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J. Biol. Chem., Vol. 279, Issue 13, 12456-12461, March 26, 2004

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Calcium Current in Rat Cardiomyocytes Is Modulated by the Carboxyl-terminal Ahnak Domain^*

Julio Alvarez¶, Jana Hamplova||, Annette Hohaus**, Ingo Morano**, Hannelore Haase**, and Guy Vassort

From the Physiopathologie Cardiovasculaire, INSERM U-390, CHU Arnaud de Villeneuve, F-34295 Montpellier Cedex 5, France, ^**Max Delbrück Center for Molecular Medicine (MDC), D-13125 Berlin, Germany, Institute of Cardiology, La Habana, Cuba, and Johannes Müller Institute for Physiology, Humboldt University (Charité), D-10117 Berlin, Germany

Received for publication, November 6, 2003 , and in revised form, January 5, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Ahnak, a protein of 5643 amino acids, interacts with the regulatory -subunit of cardiac calcium channels and with F-actin. Recently, we defined the binding sites among the protein partners in the carboxyl-terminal domain of ahnak. Here we further narrowed down the ₂-interaction sites to the carboxyl-terminal 188 amino acids of ahnak by the recombinant ahnak protein fragments P3 (amino acids 5456-5556) and P4 (amino acids 5556-5643). The effects of these P3 and P4 fragments on the calcium current were investigated under whole-cell patch clamp conditions on rat ventricular cardiomyocytes. P4 but not P3 increased significantly the current amplitude by 22.7 ± 5% without affecting its voltage dependence. The slow component of calcium current inactivation was slowed down by both P3 and P4, whereas only P3 slowed significantly the fast one. The composite recombinant protein fragment P3-P4 induced similar modifications to the ones induced by each of the ahnak fragments. In the presence of carboxyl-terminal ahnak protein fragments, isoprenaline induced a similar relative increase in current amplitude and shift in current kinetics. The actin-stabilizing agents, phalloidin and jasplakinolide, did not modify the effects of these ahnak protein fragments on calcium current in control conditions nor in the presence of isoprenaline. Hence, our results suggest that the functional effects of P3, P4, and P3-P4 on calcium current are mediated by targeting the ahnak-₂-subunit interaction rather than by targeting the ahnak-F-actin interaction. More specifically they suggest that binding of the ₂-subunit to the endogenous subsarcolemmal giant ahnak protein re-primes the _1C/₂-subunit interaction and that the ahnak-derived proteins relieve the ₂-subunit from this inhibition.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Ca²⁺ influx through the L-type Ca²⁺ channel (I_CaL) initiates and modulates cardiac contraction. L-type Ca²⁺ channels activate with membrane depolarization and inactivate over time. Inactivation is an important property in regulating action potential duration and intracellular Ca²⁺ transients. The Ca²⁺ channels are multimeric proteins, built up of at least three channel subunits, ₁, ₂/, and . The ₁-subunit serves as the channel pore and voltage sensor. The intracellular -subunit modifies the properties of the channel complex both by chaperoning the translocation of the ₁-subunit to the plasma membrane and by allosteric modulation of the ₁-subunit function. In cardiomyocytes, the subunit isoforms _1C (Ca_v1.2) and ₂ constitute the channel complex (reviewed in Ref. 1).

The most potent mechanism to enhance myocardial contractility occurs via sympathoadrenergic stimulation (2). Activation of the -adrenergic receptor results in an increase in peak inward current and a slowing of inactivation of (I_CaL) via protein kinase A (PKA)¹-dependent phosphorylation of the channel subunits _1C (3-5) and ₂ (6-8). In an attempt to define the molecular details of channel subunit phosphorylation, we have identified the 700-kDa ahnak protein as the prominent PKA target in mammalian cardiomyocytes that is recovered in anti-₂-subunit immunoprecipitates (9).

