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Originally published In Press as doi:10.1074/jbc.M508661200 on July 4, 2006

J. Biol. Chem., Vol. 281, Issue 35, 25560-25567, September 1, 2006
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Calmodulin Kinase II Is Involved in Voltage-dependent Facilitation of the L-type Cav1.2 Calcium Channel

IDENTIFICATION OF THE PHOSPHORYLATION SITES*

Tae-Seong Lee{ddagger}, Rosi Karl§, Sven Moosmang{ddagger}, Peter Lenhardt{ddagger}, Norbert Klugbauer{ddagger}1, Franz Hofmann{ddagger}, Thomas Kleppisch{ddagger}, and Andrea Welling{ddagger}2

From the {ddagger}Institut für Pharmakologie und Toxikologie and the §Institut für Molekulare Medizin, Technische Universität München, Biedersteiner Strasse 29, 80802 München, Germany

Received for publication, August 5, 2005 , and in revised form, May 22, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Calcium-dependent facilitation of L-type calcium channels has been reported to depend on the function of calmodulin kinase II. In contrast, the mechanism for voltage-dependent facilitation is not clear. In HEK 293 cells expressing Cav1.2, Cavbeta2a, and calmodulin kinase II, the calcium current measured at +30 mV was facilitated up to 1.5-fold by a 200-ms-long prepulse to +160 mV. This voltage-dependent facilitation was prevented by the calmodulin kinase II inhibitors KN93 and the autocamtide-2-related peptide. In cells expressing the Cav1.2 mutation I1649E, a residue critical for the binding of Ca2+-bound calmodulin, facilitation was also abolished. Calmodulin kinase II was coimmunoprecipitated with the Cav1.2 channel from murine heart and HEK 293 cells expressing Cav1.2 and calmodulinkinase II. The precipitated Cav1.2 channel was phosphorylated in the presence of calmodulin and Ca2+. Fifteen putative calmodulin kinase II phosphorylation sites were identified mostly in the carboxyl-terminal tail of Cav1.2. Neither truncation at amino acid 1728 nor changing the II-III loop serines 808 and 888 to alanines affected facilitation of the calcium current. In contrast, facilitation was decreased by the single mutations S1512A and S1570A and abolished by the double mutation S1512A/S1570A. These serines flank the carboxyl-terminal EF-hand motif. Immunoprecipitation of calmodulin kinase II with the Cav1.2 channel was not affected by the mutation S1512A/S1570A. The phosphorylation of the Cav1.2 protein was strongly decreased in the S1512A/S1570A double mutant. These results suggest that voltage-dependent facilitation of the Cav1.2 channel depends on the phosphorylation of Ser1512/Ser1570 by calmodulin kinase II.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ca2+ current (ICa)3 through L-type channels is an important determinant of intracellular Ca2+ transients that trigger transmitter release, secretion, and contraction (1). In the heart, the size of the intracellular Ca2+ transient is determined by the release of Ca2+ from intracellular stores and by the size of the L-type current (2). The availability of L-type channels to open is regulated by the membrane potential and other factors that include protein kinases, phosphatases, and Ca2+-binding proteins (3, 4). A train of repetitive depolarizations (5) or a strong depolarizing prepulse (6) drives the L-type calcium channels from their normal gating pattern into a mode of gating characterized by long opening and high open probability (7, 8), a process that has been termed "facilitation." The structural mechanism underlying facilitation is still not clear. Initially, it was suggested that facilitation required cAMP-dependent phosphorylation (9). These findings were not confirmed by others (8, 10-12). In contrast, it was shown that facilitation required elevated [Ca2+]inthe vicinity of the channel ("Ca2+-dependent facilitation") (13). Excessive calcium influx is toxic to cells. As a consequence, calcium channels have evolved voltage- and Ca2+-dependent inactivation mechanisms. Both Ca2+-dependent facilitation and inactivation require high affinity binding of calmodulin (CaM) to the IQ motif located in the carboxyl-terminal tail of the Cav1.2 channel (14-16). Zühlke et al. (14, 15) showed that mutation of the isoleucine to alanine in the IQ motif abolished Ca2+-dependent inactivation without affecting Ca2+-dependent facilitation. In contrast, the mutation of the same isoleucine to glutamate eliminated both forms of autoregulation. Several additional peptide sequences may be involved in the complex formation of CaM with the channel (17). The mechanisms behind this Ca2+/CaM-dependent regulation are reviewed in Ref. 18.

