Cardiovascular Research 73 (2007) 19–25www.elsevier.com/locate/cardiores

Review

Ahnak, a new player in β-adrenergic regulation of thecardiac L-type Ca2+ channel

Hannelore Haase ⁎

Max Delbrück Center for Molecular Medicine (MDC), Robert-Rössle-Str.10, 13125 Berlin, Germany

Received 13 July 2006; received in revised form 29 August 2006; accepted 1 September 2006Available online 14 September 2006Time for primary review 27 days

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Abstract

Ahnak, originally identified as a giant, tumour-related phosphoprotein, has emerged as an important signalling molecule in a wide rangeof physiological activities. In this article, current knowledge will be reviewed that places ahnak into the context of cardiac L-type Ca2+

channel function by its interaction with the β2 subunit. Beginning with an overview on structural and functional properties of ahnak, basicfeatures of β subunits are highlighted. The review characterizes multiple ahnak/β2 subunit binding sites and focuses on recent progress inunderstanding their functional role in Cav1.2 channel conductance (ICaL). Three main aspects of ahnak function in ICaL of cardiomyocytesemerge from available experimental data. First, ahnak acts as repressor towards ICaL by β2 subunit sequestration. Second, PKAphosphorylation relieves the inhibition imposed by the C-terminal ahnak domain, ahnak-C1. Third, this action is mimicked by ahnak-derivedfragments carrying a naturally occurring missense mutation Ile5236Thr. This paradigm introduces ahnak as a player in beta-adrenergiccontrol of ICaL and sheds new light upon the molecular mechanism underlying this fundamental process of Cav1.2 channel physiology.© 2006 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

14,

2014 Keywords: Ahnak; Cardiomyocyte; Calcium channel; Beta subunit; Protein kinase A; Gene polymorphism

1. Introduction

The influx of calcium ions (Ca2+) through the L-type Ca2+

channel (ICaL) is one major determinant of the plateau phase ofthe cardiac action potential and is crucial for excitation–contraction coupling in the heart [1–3]. The ICaL triggers therelease of Ca2+ from sarcoplasmic reticulum stores, and theresulting Ca2+ transient drives the contraction. Structurally, thecardiac L-type Ca2+ channel is a multi-subunit protein in whichthe pore-formingα1C subunit is physically associatedwith twoobligatory auxiliary subunits: α2/δ and β [3]. The α1C gene(Cav1.2) encodes L-type Ca2+ channels in cardiac and smoothmuscle [4]; alternative splicing produces multiple isoformswhich are differently distributed among different tissues [5].

In the adult mammalian myocardium, ICaL is primarilyregulated by the sympathetic (beta-adrenoceptor) signallingcascade that ultimately controls the level of contractile force

⁎ Tel.: +49 30 9406 3483; fax: +49 30 9406 2579.E-mail address: haase@mdc-berlin.de.

0008-6363/$ - see front matter © 2006 European Society of Cardiology. Publishedoi:10.1016/j.cardiores.2006.09.001

[6–10]. Defective ICaL regulation with blunted beta-adrenergicresponsiveness is one major cause for myocardial contractiledysfunction in both animal models and in human syndromes[11,12]. The textbook view of adrenergic signalling is thatcAMP-dependent protein kinase (PKA) activation transducesthe “turn-on” signal from the G-protein coupled beta-adrenoceptor via phosphorylation of the Ser-1928 locatedwithin the long intracellular tail region of theα1C subunit [13–15]. Additionally, phosphorylation of the intracellularlylocated, regulatory β2 subunit was reported to play a crucialrole in the PKA-mediated up-regulation of ICaL [16–18]. Theunderlying molecular mechanisms how phosphorylation ofsites located either on α1C or β2 results in increased channelopening probability is still incompletely understood.

Early on, the postulated direct link between channel phos-phorylation and enhanced ICaL was questioned by studiesdemonstrating the absence of a cAMP-mediated increase inICaL in some heterologous expression models [19–22].Therefore, the hypothesis was drawn that PKA-dependentregulationmay require additional associated proteins [20]. The

d by Elsevier B.V. All rights reserved.

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identification of a family of A-kinase-anchoring proteins(AKAPs) has greatly enhanced the understanding how thecardiac muscle cell is able to coordinate different PKAsignalling events in space and time. Adrenergic regulation ofICaL has been shown to require direct anchoring of PKA to theC-terminal of the α1C via a leucine zipper interaction withAKAP15 [15,23]. However, PKA-anchoring alone is obvi-ously not sufficient to reconstitute robust PKA-regulation ofICaL in heterologous expressionmodels. Searching for a further“missing link“ we identified the ahnak protein in cardiomyo-cytes that is tightly bound to the Ca2+ channel β2 subunit [24].

