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(The FASEB Journal. 2005;19:1969-1977.)
© 2005 FASEB

Ahnak is critical for cardiac Ca(v)1.2 calcium channel function and its ß-adrenergic regulation

Hannelore Haase*,1, Julio Alvarez{dagger}, Daria Petzhold*, Anke Doller{ddagger}, Joachim Behlke*, Jeanette Erdmann§, Roland Hetzer||, Vera Regitz-Zagrosek{ddagger},||, Guy Vassort and Ingo Morano*,{dagger}{dagger}

* Max Delbrück Center for Molecular Medicine (MDC), D-13092 Berlin, Germany;
{dagger} Institute of Cardiology, La Habana, Cuba;
{ddagger} Center for Cardiovascular Research, University Medicine Charité, Berlin, Germany;
§ Medizinische Klinik II, University UK-SH, Campus Lübeck, Germany;
|| Deutsches Herzzentrum, Berlin, Germany;
Physiopathologie Cardiovasculaire, INSERM U-637, Montpellier Cedex 5, France; and
{dagger}{dagger} Johannes Müller Institute for Physiology, University Medicine Charité, Berlin, Germany

1Correspondence: Robert-Rössle-Str. 10, Berlin D-13092, Germany. E-mail: haase{at}mdc-berlin.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Defective L-type Ca2+ channel (ICaL) regulation is one major cause for contractile dysfunction in the heart. The ICaL is enhanced by sympathetic nervous stimulation: via the activation of ß-adrenergic receptors, PKA phosphorylates the {alpha}1C(CaV1.2)- and ß2-channel subunits and ahnak, an associated 5643-amino acid (aa) protein. In this study, we examined the role of a naturally occurring, genetic variant Ile5236Thr-ahnak on ICaL. Binding experiments with ahnak fragments (wild-type, Ile5236Thr mutated) and patch clamp recordings revealed that Ile5236Thr-ahnak critically affected both ß2 subunit interaction and ICaL regulation. Binding affinity between ahnak-C1 (aa 4646-5288) and ß2 subunit decreased by {approx}50% after PKA phosphorylation or in the presence of Ile5236Thr-ahnak peptide. On native cardiomyocytes, intracellular application of this mutated ahnak peptide mimicked the PKA-effects on ICaL increasing the amplitude by {approx}60% and slowing its inactivation together with a leftward shift of its voltage dependency. Both mutated Ile5236Thr-peptide and Ile5236Thr-fragment (aa 5215-5288) prevented specifically the further up-regulation of ICaL by isoprenaline. Hence, we suggest the ahnak-C1 domain serves as physiological brake on ICaL. Relief from this inhibition is proposed as common pathway used by sympathetic signaling and Ile5236Thr-ahnak fragments to increase ICaL. This genetic ahnak variant might cause individual differences in ICaL regulation upon physiological challenges or therapeutic interventions.—Haase, H., Alvarez, J., Petzhold, D., Doller, A., Behlke, J., Erdmann, J., Hetzer, R., Regitz-Zagrosek, V., Vassort, G., Morano, I. Ahnak is critical for cardiac Ca(v)1.2 calcium channel function and its ß-adrenergic regulation.


Key Words: recombinant protein • missense variant • ß2 subunit • binding affinity


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
L-TYPE CA2+CHANNELS are multisubunit, transmembrane proteins controlling Ca2+ influx into cells and play a pivotal role in the physiological regulation of the cardiac, vascular, endocrine and central nervous system. Alterations in the density or function of L-type Ca2+ channels have been implicated in a variety of diseases, including atrial fibrillation (1 , 2) , ventricular hypertrophy (3) , and heart failure (4) . In the heart, the L-type Ca2+ channel is composed of the pore-forming {alpha}1C subunit (Cav1.2a) and the auxiliary subunits {alpha}2{delta} and ß2 (reviewed in ref 5 ). A crucial signaling pathway that regulates the heart beat is the sympathetic stimulation of Ca2+ channel activity, which increases the amplitude of the Ca2+ inward current (ICaL), leads to a leftward shift in current-voltage relationship and a slowing of channel inactivation (reviewed in ref 6 ). These effects are believed to result from PKA-mediated phosphorylation of the channel subunits {alpha}1C (7 , 8) and ß2 (9 , 10) or still undefined associated proteins (11) . In a previous attempt to define the molecular details of Ca2+ channel phosphorylation in response to the sympathetic agonist, isoprenaline, we identified ahnak, a 700 kDa PKA substrate (5643 aa) as Ca2+ channel-associated protein (12) .