Ca2+-dependent facilitation has been attributed to the action of Ca2+/calmodulin-dependent protein kinase II (CaM-KII) (19-22). Constitutively active CaM-KII facilitated L-type current in excised patches (23). CaM-KII was reported to phosphorylate the main subunit of an isolated L-type calcium channel (24). However, the phosphorylation site was not identified in that study. So far, the mechanism of facilitation caused by a strong depolarizing prepulse ("voltage-dependent facilitation") is unclear.

The functional significance of CaM-KII depends on its subcellular localization (13). CaM-KII has been shown to be localized closely to the Cav1.2 channel in cardiac muscle (25, 26). Despite this localization, activation of the enzyme apparently required release of Ca2+ from intracellular stores (27, 28), thereby allowing integration of spatially and temporally separated [Ca2+]i spikes. These and other studies showed that CaM-KII is an important regulator of neuronal and muscular function (29), because its activity reflects the frequency of cytosolic Ca2+ oscillations. The kinase might, therefore, be able to integrate variable Ca2+ signals over time.

Here we show that the Cav1.2 protein immunoprecipitated in a complex with CaM-KII and was phosphorylated in a Ca2+/CaM-dependent manner. Transient expression of Cav1.2 and the cardiac Cavbeta2a subunit in a cell line stably expressing CaM-KII allowed us to identify for the first time the two CaM-KII phosphorylated serines that are necessary for voltage-dependent facilitation. Interestingly, these two serines flank the EF-hand of Cav1.2.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All substances used were of the highest purity available. The Cav1.2-specific antibody used in this study has been described previously (30). CaM was detected using a monoclonal antibody (Upstate%20Biotechnology">Upstate Biotechnology, Inc., Lake Placid, NY). The CaM-KII antibody MAB8699 was from Chemicon.

cDNA Clones and Site-directed Mutations—The cDNAs of all subunits, including full-length Cav1.2b (data base accession number X55763 [GenBank] ) (31) or truncated Cav1.2b (Tr1728) (32), cardiac beta2a (X64297 [GenBank] ) (Cavbeta2a) (33), {alpha}2{delta}-1(M21948 [GenBank] ) (34), or mutants were subcloned into the pcDNA3 vector. The Cav1.2b mutants S808A, S888A, S808A/S888A, S1512A, S1570A, S1512A/S1570A, S1695A, the IQ -> EQ (I1649E) (15), and the Cavbeta2a mutant T500A were made by the use of the QuikChange II XL site-directed mutagenesis kit (Stratagene, La Jolla, CA). The primers are given in Table 1. PCR amplification was performed using pcDNA3Cav1.2b, pcDNA3Tr1728, or pcDNA3Cavbeta2a as template. For construction of the mutants S1512A, S1570A, S1512A/S1570A, and S1695A, the PCR products were digested with SacII/XbaI. For construction of the mutants S808A, S888A, and S808A/S888A, the PCR products were digested with SgrAI/PmII. The resulting PCR fragments were ligated into the corresponding sites of the digested vectors. The Cav1.2b subunit was truncated carboxy-terminally at amino acid 1728 (for details, see Ref. 32). The GST fusion proteins of the truncated COOH terminus were cloned in the pGEX6P-1 (Amersham Biosciences) vector and mutated using overlap PCR. Each construct was sequenced to verify the correctness of the sequence around the mutation and the ligation (SEQLAB, Göttingen, Germany).


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  		TABLE 1
Sequences of forward and reverse primers for the design of Cav1.2 and Cavbeta mutants Shaded triplets correspond to intended mutations. Underlined boldface letters correspond to mutated amino acids.