Ahnak, a protein of 5643 amino acids, has been implicated in different cell type-specific functions as diverse as cell differentiation (10, 11), signal transduction (9, 12-14), Ca²⁺ homeostasis (15), and regulated exocytosis (16). The ahnak protein can be divided into three regions: unique amino-terminal and carboxyl-terminal portions flank a large central region with multiple repeated units (10, 17). The carboxyl-terminal ahnak domain encompassing 1002 amino acid residues contains both nuclear localization signals and nuclear export signals that are believed to determine the subcellular distribution of ahnak (14, 18). We next characterized ahnak in normal human myocardium as a peripheral membrane protein associated with the cytoplasmic side of the sarcolemma including T-tubular structures (19). By using truncated ahnak fragments, we demonstrated that the high affinity interaction (K_d 50 nM) between ₂-subunit and ahnak is mediated by the most carboxyl-terminal 382 amino acids of ahnak, designated as the ahnak-C2 domain. This ahnak-C2 domain was also defined to be responsible for F-actin binding (19). Together, the localization and interaction partner suggest a role of cardiac ahnak in the regulation of Ca²⁺ channel activity either directly via ₂-subunit interaction or indirectly via F-actin interaction. In fact, recent studies (20-22) revealed the cytoskeletal actin filament organization as an important regulator of the Ca²⁺ channel inward current.

The goal of the present study was to elucidate whether ahnak affects Ca²⁺ channel gating properties. Small ahnak fragments were expressed encompassing the 188 carboxyl-terminal amino acid residues of human ahnak (P3, aa 5456-5556, and P4, aa 5556-5643); their effects on Ca²⁺ current, I_CaL, were investigated under whole-cell patch clamp conditions on ventricular rat cardiomyocytes. The rationale for this approach was that we narrowed down the interaction sites between ahnak and ₂-subunit to the composite ahnak protein fragment, P3-P4. Here we demonstrate for the first time that carboxyl-terminal ahnak-derived fragments modulate specific aspects of I_CaL gating such as an increase in I_CaL amplitude and a slowing of inactivation. Our results suggest a role of cardiac ahnak as an intrinsic regulatory protein of the L-type Ca²⁺ channel complex.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—Plasmid DNA of human ahnak was kindly provided by Dr. Emma Shtivelman (University San Francisco). The ahnak-C2 fragment encompassing the carboxyl-terminal amino acids 5262-5463 was constructed from the plasmid z7 (10) and was expressed as GST fusion protein as described (19). The construct for the Ca²⁺ channel ₂-subunit was prepared from the expression plasmid pcDNA_2a encoding the rabbit _2a (23) kindly provided by Dr. Franz Hofmann (Technical University, München, Germany). The _2a-subunit was expressed as GST fusion protein as described previously (19). The antibody against the ₂-subunit was characterized previously (9, 24).

Recombinant Ahnak Fragments—The carboxyl-terminal ahnak fragments were generated by PCR using z7 as template and the following primers: P1-P2, forward, 5'-acgtgaattctgtgatgtgaacctgccag-3', and reverse, 5'-gatctcgagcagtttgacttcagactc-3'; P2-P3, forward, 5'-acgtgaattcgacactctaagtttg-3', and reverse, 5'-gatctcgagaccttcaaactccagcgt-3'; P3-P4, forward, 5'-acgtgaattctctgaagtcaaactg-3', and reverse, 5'-gatctcgagctactctttctttgt-3'; P3, forward, 5'-acgtgaattctctgaagtcaaactg-3', and reverse, 5'-gatctcgagaccttcaaactccagcgt-3'; P4, forward, 5'-acgtgaattcggtggggaagtgtc-3', and reverse, 5'-gatctcgagctactctttctttct-3'. EcoRI-XhoI restriction sites were incorporated into primers (underlined letters) to facilitate subsequent subcloning to EcoRI-NotI sites of the expression vector pGEX-4T1. All constructs were checked by restriction site mapping and sequencing. Expression of the GST-ahnak fragments was performed in BL21-Codon Plus (DE3)-RIL cells (Stratagene). The bacteria were induced with 0.1 mM isopropyl-1-thio--D-galactopyranoside for 3-4 h at 37 °C. The cells were collected, and the GST-ahnak fragments were solubilized and purified on glutathione-Sepharose beads as described (19). The protein concentration was determined according to Bradford. For electrophysiological experiments, buffer constituents were removed from the ahnak fragments by gel filtration on Sephadex G-25 columns (PD-10, Amersham Biosciences), and the fragments were lyophilized.