2. Overview of ahnak properties and function

2.1. Molecular structure

Ahnak has been originally identified in 1992 as tumour-related gene by Shtivelman and coworkers [25]. Theseauthors introduced the name “AHNAK” (meaning giant inHebrew) to convey the large size of the encoded ∼700 kDaprotein. The ahnak genewas independently identified in 1993by Hashimoto and coworkers [26], who demonstrated thatthis gene also codes for desmoyokin, a protein first isolated in1989 as a desmosomal plaque protein from bovine muzzleepidermis [27]. Ahnak/desmoyokin is encoded by anintronless gene located on human chromosome 11q12-13[28]. Hence, no ahnak isoforms arise from alternative splicingprocesses. The deduced amino acid sequence of human ahnakpredicts a protein of 5643 amino acids with a tripartitestructure: a relatively short N-terminus of 251 amino acids, alarge central region of ∼4400 amino acids composed ofhighly conserved repeated units, and a unique C-terminus of1002 amino acids [25].

2.2. Structure/function: the ahnak central repeated units

The repeated units are believed to form a propeller-likestructure that enables ahnak to “associate with Ca2+ channelsof cardiomyocytes and other cells” [29]. This notion is notsupported by data from our group, since we defined multipleCa2+ channel interaction sites within the C-terminal ahnakdomain [30]. Further work is needed to elucidate whether thepropeller-like ahnak repeats interact also with the Ca2+

channel and what is the functional consequence. An inter-esting example for the action of ahnak's central repeated unitsas a scaffolding motif has been recently described by Lee etal. [31]. The authors elaborated a PLC-γ1 activation pathwaythat includes the concerted interaction of four repeated ahnakunits networking PLC-γ and PKC-α.

2.3. Structure/function: the ahnak C-terminal determinessubcellular location

The C-terminal ahnak domain contains putative nuclearlocalization signals, and the ahnak protein identified byShtivelman's group located indeed primarily in the nucleus

[32]. But, ahnak/desmoyokin distributes also to the plasmamembrane and the cytoplasm. This ambiguity is explained byahnak's potential for subcellular translocation first describedby Hashimoto et al. [33]. This group demonstrated that the C-terminus is necessary and sufficient to induce plasma mem-brane shuttling of ahnak from the nucleus [34]. Furthermore,Sussman et al. [35] reported on the nuclear exclusion of ahnakmediated through an export signal that involves thephosphorylation of Ser-5335 by PKB/Akt. The same studydemonstrated an interesting cross-talk between the formationof cell-cell contacts and nuclear exclusion of ahnak inepithelial cells. Hence, the hypothesis was drawn that plasma-membrane-anchored ahnak is involved in the growth arrest ofnormal epithelial cells [35]. It is tempting to speculate thatahnak serves a similar function in other cells includingterminally differentiated cardiomyocytes that withdraw fromthe cell cycle early during postnatal development.

Plasma membrane location of ahnak has been observed inrodent and human cardiomyocytes as well as in smoothmuscle and skeletal muscle studied so far [24,30,36,37]. Atthe subcellular level, ahnak of cardiomyocytes is strictlylocalized to the inner aspect of all three sarcolemma com-partments: surface sarcolemma, intercalated disc and T-tubular system [30]. Ahnak does not have membrane-span-ning regions. Thus, ahnak is considered as plasma membranesupport protein in muscle and lining epithelium [30,36].

2.4. Structure/function: the ahnak C-terminal interacts withthe cytoskeleton

Ahnak's C-terminal interacts with actin and bundles actinfilaments into paracrystalline-like structures whichwe proposeto serve structural and functional roles as an important linkbetween T-tubular Cav1.2 channels and the actin-basedcytoskeleton [30,37]. A further example for the formation ofmulti-protein complexes is the interaction of ahnak's C-terminal domainwith annexin2 via S100A as an adaptor whichhas been functionally related to the cytoskeleton of Madin–Darby canine kidney cells. In that cellular model, specificahnak down-regulation prevented the cortical actin cytoskel-eton reorganization required to support the cell height [38].

2.5. Ahnak knockout mice

Given ahnak's implication in fundamental biologicalprocesses, such as Ca2+ signalling [31–34], plasma mem-brane support [30,36–38], regulated exocytosis [39], andblood brain barrier [40], one would expect that targetedablation of ahnak gene results in a severe phenotype showingmulti-organ damage. Surprisingly however, ahnak-deficientmice are viable, fertile, and show no cross-abnormalitiessuggesting that other molecules compensate for the loss offunction of ahnak in mice [29,41]. A search performed byKomuro et al. [29] revealed an ahnak-like protein encoded onhuman chromosome 14q32 (designated as ahnak2). Thisprotein displays an internal repeat structure like ahnak/

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desmoyokin, while its N-terminal and C-terminal showessentially no similarities. Hence, ahnak2 is not considered tocompensate for ahnak's C-terminal, i.e. for interaction withthe actin-based cytoskeleton and the β subunit. Presently, noexperimental data are available on cardiomyocytes structureand function in ahnak-deficient mice.

