the transfected L-type Ca2+ channels was analyzed by gramicidin-perforated patch clamping. Time courses of ICaL for transfected L-type Ca2+ channels (wild-type [A] and mutant [B]) in HEK293 cells are shown. The points in the figures are peak inward current amplitudes at 0 mV every 10 seconds. Voltage protocol is shown in the inset. Solid bar at the top of the figure indicates the duration of exposure to LIF. Current trace for each time point was superimposed for each corresponding symbol and is shown as an inset in the top right corner. Current amplitude peaked at 20 to 25 minutes in wild-type L-type Ca2+ channels but did not increase or show a natural rundown in mutant L-type Ca2+ channels. Amplitude of inward currents before (baseline) and after LIF administration was normalized to the maximal inward current at the baseline. Averaged data are plotted against the test membrane potential in right panels (wild-type [C] and mutant [D]). LIF increased the ICaL only in wild-type L-type Ca2+ channels, without changing the current-voltage relationship. Mutant L-type Ca2+ channels were unaffected.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This study investigated the molecular mechanisms of the LIF-induced increase in I_CaL. LIF was found to induce specific phosphorylation of serine 1829 of rabbit Ca_v1.2, the _1c subunit of the cardiac L-type Ca²⁺ channel. Because LIF failed to induce any increase in current when the subunit contained an S1829A point mutation, we have shown that this phosphorylation event is necessary for the observed increase in I_CaL. The serine residue at position 1829 lies within an ERK1/2 consensus phosphorylation sequence and is therefore phosphorylated either by ERK1/2 or kinases activated by ERK1/2. In agreement with a previous study,²³ PKA activation by the cell-permeable cAMP compound failed to increase I_CaL in HEK293 cells because of the lack of A-kinase anchoring protein. This also suggests that the LIF-induced increase in I_CaL in this system follows a pathway independent of the PKA pathway.

Ca_v1.2, which is critical for the modulation of the I_CaL, is regulated via phosphorylation by various kinases at different positions in the intracellular domains, especially the long carboxyl-terminal domain. De Jongh et al³ showed that PKA phosphorylates the serine residue at position 1928 of the carboxyl terminal of Ca_v1.2 and that this phosphorylation enhances cellular Ca²⁺ entry in response to ß-adrenergic receptor stimulation. Leach et al²⁴ reported that PKA also phosphorylates serine 1627 and serine 1700 in the carboxyl terminal of Ca_v1.2 in response to ß-adrenergic stimulation. These findings suggest that PKA-activating pathways modulate the I_CaL in cardiac muscle and that the C-terminal of Ca_v1.2 is a substrate for PKA. Shistik et al⁵ demonstrated that PKC inhibits I_CaL by phosphorylating threonine 27 and threonine 31 at the N-terminal of Ca_v1.2, whereas Jiang et al²⁵ reported that cGMP inhibits I_CaL by phosphorylating serine 533 of Ca_v1.2 via the action of protein kinase G. Recent studies have revealed that the ß subunit of the L-type Ca²⁺ channel also plays an important role in modulating the I_CaL. Haase and colleagues^26,27 reported that PKA phosphorylates the ß subunit and increases Ca²⁺ entry in response to ß-adrenergic stimulation both in vivo and in vitro. Bünemann et al²⁸ showed that phosphorylation of serine 478 and serine 479 of the ß₂ subunit is involved in this PKA-dependent augmentation of I_CaL.