Immunocapture Assay—Wells of microtiter plates (MaxiSorp, Nunc) were coated with GST-ahnak-C2 by incubating with 100 µl of a 10 µg/ml protein solution (total of 14 pmol ahnak-C2) in 50 mM NaHCO₃ buffer, pH 9.6, at 4 °C overnight. The wells were blocked by incubation with 250 µl of buffer A (phosphate-buffered saline, 0.1% Tween 20) containing 1% bovine serum albumin for 40 min and washed three times with buffer A. Incubation with the recombinant ₂-subunit (14 pmol) was performed in a total volume of 100 µl of buffer A containing 1% bovine serum albumin in the absence or presence of the inhibitors (ahnak-C2, P1-P4) at room temperature for 1 h. Wells were subsequently washed twice with 200 µl of buffer A. Bound ₂-subunit was monitored by immunoreaction with our ₂-subunit-specific antibody (0.5 µg IgG/ml) in combination with the second peroxidase-coupled anti-rabbit IgG. Quantification was done by measuring the optical density after development with o-phenylenediamine as substrate of the peroxidase. For the competition experiments, a 10-fold molar excess of ahnak-C2 or its truncation mutants, P1-P2, P3-P4, P3, or P4 was incubated with the _2a-subunit for 60 min at 4 °C prior to the incubation on wells captured by ahnak-C2.

Isolation of Adult Ventricular Cardiomyocytes—Ventricular myocytes were isolated enzymatically from male Wistar rats (200-300 g) as described previously (25). Briefly, excised hearts were perfused retrogradely at a constant flow of 6 ml min^-1 with a nominally Ca²⁺-free Hepes solution (composed of (mM): 137 NaCl, 5.7 KCl, 1.5 KH₂PO₄, 4.4 NaHCO₃, 1.7 MgCl₂, 21 Hepes, 20 taurine, 10 glucose, pH 7.15, at 35-36 °C) containing collagenase (Worthington type II, 60 IU ml^-1). The freshly dissociated cells were kept in the physiological solution with 0.3 mM Ca²⁺ and 0.5% bovine serum albumin at room temperature (23-24 °C) and used within 6-8 h. In an experimental series, cells were incubated in phalloidin (100 µM) for 5 h in this solution before attempting the patch clamp.

Patch Clamp Recordings—For recording the L-type Ca²⁺ current (I_CaL), the "whole-cell" variant of the patch clamp method was used (25, 26) at room temperature (22 ± 2 °C). Electrode resistance was 0.9-1.1 megohms. K⁺ currents were blocked by Cs⁺ (intracellular and extracellular; see below). The fast inward Na⁺ current was blocked with 50 µM tetrodotoxin. The composition of the standard extracellular solution was (mM): 117 NaCl, 20 CsCl, 2 CaCl₂, 1.8 MgCl₂, 10 glucose, 10 Hepes, pH 7.4. The pipette ("intracellular") solution contained (mM): 130 CsCl, 0.4 Na₂-GTP, 5 Na₂-ATP, 5 Na₂-creatine phosphate, 11 EGTA, 4.7 CaCl₂ (free Ca²⁺ 108 nM); 10 Hepes; pH was adjusted to 7.2 with CsOH. In most cases, the alterations of the Ca²⁺ current were analyzed by comparing the quantity of charges entering the cell during a 200-ms depolarizing pulse to 0 mV after scaling to cell capacitance.