Ahnak has been implicated in essential biological functions such as cell differentiation (13 , 14) , organization of the cell membrane cytoarchitecture (15 , 16) and diverse signal transduction processes (17 18 19) . In the myocardium, ahnak was localized to the cytoplasmic side of the sarcolemma including transverse tubules (20 , 21) . Ahnak interacts with the intracellularly located, Ca2+ channel ß2 subunit via multipoint attachment mediated by ahnak's carboxyl-terminal domains, ahnak-C1 (aa 4646-5288) and ahnak-C2 (aa 5262-5643) (20) . Thus, cellular topology, interaction with the Ca2+ channel ß2 subunit, and post-translational modification by PKA phosphorylation suggested a key role of ahnak in the regulation of Ca2+ channel activity. In fact, recent patch-clamp experiments on rat ventricular cardiomyocytes showed that targeting the high-affinity ahnak-C2/ß2 subunit interaction by a peptide competition approach leads to an increase in the Ca2+ current amplitude and a slowing of channel inactivation (22) . Taking into account that the presence of the ß2 subunit increases ICaL (reviewed in ref 5 ), the results suggested that endogenous ahnak exerts a sustained inhibitory effect on the Ca2+ channel by ß2 subunit binding via the ahnak-C2 domain. Since none of the isoprenaline effects on ICaL were affected in this competition approach, the ahnak-C2 domain is apparently not important for the sympathetic signaling pathway (22) .

Herein, we describe that the interaction between ahnak-C1 and ß2 subunit plays a critical role for the sympathetic regulation of L-type Ca2+ channel activity. Since altered Ca2+ channel function with blunted ß-adrenergic responsiveness is observed in hypertrophied cardiomyocytes of animal models and in human syndrome (3 , 4) , we initially screened a patient cohort with hypertrophic cardiomyopathy (HCM) in order to identify naturally occurring, genetic ahnak variants. The identification of the coding genetic variant Ile5236Thr-ahnak led us to study potential effects of this mutation on ß2 subunit binding and Ca2+ channel function. We found that Ile5236Thr ahnak interfered with the classic ß-adrenergic regulation of ICaL that triggers the fight-or-flight response of the heart.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Study subjects and genetic analysis
For systematic mutation screening, 96 consecutive, unrelated patients presenting in the German Heart Center, Berlin, from 1992–2001 with the clinical diagnosis of hypertrophic cardiomyopathy (HCM) according to internationally recognized echocardiographic and clinical criteria (23) were used. Family members of heterozygotes were invited to undergo genetic analysis and clinical investigation. DNA isolation and SSCP analysis was carried out as described (23) . A total of 7 overlapping fragments encoding the carboxyl-terminal 440 amino acids of human ahnak were analyzed. All primer sequences are available on request from the authors. The mutation was confirmed in DNA samples by restriction-fragment polymorphism (RFLP) analysis with AlwNI. To determine allelic frequencies of the detected variant in other cohorts, RFLP analysis with AlwNI was done in 96 patients with dilated cardiomyopathy (DCM) presenting in the transplant unit of the German Heart Center 1992–2001 and in 96 healthy, unrelated control individuals recruited from the blood bank of the Institute for Haematology/Oncology, Charité, Berlin, without a history or clinical signs of cardiac disease. DCM was diagnosed by clinical and echocardiographic criteria after exclusion of coronary artery disease, valvular heart disease, and HCM. The study was approved by the ethical committee of the Charite, Humboldt University Berlin, and written informed consent was obtained from all patients.

Peptides and recombinant proteins
The synthetic peptides corresponding to amino acid position 5231-42 of ahnak or to the {alpha}1C interaction domain described previously (24) were purchased from Biosyntan GmbH (Berlin-Buch, Germany). Plasmid DNA of human ahnak (13) was kindly provided by Dr. Emma Shtivelman (UCLA, San Francisco) and the expression plasmid for the rabbit cardiac ß2a subunit (25) , was generously supplied by Dr. Franz Hofmann (TU München, Germany). The recombinant proteins, ahnak-C1 and ß2 subunit were expressed as GST fusion proteins as described (20) . The carboxyl-terminal ahnak-C1 fragment, ahnak-C1/C (aa 5215-5288) was expressed as HIS-tagged protein in pRSET A (Invitrogen, San Diego, CA, USA). The Ile5236Thr mutation was introduced into ahnak-C1 and ahnak-C1/C by using the QuickChange site-directed mutagenesis kit (Stratagene, San Diego, CA, USA). Phosphorylation of the proteins was done as in ref 20 using 0.5 µM purified catalytic subunit of PKA.