 
Transfection of HEK 293 Cells—Human embryonic kidney cells (HEK 293 cells) stably expressing Cav1.2b and the cardiac Cavbeta2a subunit were transiently transfected with the expression vector for CaM-KII (total cDNA 0.3 µg) by a lipofection method using Lipofectamine 2000 according to the manufacturer's guide (Invitrogen). HEK 293 cells stably expressing CaM-KII were transiently transfected with the expression vectors for wild type and the various Cav1.2b chimeras and the cardiac Cavbeta2a subunit (0.15 µg of each). After transfection, the cells were grown for 40-48 h before beginning the electrophysiological experiments.

Electrophysiological Recordings—Whole-cell ICa was measured at room temperature. Stimulation and data acquisition were performed through EPC 9 and HEKA PULSE (version 8.54; HEKA Electronic, Lambrecht, Germany). Data were sampled at 5 kHz and filtered at 1.67 kHz. The pipette had a resistance of 2-3.5 megaohms when filled with intracellular solution. Series resistance and capacitive components were compensated by circuitry equipped in EPC 9. Extracellular solution was composed of 82 mM NaCl, 20 mM triethanolamine-Cl, 30 mM CaCl2, 5 mM CsCl, 1 mM MgCl2, 0.1 mM EGTA, 10 mM glucose, 5 mM HEPES, pH 7.4, adjusted with NaOH. Equimolar BaCl2 was used instead of CaCl2 in the case of barium current (IBa) measurement. Intracellular solution was composed of 102 mM CsCl, 10 mM triethanolamine-Cl, 10 mM EGTA, 1 mM MgCl2, 3 mM Na2ATP, 5 mM HEPES, pH 7.4, adjusted with CsOH. To measure current-voltage (I-V) relations, the cell was stimulated with 300-ms square pulses of -50 to 80 mV in increments of 10 mV from a holding potential of -80 mV. Facilitation of ICa was measured during a triple pulse protocol with a 100-ms control pulse to +30 mV (TP1) followed by a 200-ms prepulse to +160 mV followed by a 100-ms test pulse to +30 mV (TP2) (Fig. 1a). The extent of voltage-dependent facilitation was calculated as the ratio of the peak current during TP2 and TP1. The extent of inactivation was evaluated by the residual current fraction at the end of a 300-ms square pulse at +30 mV (R300). 0.5 µM KN-93 (Calbiochem) was applied in extracellular solution, or 10 µM autocamtide-2-related inhibitory peptide (Calbiochem) was added to intracellular solution to inhibit CaM-KII. KN-92 (1 µM; Calbiochem) was used as control of KN-93. Data plotting and statistical analysis was carried out using ORIGIN software (version 6.1052; Microcal, Northampton, MA). Pooled data are given as mean ± S.E.


Figure 1
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  		FIGURE 1.
CaM-KII is necessary for ICa facilitation. a, the triple pulse protocol to induce voltage-dependent facilitation. TP1, 100-ms control pulse to +30 mV, followed by a 200-ms prepulse to +160 mV; TP2, 100-ms test pulse to +30 mV. b, HEK 293 cells were stably or transiently transfected with Cav1.2 or the mutant Cav1.2 (I1649E) and the Cavbeta2a subunit. CaM-KII was co-transfected. Prepulse facilitation was measured as described under "Materials and Methods" and for a. Current traces from TP1 and TP2 were superimposed. Black line, control; red line, current after prepulse; KN-93, CaM-KII inhibitor (0.5 µM); scale bars, 20 ms. (a), 20 pA/pF; (b), 10 pA/pF; (c), 5 pA/pF; (d), 2 pA/pF. c, statistical analysis of examples shown in b. The peak ICa amplitude from TP2 was normalized to the peak ICa amplitude from TP1. **, p < 0.005; ***, p < 0.0005 versus without CaM-KII.

 


Figure 2
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  		FIGURE 2.
Putative CaM-KII phosphorylation sites in the Cav1.2 subunit. According to the minimum consensus sequence of Arg-X-X-Ser/Thr in Ref. 43, 16 sites were identified as putative phosphorylation sites on the Cav1.2 subunit and Cavbeta subunit (open and filled circles). These sites were restricted to eight sites according to the consensus sequence of "Hyd-X-Arg-NB-X-Ser/Thr-Hyd" reported in Ref. 44 (filled circle). The arrows mark the truncation at amino acid 1728 (Tr1728). Hyd, hydrophobic amino acid residue; X, any amino acid residue; NB, nonbasic amino acid residue.