Statistical Evaluation—When possible, results were analyzed by the Student's paired t test and are expressed as means ± S.D. The criterion for significance was p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Dissection of Carboxyl-terminal Ahnak-binding Sites to the Ca²⁺ Channel ₂-Subunit—In a recent study, we demonstrated that the carboxyl-terminal 382 amino acids of ahnak, designated as ahnak-C2, are important for the high affinity interaction with the Ca²⁺ channel ₂-subunit (19). To further narrow down the interaction sites, the ahnak-C2 deletion mutants depicted in Fig. 1A were expressed as GST fusion proteins: P1-P2 (aa 5255-5456), P3-P4 (aa 5456-5643), P3 (aa 5456-5556), and P4 (aa 5556-5643). These recombinant proteins showed the expected molecular masses (Fig. 1B).

View larger version (18K): [in this window] [in a new window]   FIG. 1. Molecular structure of ahnak and dissection of carboxyl-terminal binding sites to the Ca2+ channel 2-subunit. A, scheme of ahnak indicating the molecular structure and the location of the carboxyl-terminal fragments P1-P2, P3-P4, P3, and P4. B, recombinant proteins used in the present study. The proteins (5 µg) were analyzed by SDS-PAGE and Coomassie Blue staining. C, competition assay for Ca2+ channel 2-subunit binding. The competitors are truncation mutants of ahnak-C2 shown in A. The optical densities measured for bound 2-subunit in the absence of competitors were 0.513 ± 0.011 (X ± S.E., n = 3). Results of a representative experiment are shown out of three similar experiments.

Effects of Ahnak Fragments on Ca²⁺ Current—Cardiac myocytes were investigated under whole-cell patch clamp conditions using a pipette solution that was added with 10 µM of one of the short carboxyl-terminal sequences of ahnak P3, P4, or P3-P4, or with GST as a control, such a concentration was far above the apparent K_d of 50 nM for binding to ahnak-C2 domain. The intracellular perfusion of a cell with a pipette solution that contained the combined P3-P4 ahnak protein fragment induced a slowly developing increase in peak I_CaL elicited at 0 mV and reached a maximum within about 3-4 min, a time needed for diffusion of the internal pipette solution. I_CaL inactivation kinetics were simultaneously modified showing an increase in both _f and _s, the fast and slow time constants that described the two components of I_CaL inactivation (Fig. 2A). The results of similar experiments with adding 10 µM GST, P3, P4, or P3-P4 ahnak protein fragments are summarized in Fig. 2B (see also Table I). The addition of GST had no effect on I_CaL amplitude and kinetics. Thus in some cases, data are pooled results of about the same number of cells investigated in control conditions or with GST-added pipette solution. Added at 10 µM, P4 had a significant positive effect on I_CaL amplitude that was increased by 22.7 ± 5.0% from a control value of 13.2 ± 1.1 pA/pF (n = 23, 16; p < 0.01). The combined ahnak fragment P3-P4 also significantly increased peak I_CaL by 34.8 ± 7.1%, whereas fragment P3 had no significant effect. All fragments significantly slowed down both the fast and slow components of I_CaL inactivation except P4 that did not significantly alter the fast component of inactivation (Table I). Altogether, in the presence of one of the ahnak protein fragments, the increase in current amplitude or slowing of its inactivation led to a 50% increase in the amount of charges carried by Ca²⁺ (see also Fig. 3).

View larger version (19K): [in this window] [in a new window]   FIG. 2. Carboxyl-terminal ahnak fragments increased Ca2+ current and slowed its kinetics. A, time course of peak ICaL and f and s, the time constant of the fast and slow components of inactivation shown here following a 1-min delay after breaking the patch with a pipette that contained the P3-P4 ahnak protein fragment. Stimulation frequency was 0.125 Hz. Inset, typical recordings of the Ca2+ current elicited at 0 mV at the indicated time. B, peak ICaL densities elicited at 0 mV depolarization in control conditions and with a pipette solution added with 10 µM GST, P3, P4, or P3-P4 protein fragment. *, p < 0.05 compared with control.