Overlay binding assays
The purified fusion proteins, GST-ß2 and GST-ahnak-C1, as well as unfused GST (2.5–10 µg) were separated by SDS-PAGE on 10% polyacrylamide gels and blotted onto nitrocellulose membranes. The blots were incubated for 1 h in blocking solution containing 5% bovine serum albumin, 0.5% nonfat dry milk in PBS, pH 7.5. Blocked membranes were washed and incubated with 3–100 µM of N-terminally biotinylated ahnak peptides either wild-type (GGLPGIGVQGLE) or mutated (GGLPGTGVQGLE), or with biotinylated {alpha}1C interaction peptide (24) for 2 h at room temperature. After washing steps, the membranes were incubated for 30 min with 2 µg/mL horseradish peroxidase-linked avidin. Reactive bands were visualized using the ECL detection system.

Analytical ultracentrifugation
Molecular mass studies on dissolved ahnak-C1 and ß2 subunit and were performed in a XL-A type analytical ultracentrifuge (Beckman, Palo Alto, CA, USA) equipped with UV absorbance optics. Sedimentation equilibrium experiments were analyzed using externally loaded six-channel cells with 12 mm optical path length and the capacity to handle three solvent solution pairs of ~70 µL liquid. Sedimentation equilibrium was reached after 2 h of overspeed at 16,000 rpm, followed by an equilibrium speed of 12,000 rpm for ~30 h at 10°C. Depending on the loading concentration the radial absorbance in each compartment was recorded at three different wavelengths between 240 and 295 nm using the molar absorbance coefficients. Molecular mass calculations employed the global fit of three radial distribution curves described by

using our program POLYMOLE (26) . In these equations, Ar is the radial absorbance, Arm is the corresponding absorbance at meniscus position, {rho} is the solvent density, -{nu} is the partial specific volume, {omega} is the angular velocity, R is the gas constant, and T is the absolute temperature, M is the molecular mass, and rm is the radius at meniscus point. Determination of molecular masses and analyzing absorbance profiles at three different wavelengths allowed the estimation of the partial concentration (ci) of complexes and free reactants. Dissociation constants and stoichiometry for the reacting components were derived by fitting the sum of exponential functions (considering molecular mass, loading concentration and extinction coefficient of the reactants) to the experimentally obtained radial distributions as described in detail in ref 26 . By using the discrete molecular weights (Mi) and the partial concentrations (ci) of each component the weight average molecular mass (Mw) was determined according to Mw = ({Sigma}ci · Mi)/{Sigma}ci.

The latter equation was applied to describe ß2 subunit interaction with ahnak-C1/C.

Electrophysiological measurements
Ventricular myocytes were isolated from adult male Wistar rats (200–300 g) as described previously (22) . The freshly dissociated cells were kept in physiological solution with 1mM Ca2+ at room temperature (23°–24°C) and used within 6–8 h. L-type Ca2+ current (ICaL) was recorded using the "whole-cell" variant of the patch-clamp method (27) at room temperature (22±2°C). K+-currents were blocked by Cs+ (intracellular and extracellular; see below). The fast inward Na+ current was blocked with 50 µM tetrodotoxin (TTX). 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 ethyleneglycol-bis-(-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA), 4.7 CaCl2 (free Ca2+ 108 nM); 10 HEPES; pH was adjusted to 7.2 with CsOH. The alterations of the Ca2+ current were analyzed during a 200 ms depolarizing pulse to 0 mV applied from a –80 mV holding potential after scaling to cell capacitance. Results were analyzed by the Student’s paired t test and are expressed as means and standard deviations. The criterion for significance was P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of the missense variant, ahnak Ile5236Thr
The carboxyl-terminal ahnak domain encompassing amino acids 5200 to 5643 was screened for mutations in 96 unrelated patients with HCM using PCR-SSCP analysis. Two individuals were identified with a heterozygous T>C nucleotide transition (Fig. 1 A). The mutation introduces a new AlwNI restriction site (Fig. 1B ) and converts ATT to ACT, which results in the substitution of the hydrophobic amino acid isoleucine by the polar amino acid threonine at position 5236 (Fig. 1C ). The female index patient with Ile5236Thr presented at age of 60 years had a typical HCM with septal thickness above 17 mm. This woman conducted the mutated allele to two of her four children (Fig. 1B ). Both children heterozygous for ahnak Ile5236Thr showed no HCM phenotype at the age of 43 and 48 years and presented normal values in ECG and echocardiography. The second unrelated heterozygous individual was diagnosed for HCM at the age of 45 years. First-degree relatives were not available for this patient. Mutations of ß-myosin heavy chain, troponin T, and myosin binding protein C genes, which are the most frequent causal genes of HCM were excluded in both index patients with ahnak Ile5236Thr (not shown). To determine whether or not the identified genetic variant is associated with HCM, the allele frequencies were studied by restriction site analysis with AlwNI in a cohort of 96 index patients with DCM and in 96 anonymous blood donors. Ahnak Ile5236Thr was detected as heterozygous variant in three unrelated patients with DCM and in two individuals of the control group. These DCM patients exhibited severe heart failure with ejection fractions below 20% and conduction abnormalities (bundle branch block or AV block). One female patient and one male patient died at the age of 55 and 47 years, respectively while being on the waiting list for transplantation. Family members of the DCM patients with ahnak Ile5236Thr could not be recruited. Taken together, our data suggest that ahnak Ile5236Thr represents a rare genetic variant with no obvious differences in allele frequency among patients with either HCM or DCM or the control population.