 
Generation of Cell Lines—The cDNA for CaM-KII{alpha} (AK083245 [GenBank] ) was cloned from a murine embryonal cDNA library using a PCR strategy and subcloned into the EcoRI site of pcDNA3 (Invitrogen). The 5'-PCR primer contains an additional consensus sequence for initiation of translation (GCC ACC). HEK 293 cells were transfected by electroporation (Gene PulserTM; Bio-Rad) with the expression vectors pcDNA3Cav1.2b plus pcDNA3Cavbeta2a or pcDNA3CaM-KII using linearized DNA. The HEK 293 cells were grown in minimal essential medium supplemented with 5-10% fetal calf serum, streptomycin (100 µg/ml), and penicillin (100 units/ml). After electroporation, the cells were grown for 48 h without selection medium. Thereafter, the medium was supplemented with 400 µg/ml G418 to select for CaM-KII or with 400 µg/ml G418 plus 200 µg/ml hygromycin B to select for Cav1.2b plus Cavbeta2a. The medium was changed every third day. Three weeks after the electroporation, individual cell clones were isolated with the aid of a cloning ring. Stable expression of the calcium channel subunits was confirmed by patch-clamp experiments; stable expression of CaM-KII was confirmed by Western blotting with a monoclonal CaM-KII antibody (MA1-048; Dianova, Hamburg, Germany) and a goat secondary antibody (IgG (H + L) 112-055-062; Dianova). Untransfected HEK 293 cells expressed neither L-type calcium channels nor CaM-KII.

Preparation and Solubilization of Membranes and GST Fusion Proteins—All preparative steps were performed at 4 °C using precooled solutions containing the protease inhibitor mixture P8340 (2 µl/ml; Sigma), phenylmethylsulfonyl fluoride (200 nM), calpain inhibitor I (8 µg/ml), and calpain inhibitor II (8 µg/ml). Hearts from 6-12-week-old male mice or HEK 293 cells were frozen and pulverized under liquid N2 in a porcelain mortar and then resuspended in membrane preparation buffer containing 300 mM sucrose, 75 mM NaCl, 20 mM EDTA, 20 mM EGTA, 10 mM Tris-HCl, pH 7.4 (1 ml/100 mg of tissue or 3 x 106 cells, respectively). Homogenates were centrifuged at 4,500 rpm for 10 min at 4 °C to remove larger cell fragments, including nuclei. Membranes were collected by ultracentrifugation (50,000 rpm at 4 °C) for 30 min, and channels were solubilized for 20 min on ice with 1% deoxycholate, 10 mM EDTA, 10 mM EGTA, 50 mM, Tris-HCl, pH 7.4, containing protease inhibitors. The following buffer was used for solubilization in the presence of calcium: 150 mM NaCl, 20 mM Tris-HCl, pH 7.5, 0.5 mM CaCl2, 1 mM dithiothreitol, 1% Triton, and EDTA-free protease inhibitor mixture. Nonsoluble material was removed by a second ultracentrifugation step (50,000 rpm at 4 °C for 30 min). GST fusion proteins were expressed in BL21 E. coli according to the manufacturer's instructions (Amersham Biosciences).

Immunoprecipitation and Immunoblotting of Cav1.2 and CaM-KII—The solubilized membranes from heart and HEK 293 cells were preincubated with protein A-Sepharose (Sigma) or protein G-Sepharose (Amersham Biosciences) to remove proteins that bind to the resin nonspecifically. After removal of the Sepharose beads by centrifugation, the supernatant was incubated on ice with antibodies. After 2.5 h, protein A-Sepharose or protein G-Sepharose was added, samples were tilted for 1 h, and the resins were washed and extracted with 1:6 (v/v) SDS sample buffer (35). Eluted proteins were separated on a 10% SDS gel. The separated proteins were immunoblotted as described in Ref. 30. All experiments were repeated three times with comparable results.