View this table: [in this window] [in a new window]   TABLE I Effects of the carboxyl-terminal fragments of ahnak on the amplitude and kinetics of the L-type Ca;2+ current in rat ventricular cardiomyocytes The L-type Ca2+ current, ICaL (pA/pF), was elicited by depolarization to 0 mV from a - 80-mV holding potential. f and s are the fast and slow time constants (ms) of Ca2+ current inactivation, and Af and As are their respective maximal initial amplitudes (pA/pF).

View larger version (19K): [in this window] [in a new window]   FIG. 3. -Adrenergic stimulation of ICaL in the presence of carboxyl-terminal ahnak fragments. The -adrenergic stimulatory effect on ICaL induced by ISO (1 µM) was not altered by the carboxyl-terminal ahnak protein fragments, all added at 10 µM to the pipette solution. Note that the changes in ICaL are expressed as the quantity of charges entering the cell relative to its capacitance. * and **, p <0.05 with respect to control cells under control or ISO conditions, respectively. Inset, typical ICaL recordings with 10 µM GST or P4 fragment before and after ISO application.

The actin-stabilizing agents, a 5-h incubation with 100-µM phalloidin as well as the acute application of 10 µM jasplakinolide did not modify the effects of the two P3 and P4 fragments on I_CaL amplitude or inactivation kinetics, upon a depolarization at 0 mV in control conditions as well as in the presence of isoprenaline (not shown).

Effects of Ahnak Protein Fragments on Voltage-dependent Ca²⁺ Current Facilitation—High voltage pre-depolarizing steps are inducing a partial recovery from inactivation or voltage-dependent facilitation of I_CaL that is accompanied by a slowing of I_CaL inactivation kinetics. I_CaL voltage-dependent facilitation had similar relative amplitude in each experimental condition with pipette solution containing 10 µM GST, P3, P4, or P3-P4 fragment. Voltage-dependent facilitation was not affected by ISO (not shown). The changes in current inactivation kinetics were further analyzed following a depolarizing prepulse to +60 mV (Fig. 4). The depolarizing prepulse induced an increase in both _f and _s characterizing current inactivation during the test pulse to 0 mV. Nevertheless, _s was further increased in the presence of each ahnak fragment, and the effect was slightly more marked in the presence of isoprenaline.

View larger version (21K): [in this window] [in a new window]   FIG. 4. Voltage-dependent facilitation of ICaL in the presence of carboxyl-terminal ahnak fragments. The voltage-dependent facilitation of ICaL was altered by the carboxyl-terminal ahnak fragments. Bar graph shows that P3, P4, and P3-P4 peptides increased f and s, the time constant of the fast and slow inactivation components of ICaL recorded at 0 mV after a depolarizing pre-pulse to +60 mV. These effects were similar after -adrenergic stimulation (ISO, 1 µM). n = 12, 8, 11, 12, and 4 in control conditions or in the presence of 10 µM GST, P3, P4, or P3-P4 fragment. * and **, p < 0.01 in the absence or presence of ISO, respectively.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
This study demonstrates that carboxyl-terminal ahnak protein fragments modulate Ca²⁺ channel properties. Reversible binding between the protein fragments and the cardiac ₂-subunit suggests this interaction to be responsible for Ca²⁺ channel modulation. Carboxyl-terminal fragment addition to the pipette solution induces an increase in I_CaL amplitude and a slowing of I_CaL inactivation. Furthermore, voltage-dependent but not use-dependent I_CaL facilitation is affected. None of the isoprenaline effects on Ca²⁺ current are affected. Taking into account that the presence of the ₂-subunit increases I_CaL, these results suggest a stronger interaction of endogenous ₁- and _2-Ca²⁺ channel subunits occurs in the presence of carboxyl-terminal ahnak protein fragments by preventing the ahnak-₂-subunit binding. The results are thus consistent with the hypothesis that endogenous ahnak exerts a sustained inhibitory effect on the cardiac Ca²⁺ channel by binding to the ₂-subunit.