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  		Figure 1. Ahnak missense mutation Ile5236Thr. A) Chromatogram demonstrating the mutation in female mutation carrier. Heterozygous mutation T2611C (GenBankTM accession number M80899 that encodes the C-terminal 1277 amino acids) appears as overlap of T (red) and C (blue) marked with arrowhead. B) Restriction fragment analysis of 219-bp amplicons with AlwNI confirms introduction of a new AlwNI site due to T->C transition resulting in ATT->ACT (lane 1, undigested amplicons, lanes 2, 3, 5 heterozygous mutation carriers, lane 4, carrier homozygous for the wild-type allele. C) Amino acid sequence of full-length ahnak deduced from the sequence of chromosome band 11q12 (GenBankTM accession number AC004230) includes the sequences of two cDNAs (accession numbers M80902 and M80899) identified by Shtivelman et al. (13) . Ahnak can be divided into three structural regions, the globular head, a large central region with multiple repeated motifs, and the carboxyl-terminal domains, ahnak-C1 and ahnak-C2. Ahnak-C1 spanning aa 4646-5288 contains a leucine zipper motif (aa 5011-40) and the Ile5236Thr mutation. Synthetic ahnak peptides corresponding to aa 5231-42 and the recombinant ahnak-C1/C fragment corresponding to aa 5215-88 were used as tools in this study.

The mutated ahnak peptide mimics the isoprenaline effects on the Ca2+ inward current
To get insights into a possible functional significance of the ahnak variant, Ile5236Thr, we designed synthetic peptides corresponding to ahnak’s amino acid sequence 5231-5242 with either isoleucine (wild-type) or threonine (Ile5236Thr mutated) at position 5236. The effects of the addition of these ahnak-derived peptides to the pipette solution were investigated on the Ca2+ current, ICaL, elicited under whole-cell patch-clamp conditions on ventricular rat cardiomyocytes. Added intracellularly at 10 µM, the wild-type peptide had no effect on ICaL amplitude or its kinetic. However, the addition of the Ile5236Thr mutated ahnak peptide (10 µM) induced within 2–3 min, a time requested for cell dialysis by pipette solution, a marked increase in ICaL from 11.9 ± 0.8 to 18.8 ± 1.4 pA/pF (P<0.05; 18/20 cells) at 0 mV depolarization (Fig. 2 A, B).



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  		Figure 2. Effects of ahnak-derived peptides on Ca2+ inward current of rat cardiomyocytes. A) Representative traces of ICaL under control conditions and in the presence of wild-type ahnak peptide (GGLPGIGVQGLE) or mutated ahnak peptide (GGLPGTGVQGLE) added to the pipette intracellular solution at 10 µM each. B) Bar graphs demonstrating ICaL density at zero mV under control conditions and in the presence of either 10 µM wild-type ahnak peptide or 10 µM mutated ahnak peptide in the pipette solution. The additional effects of isoprenaline (Iso, 1 µM) are shown by filled bars. C) Current-voltage relations established in control conditions (n=12), in the presence of 1 µM isoprenaline (Iso, n=12) and with a pipette solution added with 10 µM mutated ahnak peptide (n=10, left panel). Availability curves of ICaL established in the same experimental conditions (right panel). The steady-state inactivation or availability curves are fits of mean values to Boltzmann functions with V1/2 = –32.6 ± 1.3, –37.1 ± 1.5 and –36.8 ± 1.8mV and s = 5.1 ± 0.6, 5.2 ± 0.5, and 5.3 ± 0.5 mV in the same cells.

Similarly to the control L-type Ca2+ current, the Ca2+ current increased by Ile5236Thr mutated ahnak peptide could be inhibited by Cd2+ (100 µM) or nifedipine (1 µM) and was carried by Ba2+ after equimolar substitution for Ca2+ (not shown). The current/voltage relationship demonstrated an increase in ICaL at eachmembrane potential together with a slight leftward shift of its voltage dependence (Fig. 2C , left panel). A similar shift was observed for the availability curve (Fig. 2C , right panel). Both of these effects, an increase in ICaL and a shift in voltage dependence, are reminiscent of ß-adrenergic stimulation. Indeed, applying isoprenaline (Iso, 1 µM) in the presence of the Ile5236Thr mutated ahnak peptide had no more effect on ICaL amplitude and its voltage dependence (Fig. 2B, C ). A close examination of the Ile5236Thr mutated ahnak peptide effects reinforces the similarities with the Iso-induced effects. Both experimental conditions increased the fast inactivation time constant and induced similar increases in the amplitude of the two-inactivation components (Table 1 ).