Figure 3
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  		FIGURE 3.
I-V relation and inactivation properties of the S1512A/S1570A mutated Cav1.2 channel. a, current-voltage relation of Tr1728 (truncated at amino acid 1728; open circles) and the S1512A/S1570A mutant (filled circles) co-transfected with Cavbeta2a in a CaM-KII-expressing HEK 293 cell. b, comparison of voltage-dependent facilitation between Tr1728 (open bar) and S1512A/S1570A (filled bar). c, superimposed IBa and ICa current traces of wild type (WT) and S1512A/S1570A at +30 mV without a prepulse. Both traces are scaled to the peak current. d, residual current fraction (R300)of IBa (black) and ICa (white) for WT and S1512A/S1570A mutant. **, p < 0.005 versus Tr1728; *, p < 0.05 S1512/1570A versus WT.

 
Phosphorylation with CaM-KII—For CaM-KII kinase reactions, full-length Cav1.2 was immunoprecipitated with the Cav1.2-specific antibody, and immune complexes were captured on protein A-Sepharose. GST fusion proteins were purified on glutathione-Sepharose. Precipitated complexes were resuspended in phosphorylation buffer (0.1% Triton X-100, 50 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 0.1 mM EDTA). Samples were incubated with 250 units of CaM-KII (New England Biolabs) (preincubated in the presence or absence of Ca2+/CaM), and phosphorylation was initiated at 30 °C with 33 µM Mg-[{gamma}-32P]ATP (54 µCi/µmol) (PerkinElmer Life Sciences) and terminated after 10 min. Proteins were separated on a 10% SDS gel, and incorporated 32Pi was detected by autoradiography.


Figure 4
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  		FIGURE 4.
Co-immunoprecipitation of Cav1.2 and CaM-KII. Shown are representative Western blots of the immunoprecipitated Cav1.2 complex. All experiments were repeated at least three times. Blots were probed with specific antibodies as indicated. In some experiments, a HEK 293 cell membrane preparation (MP) was immunoblotted as positive control. a, Western blots of immunoprecipitated (IP) murine cardiac homogenates using an anti-CaM-KII antibody and immunoblotted with specific antibodies as indicated. Ctr, immunoprecipitation with an unrelated antibody. b, Western blots of immunoprecipitated murine cardiac homogenates using an anti-Cav1.2 antibody either in the presence of 0.5 mM Ca2+ (+Ca2+) or under nominally calcium-free conditions (-Ca2+). c, reconstitution of CaM-KII-Cav1.2 association in HEK 293 cells. The Cav1.2 and Cavbeta2a subunits were expressed in HEK 293 cells stably expressing CaM-KII. Lysates were prepared, and the Cav1.2 was immunoprecipitated with an Cav1.2 antibody. d, the WT or mutant Cav1.2 (S1570A, Tr1728, and I1649E) subunits were coexpressed with the beta2a subunit in HEK 293 cells stably expressing CaM-KII. Membranes were prepared, and the Cav1.2 was immunoprecipitated with an Cav1.2 antibody. e, association of CaM-KII with the S1512/1570A Cav1.2 mutant. The WT or the S1512/1570A Cav1.2 mutant and beta2a subunits were expressed in HEK 293 cells stably expressing CaM-KII. Lysates were prepared, and the Cav1.2 was immunoprecipitated with an Cav1.2 antibody.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Numerous experiments have shown that the cardiac L-type ICa can be facilitated by a strong prepulse to positive membrane potentials or by Ca2+ released from intracellular stores (for a review, see Ref. 13). Initial experiments with an HEK 293 cell line stably expressing the Cav1.2 and the cardiac Cavbeta2a subunits of the L-type calcium channel in the presence or absence of the {alpha}2{delta} subunit did not result in voltage-dependent facilitation of ICa (Fig. 1b). Similar findings have been reported by other groups (13, 14). The failure to induce facilitation in HEK 293 cells was not caused by a lack of CaM, which is expressed in sufficient amounts, but was rather due to a deficiency of CaM-KII, which we were unable to detect in these cells with the antibodies used. In line with this view, facilitation was readily observed following coexpression of the Cav1.2 and the Cavbeta2a subunit with CaM-KII (Fig. 1b). Moreover, superfusion of the cells with the CaM-KII inhibitor KN-93 (0.5 µM) abolished ICa facilitation (Fig. 1b), whereas it was not affected by the inactive analogue KN-92 (1 µM). The CaM-KII specificity of the reaction was substantiated further by the finding that inclusion of the CaM-KII inhibitor peptide, autocamtide-2-related inhibitory peptide (10 µM), in the pipette solution suppressed ICa facilitation. As reported by Zühlke et al. for calcium-dependent facilitation (14, 15), voltage-dependent facilitation was abolished by mutation of the IQ motif to EQ in the Cav1.2 subunit (Fig. 1b). The relative small current was not unexpected (see also Ref. 36), because mutations in this region of Cav1.2 affect trafficking of the channel (37). The importance of an intact IQ motif for calcium-dependent inactivation of the channel has also been reported by several other groups (38-41). The depolarizing prepulse significantly inhibited ICa through the mutated channel (Fig. 1, b and c), supporting the notion that voltage-dependent inactivation is intact in the I1649E mutant. Similar results have been reported by others (16, 38). The lack of facilitation of the I1649E channel was not caused by an impairment of the interaction between the channel and the CaM-KII, because CaM-KII immunoprecipitated with the mutated channel (see Fig. 4d). Complex formation of CaM-KII with the channel outside of the IQ motif has been reported also by Hudmon et al. (42) during the processing of this manuscript.