This study has identified a novel regulatory mechanism of cardiac I_CaL. To our knowledge, this is the first report of ERK1/2 involvement in the regulation of cardiac I_CaL. There seem to be several reasons why this involvement has not been previously recognized. After ligand stimulation, PKA, PKC, and protein kinase G are rapidly activated following the rapid increase of the upstream second messengers cAMP, DG, and cGMP. As a result, these kinases can phosphorylate Ca_v1.2 and modulate the I_CaL at an early stage, eg, 1 to 5 minutes after the stimulus. By contrast, ERK1/2 is activated through various signaling molecules, such as sos, shc, Grb2, raf1, and MEK, and its activation does not peak until 8 to 15 minutes after the stimulus. The activation by LIF peaked at 15 minutes. In addition, the ERK-mediated increase in I_CaL was only 25% to 30% greater than baseline, far smaller than the increase by PKA. Presumably, this relatively small and slow increase in Ca²⁺ current caused by ERK or its downstream kinases was easily masked by the other stimuli. For example, endothelin-1 is known to increase I_CaL in cardiomyocytes. Because the ET-A receptor is linked to G_s/G_i/G_q, endothelin-1 stimulation rapidly increases cAMP and IP3, resulting in rapid activation of PKA and PKC at as early as 1 to 3 minutes.^29,30 Endothelin-1 also activates ERK, but its activation peaks at 8 to 10 minutes,³¹ and thus the rapid activation of PKA or PKC may mask the ERK-mediated augmentation of I_CaL by endothelin-1. By contrast, the signaling pathway via LIF uses raf1/MEK/ERK, JAK/STAT, and phosphatidylinositol 3-kinase/ACT pathways through gp130 and not the PKA or PKC pathways. Presumably we were able to detect ERK1/2-mediated modification of cardiac L-type Ca²⁺ channels because LIF activates ERK and not PKA or PKC.

ERK1/2 is a ubiquitously expressed member of the mitogen-activated protein kinase family, which is activated in response to a variety of extracellular stimuli. It has been implicated in both growth and apoptosis in the cardiovascular system. The downstream substrates of ERK include other kinases, transcription factors, and membrane receptors and other cell mediators. The only evidence of ERK functioning in the regulation of the activity of membrane ion channels has come from neurological studies. Adams and colleagues^32,33 reported that the A-type potassium channel Kv4.2 is a substrate for ERK in hippocampal neurons and demonstrated that ERK phosphorylates threonine 602, threonine 607, and serine 616 within the cytoplasmic domain. Shi et al³⁴ demonstrated that ERK phosphorylates the carboxyl terminal of the ß (threonine 613) and (threonine 623) subunits of the epithelial Na⁺ channel, thereby facilitating their interactions with the ubiquitin ligase Nedd4, which ultimately inhibits channel activity. These findings indicate that the phosphorylation of membrane ion channels by ERK1/2 is perhaps not an uncommon regulatory mechanism. This study is the first to demonstrate involvement of ERK1/2 in modifying cardiac ion channels.

In the present study, the phosphorylation status of the ß_2b subunit was not investigated, because preliminary experiments showed that LIF does not cause phosphorylation of this subunit. Our finding that the point mutation of Ca_v1.2 at serine 1829 completely abolishes the increase in I_CaL suggests that it is unlikely that phosphorylation of the ß_2b subunit plays a critical role in the regulation of the LIF-induced increase in I_CaL. Because cardiac L-type Ca²⁺ channels are critical ion channels in the control of cardiac function, additional investigations are needed to precisely identify all of the regulatory mechanisms involved.

	Acknowledgments

 
This study was supported in part by research grants from the Ministry of Education, Science and Culture, Japan (to K.F. and E.T.), the Health Science Research Grants for Advanced Medical Technology from the Ministry of Welfare, Japan (to K.F.), the Japan Heart Foundation (Molecular Cardiology Fund by Zeria New Drug Inc) (to E.T.), and the Takeda Science Foundation (to E.T.). We also thank Haruko Kawaguchi and Yasuyo Hisaka for their technical help.

	Footnotes

 
This manuscript was sent to Stephen F. Vatner, Consulting Editor, for review by expert referees, editorial decision, and final deposition.

Original received June 10, 2003; resubmission received November 6, 2003; revised resubmission received March 11, 2004; accepted March 11, 2004.

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