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  		Table 1. Effects of synthetic ahnak-derived peptides (wild-type, Ile5236Thr mutated) on the amplitude and kinetics of the L-type Ca2+ current in rat ventricular cardiomyocytesa

To test whether the effects of the Ile5236Thr mutated ahnak peptide were mediated by the PKA signaling pathway, carbachol (Cch) was used since most of the muscarinic effects of Cch on cardiac ICaL result from antagonizing the ß-adrenergic effects (28) . The application of Cch (10 µM) on Ile5236Thr mutated peptide perfused cells hardly reduced ICaL in control conditions (7.2±2.1 and 16.3±1.8%, respectively; mutated peptide: n=7, control: n=17) and was much less effective after Iso-stimulation (12.3±4.2 instead of 83.5±1.8%; same cells). These data suggest that the increase in ICaL exerted by the mutated ahnak peptide imitates the Iso stimulation without being mediated by PKA.

Ahnak Ile5236Thr is critical for Ca2+ channel ß2 subunit binding
In a recent study we demonstrated the interaction between the Ca2+ channel ß2 subunit and the carboxyl-terminal ahnak-C1 domain (20) , which contains the genetic variant Ile5236Thr (Fig. 1C ). Given our patch clamp results, we examined whether the Ile5236Thr ahnak mutation affects the ß2 subunit interaction. To address this, we used three experimental contexts of Ile5236Thr ahnak presentation to the ß2 subunit. The first used the synthetic ahnak peptides (wild-type, Ile5236Thr mutated) in overlay binding assays. Different amounts of purified recombinant ß2 subunit and ahnak-C1 were subjected to SDS-PAGE (Fig. 3 A, upper panel, lanes 1–3 and 4–6, respectively), blotted on nitrocellulose and incubated with biotinylated ahnak-derived peptides to monitor the binding by subsequent horseradish peroxidase-linked avidin reaction. As presented in Fig. 3A (lanes 1–3), neither the wild-type peptide (middle panel) nor the Ile-5236Thr mutated ahnak peptide (lower panel) revealed ß2 subunit binding. As a positive control, we used the cardiac {alpha}1 interaction domain peptide (AIDc) that is known for high-affinity ß2 subunit binding (24) . When blotted ß2 subunit was overlaid with 3 µM of the biotinylated AIDc peptide, a faint signal was obtained (data not shown) indicating that albeit the binding potential of the ß2 subunit may be reduced upon SDS-PAGE and blotting, high-affinity interactions can be detected. The ahnak peptides reacted with ahnak-C1 blotted on the same membranes. As demonstrated in Fig. 3A (lanes 4–6), they differed tremendously in their binding potential: the wild-type peptide showed weak interaction at 100 µM (middle panel), whereas the Ile5236Thr mutated peptide bound strongly at 10 µM (lower panel).



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  		Figure 3. Ahnak Ile5236Thr affects the ß2 subunit binding. A) The ß2 subunit and ahnak-C1 were expressed in E. coli as GST fusion proteins. Decreasing amounts of ß2 subunit (10, 5, 2.5 µg at lanes 1–3, respectively) as well as ahnak-C1 (10, 5, 2.5 µg at lanes 4–6, respectively) were subjected to SDS-PAGE and stained with Coomassie blue (upper panel). Proteins from identical gels were blotted to nitrocellulose and overlaid with biotinylated wild-type ahnak peptide (middle panel) or mutated ahnak peptide (lower panel). B) Equilibrium sedimentation analysis demonstrating the ahnak-C1/ß2-complex formation. The ß2 subunit (0.16 µM) and ahnak-C1 (0.185 µM) dissolved in 50 mM Tris-HCl (pH 7.4), 500 mM NaCl were centrifuged at 14,000 rpm and 10°C. Radial concentration distribution curves are recorded at 270 nm ({circ}), 275 nm (•), or 280 nm ({square}) were globally fitted using the POLYMOLE program (26) . The solid curves below represent the free reactants and a 1:1 complex resulting in a dissociation constant (Kd) of 152 ± 17 nM. As an indicator of the fit quality, residuals are given (upper part) that are statistically distributed around the mean values. C) Different mixtures consisting of 0.16 µM ß2 and variable amounts of ahnak-C1 with either isoleucine (wild-type, circle) or threonine (mutated; triangle) at amino acid position 5236 were centrifuged to sedimentation equilibrium and analyzed for complex formation. The Kd values were 158 ± 26 nM (n=20) and 96 ± 17 nM (n=9) for wild-type ahnak-C1 and Ile5236Thr mutated ahnak-C1, respectively. The values are means ±SD with the number of independent experiments in parenthesis. D) Bar graphs demonstrating the effects of synthetic ahnak peptides (wild-type, Ile5236Thr mutated; as in legend to Fig. 2 ) on ahnak-C1/ß2 subunit binding expressed as Kd values of ahnak-C1/ß2-complexes (means±SD for at least 3 independent experiments). Kd was solely increased by the mutated ahnak peptide demonstrating its specific uncoupling effect.