The results above strongly support the hypothesis that CaM-KII is activated during the prepulse and that activated CaM-KII phosphorylates either the Cav1.2 or the Cavbeta2a subunit. To substantiate this hypothesis, we analyzed the sequence of both proteins for potential CaM-KII phosphorylation sites using the prediction algorithm of Refs. 43 and 44. Fifteen putative phosphorylation sites were identified in the Cav1.2 sequence (Fig. 2). Ser249, Thr713, and Thr1439 were not considered further, because it is rather unlikely that these residues are accessible from the intracellular space. The remaining sites were analyzed in a HEK 293 cell line stably expressing CaM-KII. Six of the sites were located carboxyl-terminally from amino acid 1728 (Fig. 2). Truncation of the Cav1.2 subunit at amino acid 1728 did not affect facilitation (Table 2), ruling out that these putative phosphorylation sites were involved in the facilitation process. In agreement with previous findings (12, 32, 45), the current voltage relation of the truncated channel was also not affected (Fig. 3a).


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  		TABLE 2
Summary of current density and facilitation obtained from each Cav1.2 mutant (see Fig. 2) co-transfected with wild type (betaWT) or mutant (betaT500A) Cavbeta The cell line expressing stably CaM-KII was used.

 
Further experiments were carried out using the truncated channel (Tr1728) as template. We investigated the role of serine residues Ser808, Ser888, Ser808/888, or Ser1695 by mutating them to alanine. ICa through the Cav1.2 subunit containing the mutation S808A, S888A, S808A/S888A, or S1695A still showed significant facilitation following the prepulse protocol (Table 2). ICa facilitation was also observed in HEK 293 cells coexpressing the truncated Cav1.2 subunit and the Cavbeta2a subunit with a mutation of its single putative phosphorylation site (T500A) (Table 2). In sharp contrast to these "silent mutations," mutations of either S1512A or S1570A almost completely abolished voltage-dependent facilitation of ICa (Table 2), suggesting their critical function for this process. The same result was obtained when both sites were mutated concurrently. Remarkably, the two serine residues involved in facilitation flank the EF-hand of the Cav1.2 subunit, a domain that has been implicated in the Ca2+-dependent inactivation of the Cav1.2 channel (46-48). It is noteworthy that the loss of facilitation by the mutation S1512A/S1570A was not accompanied by any alteration in the voltage dependence of the channel (Fig. 3, a and b) and of its voltage-dependent inactivation measured with IBa (Fig. 3, c and d). As expected, Ca2+-dependent inactivation was not abolished but was slightly increased by this mutation (Fig. 3, c and d).