-Subunits induce generic as well as specific modifications in ₁-subunit behavior. Increased channel expression, activity, and membrane targeting of the ₁-subunit are common modifications induced by all -subunits, whereas changes in the voltage dependence and kinetics of activation and inactivation are more specific of a given pair of ₁-/-subunits. These effects appear to be mediated by high affinity interactions between the AID (-interaction domain; located on the loop connecting domains I and II of each ₁-subunit) (28) and the BID (-interaction domain; located on the beginning of the second conserved region of the -subunit) (29). Additional interaction sites of lower affinity (on the amino- and carboxyl-terminal domains of the -subunits and ₁-subunits) may also contribute to these regulations (30-33). Previously, we proposed potential regulation of Ca²⁺ channel activity by the interaction of the ₂-subunit with ahnak, a 700-kDa PKA substrate (9) originally identified as neuroblast differentiation-associated protein (10). The most carboxyl-terminal 382 amino acids of ahnak, designated as the ahnak-C2 domain, were identified to mediate high affinity (K_d 50 nM) ₂-subunit binding that do not interfere with _1C-₂-subunit interaction mediated by the AID (19). The first part of this work more precisely determines the interaction of carboxyl-terminal ahnak with the ₂-subunit by using the amino- and carboxyl-terminal truncation mutants of ahnak-C2, P1-P2, and P3-P4, respectively. In vitro binding experiments revealed that P3-P4 fully accounts for the reversible ₂-subunit interaction observed with ahnak-C2. Because P3 and P4 alone showed partial ₂-subunit binding, we propose the existence of two binding sites within the 188 carboxyl-terminal amino acid residues of ahnak.

The increase in Ca²⁺ current amplitude induced by applying the carboxyl-terminal fragments indicates changes in channel gating. This observation could be accounted for by suggesting that -subunits are made more available because co-expression of the -subunit with the ₁-subunit enhances charge movement (34-36). -Subunits are also known to modulate Ca²⁺ channel kinetics. Ca²⁺ channels are capable of undergoing three different types of inactivation processes: Ca²⁺-dependent inactivation, fast voltage-dependent inactivation, and slow inactivation (37). Increasing the depolarization voltage progressively replaced Ca²⁺-dependent inactivation in the fast phase of the decay of the Ca²⁺ current with rapid voltage-dependent inactivation. Ba²⁺ current, through the L-type Ca²⁺ channel, inactivates essentially by voltage-dependent mechanisms with fast and slow kinetics. Furthermore, the fractional inhibition of slow inactivation in mutants causes an acceleration of fast inactivation suggesting that fast and slow inactivation mechanisms are linked. Two major mechanisms have been implicated in voltage-dependent inactivation. The ball-and-chain mechanism or hinged-lid of an ion pore implies the occlusion by a positively charged segment that could be the amino terminus in K⁺ channel or, in Ca²⁺ channel, the I-II linker region that docks to a site comprising at least the domain II and III of S6 segments (38). The second C-type mechanism of slower K⁺ channel inactivation was found to involve a constriction of the pore by S6 segments (39). Recent results suggest that the slow inactivation of _1C-channel is mediated by an annular determinant composed of amino acid residues situated in the cytoplasmic ends of transmembrane segments S6 in repeats I-IV (40). This slow phase was not investigated in this study that uses depolarizing pulses in the hundreds millisecond range. Voltage- and Ca²⁺-dependent inactivation are intrinsic properties of the ₁-subunit. It was proposed that voltage and Ca²⁺ inactivate by using a "ball-and-chain" mechanism with blocking particles and binding sites encoded by homologous sequences therefore sensitive to the same molecular interactions with the ₂-subunit, with binding ensuring channel inactivation. The I-II loop represents an attractive candidate (38, 41, 42). The three ahnak protein fragments that are shown in this work to bind with the ₂-subunit induce an increase in I_CaL amplitude as well as a slowing of both components of I_CaL inactivation. Note, however, that these effects are not directly correlated because a lesser increase in peak I_CaL is observed when adding fragment P3 that also induces a more marked slowing of the fast inactivation component than P4.