In a second experimental context, the Ile5236Thr was introduced into the whole recombinant ahnak-C1 protein by mutagenesis and equilibrium sedimentation analyses were performed with recombinant cardiac ß2 subunit. This method allows the determination of the apparent dissociation constant for the ahnak-C1/ß2 subunit complex (Kd value) in solution without any labels and gives an estimate for the molecular mass of the reactants. The molecular masses obtained for recombinant (GST) ß2 subunit and (GST)-ahnak-C1 were 95 kDa and 94 kDa, respectively, indicating that both proteins are monomers under the experimental conditions. Figure 3B presents a typical plot demonstrating the complex formation between wild-type ahnak-C1 and ß2 subunit. Radial concentration distributions were recorded at three different wavelengths. The data were fitted to determine the Kd value from the mixture of both proteins assuming a 1:1 complex using the POLYMOLE program (26) . To examine whether the proposed stoichiometry of complex formation is correct, different mixtures consisting of 0.16 µM ß2 subunit and increasing amounts of ahnak-C1 were centrifuged to the sedimentation equilibrium and analyzed as described. Twenty and eight different molar ratios were used for wild-type ahnak-C1 and Ile5236Thr mutated ahnak-C1, respectively. In both cases the optimal fit yielded the formation of 1:1 complexes for the concentration range tested (Fig. 3C ). The substitution of isoleucine (wild-type) by threonine (mutated) at position 5236 in ahnak-C1 conferred an increase in ß2 subunit binding affinity expressed by a decreased dissociation constant: the Kd values were 158 ± 26 nM (n=20) and 96 ± 17 nM (n=8) for wild-type ahnak-C1 and Ile5236Thr mutated ahnak-C1, respectively (Fig. 3C ).

Finally, in a third context, equilibrium binding experiments with wild-type ahnak-C1 and ß2 subunit were performed in the presence of synthetic ahnak peptides (either wild-type, or mutated) in an ~100-fold molar excess. If the ahnak region around the mutation is directly involved in reversible ß2 subunit binding, the peptides are expected to compete with ahnak-C1 for ß2 subunit complex formation. Analysis of equilibrium binding data revealed that the wild-type ahnak peptide at 10 µM and 20 µM had minor effects on the ahnak-C1/ß2 subunit complex formation expressed as a slight but not statistically significant increase in Kd (Fig. 3D ). However, the parallel inclusion of Ile5236Thr mutated ahnak peptide attenuated efficiently the ahnak-C1/ß2 subunit binding expressed by increased Kd values (1.7-fold at 10 µM; Fig. 3D ). This in vitro binding approach resembles most closely the electrophysiological experiments documented in Fig. 2 , in which the same ahnak peptides were targeted to native ahnak/Ca2+ channel complexes. Taken together, these results indicate that the genetic variant Ile5236Thr ahnak is critical for ahnak-C1/ß2 subunit binding and that the mutated peptide is a useful tool for specific attenuation the ahnak-C1/ß2 subunit binding.

Attenuation of ahnak-C1/ß2 subunit binding affinity accompanies increased ICaL
Since the Ile5236Thr mutated ahnak peptide mimics the PKA-mediated increase on ICaL and weakened ahnak-C1/ß2 subunit interaction, we supposed a relationship between both events. Consequently, we asked whether PKA-mediated phosphorylation by itself induces changes in ahnak-C1/ß2 subunit binding. To address this question, we phosphorylated both recombinant proteins in vitro with the catalytic subunit of PKA, and performed subsequent equilibrium binding analyses with different input ratios of phosphorylated ahnak-C1/ß2 subunit. The data were analyzed with respect to complex formation and yielded a Kd value of 339 ± 77 nM (mean±SD, n=10). Thus, PKA-mediated phosphorylation of both protein partners led indeed to a significant attenuation (~50%) of the ahnak-C1/ß2 subunit binding. This partial ß2 subunit uncoupling from ahnak-C1 upon PKA phosphorylation was very similar to that induced by the addition of micromolar concentrations of Ile5236Thr mutated ahnak peptide (Fig. 3D ).