Figure 5
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  		FIGURE 5.
CaM-KII phosphorylates Ser1512 and Ser1570. a and b, full-length Cav1.2 immunoprecipitated from mouse heart was incubated with exogenous CaM-KII and [{gamma}-32P]ATP. The proteins were separated on a 10% SDS gel, transferred to nitrocellulose, and Western blotted followed by autoradiography. The specificity of the CaM-KII phosphorylation was verified by a phosphorylation reaction in the absence of CaM and Ca2+. a, an autoradiograph of a whole gel demonstrating CaM-KII autophosphorylation and phosphorylation of the WT Cav1.2 protein. b, upper panels, region between 150 and 300 kDa cut from a whole gel as shown in a and exposed five times longer to facilitate visualization of the Cav1.2 signal without the very strong background of the autophosphorylated CaM-KII. Lower panels, Cav1.2 immunoblot demonstrating equivalent Cav1.2 input into the phosphorylation assay. c, identification of Ser1512 and Ser1570 as the CaM-KII phosphorylation sites on full-length Cav1.2 by alanine substitution (S1512A/S1570A). WT Cav1.2 and Cav1.2-S1512A/S1570A coexpressed with Cavbeta2a in HEK 293 cells were immunoprecipitated with anti-Cav1.2 and incubated with CaM-KII (250 units) and [{gamma}-32P]ATP. The proteins were separated on a 10% SDS gel and Western blotted followed by autoradiography. The specificity of the CaM-KII phosphorylation was verified by phosphorylation reactions in the absence of CaM and Ca2+. All blots are representative of three experiments. d, the GST-fused Cav1.2 WT and (S1512A/S1570A) COOH fragments 1492-1728 were phosphorylated by CaM-KII kinase in the presence of [{gamma}-32P]ATP. The reaction mixture was separated on SDS gels and autoradiographed (upper panel). CaM-KII phosphorylated the wild type but not the S1512A/S1570A mutated fragment. Lower panels, loading controls using an antibody against the GST fusion part. All blots are representative of three experiments.

 
CaM-KII-dependent facilitation is well described in cardiac myocytes (21-23). In rat ventricular myocytes, facilitation requires translocation of activated CaM-KII to the inner leaflet of the transverse tubule (26), probably as a prerequisite for a close association of the kinase with the channel protein. This prompted us to investigate a physical interaction between CaM-KII and the Cav1.2 calcium channel in cardiac myocytes and HEK 293 cells. Therefore, the full-length Cav1.2 channel was immunoprecipitated from murine heart membranes and HEK 293 cells. Western blots of the precipitated proteins showed that CaM-KII was co-immunoprecipitated with the Cav1.2 protein in the presence and absence of Ca2+ (Fig. 4, a-c). The same two proteins precipitated when an antibody against CaM-KII was used. As noted also by other groups (38, 49-52), coprecipitation of CaM with the full-length Cav1.2 channel protein was variable and not always detectable.

Based upon our electrophysiological data, the two serines Ser1512 and Ser1570 within the carboxyl terminus are expected to represent authentic CaM-KII phosphorylation sites of the Cav1.2 subunit. To substantiate this idea, we first sought to determine whether CaM-KII directly phosphorylates the full-length Cav1.2 calcium channel from cardiac tissue. In the presence of [32P]ATP, radioactive phosphate was incorporated into the Cav1.2 protein in a Ca2+/CaM-dependent manner (Fig. 5, a and b).

Having validated the specificity of our system to track kinase-dependent phosphorylation of Cav1.2, we next used it to examine the phosphorylation status of full-length recombinant WT and S1512A and S1570A Cav1.2 subunits expressed in HEK 293 cells (Fig. 5c). The extent of radioactivity associated with the full-length Cav1.2 protein was strongly reduced when the S1512A/S1570A mutant protein was used, demonstrating that these two serines in the carboxyl terminus of Cav1.2 are phosphorylated by CaM-KII. The mutations did not interfere with the interaction between the CaM-KII and the channel, as shown by the co-immunoprecipitation of both proteins (Fig. 4, d and e). These findings support the notion that Ser1512 and Ser1570 are not involved in association of CaM-KII with the calcium channel but are true phosphorylation sites.