Analysis of voltage-dependent activation shows a hyperpolarizing shift when co-expressing any ₁-subunit with any -subunit; such a -subunit-induced shift also occurs for inactivation except with ₂-subunit (33, 43). The ahnak fragment-induced shift in activation kinetics as well as their slowing of both inactivation phases (Table I) are consistent with the fact that these protein fragments favor the interaction of the -subunit with the ₁-subunit. The leftward shift of the activation and availability parameters induced -adrenergic stimulation were unaffected by the ahnak fragments. Furthermore, the fact that the changes in Ca²⁺ current kinetics induced by each ahnak fragment are additive to the changes induced by isoprenaline indicates that the two effects involve independent mechanisms. Multiple molecular mechanisms have been suggested that underlie facilitation of L-type Ca²⁺ channels. In cardiac myocytes, where the _1C-subunit is predominantly expressed, a Ca²⁺-dependent facilitation and a voltage-dependent (Ca²⁺-independent) facilitation have been described (25, 44, 45). Ca²⁺-dependent facilitation is attributed to activation of Ca²⁺-calmodulin protein kinase II that phosphorylates the Ca²⁺ channel (46, 47). The voltage-dependent facilitation has been suggested to be due to cAMP-dependent phosphorylation of the channel (48) or a voltage-dependent conformational switch of the channel protein complex leading to altered gating (44). Many neuronal non-L-type Ca²⁺ channels exhibit apparent voltage-dependent facilitation as a result of G-protein-mediated inhibition of the channels (49). Conversely, voltage-dependent facilitation of the rabbit cardiac _1C-subunit expressed in HEK-293 demonstrates a rapid onset that is independent of PKA phosphorylation and G-protein modulation; however, it requires the co-expression of an auxiliary -subunit (50). -Subunits all contain a sequence, located in the second conserved domain, that is responsible for the promotion of current facilitation; besides, another sequence of 16 amino acids, located on the amino-terminal tail of the ₂-subunit, induces a block of L-type Ca²⁺ current facilitation (31). Voltage-dependent facilitation following high pre-depolarizing pulses occurs with slower Ca²⁺ current inactivation phases. The further slowing of both phases in the presence of the three ahnak fragments is also consistent with a facilitated binding of the ₂-subunit to the _1C-subunit. On the other hand, these carboxyl-terminal ahnak fragments do not significantly modify use-dependent facilitation that is more related to Ca²⁺ ions. Under both experimental conditions there was no significant effect of the -adrenergic stimulation.

In summary, the recombinant C2-terminal ahnak protein fragments P3, P4, and P3-P4 bind to the ₂-subunit of the Ca²⁺ channel. Their addition to the pipette solution induces an increase in peak I_CaL and a slowing of its inactivation that are attributable to a stronger interaction of the _1C- and ₂-subunits. These effects are best accounted for by the fact that these ahnak fragments compete with the whole ahnak to bind to ₂-subunits. It can thus be considered that the _1C/₂-subunit interaction is under the constant repriming control of ahnak that will potentially reduce peak I_CaL and fasten its inactivation. Although ₂-subunits are phosphorylated under -adrenergic stimulation, the effects of these C2-terminal ahnak fragments are not affected.

	FOOTNOTES

 
^* This work was supported in part by Region Languedoc-Roussillon, Association Française Contre les Myopathies, Fondation de France, and Deutsche Forschungsgemeinschaft Grant Ha 1779/4-1 and 1779/-2. 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.

^¶ Visiting scientist at INSERM U-390.

^|| Supported by an INSERM "Programme Orange."

To whom correspondence should be addressed. Tel.: 33-4-67-41-52-41; Fax: 33-4-67-41-52-42; E-mail: vassort{at}montp.inserm.fr.

¹ The abbreviations used are: PKA, protein kinase A; ISO, isoprenaline; aa, amino acids; GST, glutathione S-transferase.

	ACKNOWLEDGMENTS

 
We thank Loipa Galan (La Habana, Cuba) for help during the course of some the experiments. The technical assistance of Andrea Bartsch and Christl Kemsies is greatly acknowledged.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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