To strengthen the apparent link between attenuation of ahnak-C1/ß2 subunit binding and increase in ICaL we designed ahnak fragments encompassing amino acids 5215-88, i.e., the C-terminal portion of ahnak-C1 (named ahnak-C1/C) as HIS-tagged proteins. These ahnak fragments had calculated molecular masses of 13 kDa. But, both wild-type ahnak-C1/C and Ile52336Thr mutated one revealed apparent molecular masses (Mw) of 80 kDa in sedimentation analysis indicating that the ahnak-C1/C fragments formed aggregates under the experimental conditions. Analyzing the equilibrium binding data with respect to the Mw values, we observed that Ile5236Thr mutated ahnak-C1/C influenced the complex formation between ahnak-C1/ß2 subunit whereas wild-type ahnak-C1/C did not (data not shown). The specific effect of mutated ahnak-C1/C prompted us to employ it in patch clamp experiments. Indeed, intracellular perfusion of rat cardiomyocytes with Ile5236Thr mutated ahnak-C1/C (10 µM) mimicked the ß-adrenergic stimulation on ICaL. As demonstrated in Fig. 4 , subsequent application of isoprenaline did not exert additional effects, neither on amplitude nor on kinetics, of the large ICaL elicited by repetitive depolarizations to 0 mV (Fig. 4) . These data suggest that the ahnak-C1/ß2 subunit interaction is an important determinant of ICaL in cardiomyocytes.



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  		Figure 4. Mutated ahnak fragment, Ile5236Thr ahnak-C1/C mimics ß-adrenergic stimulation of ICaL. Time course of peak ICaL (in pA/pF) in a rat cardiomyocyte intracellularly perfused with the Ile5236Thr mutated ahnak fragment, ahnak-C1/C (10 µM) under control condition and during ß-adrenergic stimulation with 1 µM isoprenaline (Iso). Inset show current traces recorded under control condition (a) and during ß-adrenergic stimulation (b). ICaL was recorded at 0 mV with 200 ms pulses from a holding potential of –80 mV. Stimulation frequency: 0.125 Hz.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study provides new insight into the role of ahnak in ß-adrenergic regulation of cardiac L-type Ca2+ channels. We propose the ahnak-C1 domain (aa 4646-5288) serves as physiological brake on ICaL through interaction with the ß2 subunit. Heterologous channel expression has shown that a common effect of the different ß subunits is to enhance the coupling between depolarization and activation. ß Subunit coexpression enhances charge movements (29 30 31) , an effect that has been attributed to {alpha}1C/ß subunit interaction (32 , 33) . Moreover, ß subunit has also been suggested to serve as voltage-dependent inactivation particle (34 , 35) . The release of the ß2 subunit from ahnak-C1 after PKA phosphorylation may account for the marked increase in Ca2+ current and its slower inactivation (Fig. 5 ).



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  		Figure 5. Proposed model for sympathetic control of ICaL by ahnak/Ca2+ channel binding. Under basal conditions, ICaL carried by the {alpha}1C subunit is reprimed by strong ahnak-C1/ß2 subunit binding (left panel). Upon sympathetic stimulation, PKA sites in ahnak (12) and Ser-478 and Ser-479 in ß2 (41) are phosphorylated. This attenuates ahnak-C1/ß2 subunit binding, resulting in increased ICaL since ß2 subunit is more available for {alpha}1C (right panel). Hence, we propose ahnak-C1/ß2 subunit binding serves as physiological brake of {alpha}1C conductance. Relief from this inhibition is proposed as a common pathway used by the sympathetic signal cascade and Ile5236Thr mutated ahnak fragments to increase ICaL. Mutated ahnak, like chronic ß-adrenergic stimulation, might lead to cardiotoxicity.

A crucial finding of the present study was that PKA phosphorylation of ahnak-C1 and ß2 subunit attenuated their interaction by ~50%. Remarkably, the Ile5236Thr mutated ahnak peptide, but not the wild-type version, induced a similar partial dissociation of ahnak-C1/ß2 subunit complexes (Fig. 3D ). Two different modes of action can be considered for the Ile5236Thr mutated ahnak peptide: a direct competition with ahnak-C1 for the ß2 subunit or an allosteric modulation of the complex formation. Several observations argue for the latter possibility. Effective competition is expected if the peptide sequence around Ile5236Thr represents the high-affinity ahnak-C1/ß2 subunit interaction site. In that case the respective wild-type ahnak peptide should also act as efficient competitor. But it had no effect on ahnak-C1/ß2 subunit binding at 100-fold molar excess. Furthermore, though the Ile5236Thr mutated ahnak peptide weakened the ahnak-C1/ß2 subunit interaction under the same conditions, it was not able for entire displacement and the effects exerted by 10 µM and 20 µM peptide were rather similar. Moreover, both ahnak peptides showed no ß2 subunit binding in overlay experiments (Fig. 3A ). Thus, we consider the string of amino acids encompassing ahnak Ile5236Thr as allosteric modulator of the high-affinity ahnak-C1/ß2 subunit interaction site. Studies are in progress to define this site in ahnak-C1 and to clarify the significance of a leucine zipper motif located at ahnak position 5012-5040. Specifically, the mutated ahnak peptide showed significant binding to ahnak-C1 (Fig. 3A ). This interaction could account for modulation of ahnak-C1/ß2 subunit complex formation. This notion is supported by the finding that solely the mutated ahnak-C1/C fragment interfered with the ahnak-C1/ß2 subunit complex formation.