We confirmed these findings utilizing wild type and S1512A/S1570A-mutated GST fusion proteins containing amino acids 1492-1728 of the Cav1.2 carboxyl terminus as substrates for in vitro CaM-KII phosphorylation. Whereas CaM-KII readily phosphorylated the wild type truncated carboxyl terminus, only very weak phosphorylation could be detected in the S1512A/S1570A mutant protein (Fig. 5d).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The cardiac Cav1.2 Ca2+ channel shows two properties mediated through an interaction of Ca2+/CaM with the carboxyl-terminal sequence of the channel protein (14, 39, 40): Ca2+-dependent inactivation and facilitation. Mechanisms supporting facilitation have not been established clearly, because the site(s) phosphorylated by CaM-KII remained unknown. The present findings strongly support the view (13, 23) that facilitation can be induced by CaM-KII-dependent phosphorylation of two amino acids flanking the EF-hand motif of the Cav1.2 channel protein.

Coimmunoprecipitation experiments with probes obtained from native tissue and HEK 293 cells suggest a close association of the kinase with the channel protein that may be functionally relevant. Phosphorylation was greatly reduced in Cav1.2 proteins with the amino acid residue Ser1512 and/or Ser1570 mutated to alanine. During the processing of this manuscript, CaM-KII-dependent phosphorylation of Ser1512 has been also reported by others (53), strengthening the finding of this study. Mutation of the two serines eliminated facilitation but did not reduce Ca2+-dependent inactivation of the Cav1.2 channel. This dissociation emphasizes that Ca2+-dependent facilitation and inactivation rely on distinct and independent structural mechanisms.

So far, only Ca2+-dependent inactivation has been observed for Cav1.2 channels expressed in HEK 293 cells (39). Our data suggest that this is due to the fact that HEK 293 cells do not express CaM-KII to an extent sufficient for supporting voltage-dependent facilitation. These findings fit well with data obtained in oocytes (14, 15) that express CaM-KII abundantly (54, 55).

The location of the two phosphorylated serines is intriguing, because the sequence between them includes an EF-hand motif. Initially, this EF-hand motif was implicated in the Ca2+-dependent regulation of the Cav1.2 channel (56), but later it was concluded that the EF-hand motif is not relevant for Ca2+-dependent inactivation (14, 15, 39, 40, 47, 57). A recent report concluded that the EF-hand interacts with CaM-KII and, thereby, mediates channel facilitation (42). The findings of the present report that mutation of Ser1512 and/or Ser1570 does slightly increase Ca2+-dependent inactivation but abolishes facilitation support the notion that the sequence flanking the EF-hand motif is required for the facilitation process. Facilitation also requires an intact IQ motif. Mutation of the IQ motif to EQ decreased 100-fold the affinity of the corresponding peptide for CaM (15), eliminated Ca2+-dependent facilitation (15), and eliminated CaM-KII dependent facilitation (see Fig. 1).

These results support the hypothesis that the IQ motif is of primary importance for Ca2+-dependent modulation of the Cav1.2 channel. However, the signaling mechanism leading to inactivation and facilitation involves different parts of the channel sequence. Apparently, facilitation needs a conformational new positioning of the EF-hand motif triggered by CaM-KII-dependent phosphorylation. Together with two reports (42, 53) published during the processing of this manuscript, these results identify the carboxyl-terminal region around the EF-hand as important for Ca2+-dependent regulation of ICa.


   FOOTNOTES
 
* This work was supported by grants from Deutsche Forschungsgemeinschaft and Fond der Chemischen Industrie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Present address: Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Universität Freiburg, Albertstr. 25, 79104 Freiburg, Germany. Back

2 To whom correspondence should be addressed: Institut für Pharmakologie und Toxikologie, TU München, Biedersteiner Str. 29, 80802 München, Germany. Tel.: 49-89-4140-3283; Fax: 49-89-4140-3261; E-mail: Welling{at}ipt.med.tu-muenchen.de.

3 The abbreviations used are: ICa, calcium current; CaM, calmodulin; CaM-KII, calmodulin kinase II; IBa, barium current; Tr1728, truncated Cav1.2 calcium channel; pF, picofarads; GST, glutathione S-transferase. Back


   ACKNOWLEDGMENTS
 
We thank U. Kremser, H. Lefrank, and S. Paparisto for expert technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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