The Ile5236Thr mutated ahnak peptide mimicked specifically the isoprenaline stimulation of ICaL in native cardiomyocytes when applied in the intracellular pipette solution. It increased ICaL, slowed its inactivation, and led to a leftward shift in the current-voltage relationship, i.e., effects that resemble those elicited by sympathetic agonists (Fig. 2 , Table 1 ). ICaL stimulated by the mutated peptide or the Ile5236Thr mutated ahnak-C1/C fragment (Fig. 4) was not further modified by isoprenaline, nor was it reduced by carbachol. Our data suggest that the Ile5236Thr mutated peptide/fragment bypassed the PKA acting directly on Ca2+ channel. Hence, we propose a common pathway for both PKA phosphorylation and the Ile5236Thr mutated ahnak peptide that consists in partial functional uncoupling of ß2 subunit from the inhibitory ahnak-C1 domain (Fig. 5) .

Functional uncoupling of inhibitory regulatory proteins upon phosphorylation by PKA is a common mechanism in ß-adrenergic signaling, e.g., uncoupling of phospholamban from SERCA II (36) and uncoupling of FKBP12.6 from RyR2 (37) . In addition, the mutated ahnak peptide/fragment may interact with as yet unknown partners in the cardiac cell via recruiting other regulators/modifiers to the channel complex. Future studies should clarify the role of ß subunit isoforms and their promiscuity in such processes. Our data do not contradict the importance of PKA anchoring proteins (AKAP) in the signal transduction pathway (8) . While the AKAPs are known to play a critical role in efficient channel phosphorylation, ahnak is considered to act downstream of PKA playing a role in transducing the phosphorylation effects on channel gating.

Very recently, Komuro et al. (38) identified ahnak2 in cardiomyocytes of mice with targeted ablation of the intronless ahnak gene (now designated as ahnak1). Both ahnak molecules have the same tripartite structure characterized by intermediate repeat segments. It has been hypothesized that the repeat segments of both ahnaks are organized as ß-propeller proteins rendering them an ideal class of scaffolding proteins that associate with Ca2+ channel proteins of cardiomyocytes and other cells (38) . According to our data ahnak’s potential to regulate cardiac ICaL requires the carboxyl-terminal domain unique to ahnak1.

We identified Ile5236Thr ahnak in humans. Since allele frequency was similar in HCM and DCM patients and in healthy controls, this missense mutation is not linked to disease. However, our data indicate the genetic variant Ile5236Thr ahnak is functional. Thus, it may cause individual differences in ICaL response upon physiological challenges or therapeutic interventions. Ahnak fragments containing this mutation critically affected ICaL of cardiomyocytes. Of note, we recently demonstrated the existence of cardiac-specific carboxyl-terminal ahnak fragments in human myocardium (39) . The occurrence of Ile5236Thr mutated ahnak fragments in human might result in sustained increased ICaL like a chronic ß-adrenergic stimulation as demonstrated experimentally in this study (Fig. 4) . Notably, the gain of function mechanism exerted by Ile5236Thr mutated ahnak fragments on Ca2+ current is mediated through both increased current amplitude and slowed channel inactivation. Prolonged Ca2+ current induces intracellular Ca2+ overload, delays cardiomyocytes repolarization (long QT), and increases risk of arrhythmias. Lethal arrhythmias caused by a nearly complete loss of Ca2+ channel inactivation due to Cav1.2 channel mutation has recently been shown for Timothy’s syndrome (40) .

Taken together, our data highlight the importance of ahnak for cardiac Ca2+ channel function. They demonstrate that Ile5236Thr is a hotspot within the carboxyl-terminal ahnak-C1 domain that interfered with the ß-adrenergic regulation of ICaL.


   ACKNOWLEDGMENTS
 
We are grateful to Gerlinde Grelle (MDC, Berlin, Germany) for determination of amino acid compositions of the recombinant proteins and to Steffen Lutter for excellent technical assistance. We thank Tobias Haase (Berlin, Germany) for performing the initial SSCP analyses during his practical training. The authors thank Dr. Peter Karczewski (MDC, Berlin, Germany) for critical reading the manuscript and valuable suggestions. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Ha 1779/ 4-2 and 1779/4-3).

Received for publication April 8, 2005. Accepted for publication July 26, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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