3. Functional consequences of ahnak/β subunitinteraction on Cav1.2 channel

3.1. Basic properties of Ca2+ channel β subunits

To understand ahnak actions on the Ca2+ channel, somebackground on the β subunit is necessary. The importance ofβ subunits for channel function has been highlighted byheterologous expression, antisense and gene knock-outexperiments which are summarized by excellent reviews[3,42–45]. In fact, the β subunit is responsible for correctplasma membrane insertion of the α1 subunit and once α1 isinserted the β subunit becomes an allosteric modulator ofchannel gating properties. As such, membrane targeting andcurrent modulation of the channel appear to be independentfunctions of theβ subunit [43,46]. Furthermore, the variousβsubunit isoforms enhance the whole cell current density atdifferent levels by increasing the channel opening probability[47,48], produce distinctive effects on channel inactivationkinetics [47–49], and induce hyperpolarizing shifts in thevoltage-dependence of channel activation [50,51].

All known four Cavβ genes are expressed in the heart[52] and can in theory bind to the Cav1.2 subunit via the α1interaction domain (AID), a highly conserved binding motifof 18 amino acid residues present in the cytoplasmic linkerbetween repeat I and II of α1 subunits [53]. Although theidentity of the assembled subunits is not entirely clarified,the β2 subunit isoform is generally believed to constitute theintracellular, accessory subunit of the Cav1.2 channel inadult mammalian myocardium [43,54].

Recent functional studies and crystallographic informationhave defined the β subunits of voltage-dependent Ca2+

channels as members of the membrane-associated guanylatekinase (MAGUK) family of proteins [55–57]. The MAGUKproteins are known to be scaffolding proteins with two well-conserved protein–protein interaction domains: a type-3 Sarchomology (SH3) domain and a guanylate kinase (GK) domain.Hence, it was suggested that the β subunit represents an idealtarget for establishing molecular interactions of the channelwith intracellular proteins required for channel regulation [55].Indeed, proteins are emerging which interact with β subunitsincluding ahnak [24,30,58,59] and various members of theGem/kir family of small Ras-like GTPases [60].

3.2. The β subunit binds to multiple ahnak sites located atthe C-terminal

β2 Subunit interaction with ahnak has been originallyidentified by co-immunoprecipitation with several cardiac

preparations derived from rodent to human heart [24]. Thisobservation suggested a tight physical association betweenahnak and the β2 subunit. Consistent with this notion,subsequent in vitro binding studies disclosed a high affinityβ2 subunit interaction site (Kd≈50 nM) within distal C-terminal ahnak domain (designated as ahnak-C2), whileapparently neither the ahnak head region nor the ahnakrepeating units contribute to ahnak interaction [30]. Furtherin-depth equilibrium binding experiments reveal a complexpicture for ahnak/β2 subunit interaction as multipleattachment sites have been defined within ahnak's C-ter-minal. In fact, one additional β2 subunit binding site with∼160 nM affinity locates in the proximal tail region,designated as ahnak-C1 [59], while a second site with∼300 nM affinity occurs in the distal ahnak-C2 region [30].At the level of the β2 subunit, ahnak interaction sites are lessdefined. A first step towards this direction is a pull-downassay with recombinant ahnak-C2 demonstrating that bothcardiac Cav1.2 and skeletal muscle Cav1.1 channel com-plexes are recovered containing the β2 subunit and β1asubunit, respectively [30]. These findings suggest that theconserved β subunit modules, SH3 and/or GK, are importantfor ahnak/Cav1.x channel interaction.

3.3. Ahnak polymorphism, I5236T, interferes withadrenergic stimulation of ICaL

In human ahnak, four polymorphic loci encoding aminoacid substitutions have been identified so far [61]. The firstcharacterized T/C polymorphism results in an Ile or Thr atamino acid 5236, whereby the Ile variant is clearly the wild-type allele. Comparison of allelic frequencies among a controlCaucasian population and patients with hypertrophic or dilatedcardiomyopathy (∼100 individuals each) reveals that themajority of individuals are homozygous for Ile5236 in eithergroup and only 2% is heterozygous Ile/Thr with no subjects todate identified as being Thr homozygous. As such, no directcorrelation can be drawn between the heterozygous genotypeand a cardiomyopathy phenotype. Nonetheless, the Ile5236Thrpolymorphism is functional as recently demonstrated by us andcollaborators [59]. A synthetic ahnak peptide carrying the Thrmutation mimicked the beta-adrenergic stimulation on ICaL,elicited under whole-cell patch-clamp conditions on ventricularrat cardiomyocytes using Ca2+ as charge carrier. Acuteintracellular perfusion with Thr-containing ahnak peptide(10 μM) yielded a robust increase in current amplitude (1.7fold at 0mV depolarization), a leftward shift in current–voltagerelationship and availability curve, and a slowing of currentdecay kinetics during the initial fast phase. All three effects aretypical of adrenergic stimulation. Moreover, ICaL enhanced byThr5236-ahnak peptide does not anymore respond to the beta-adrenergic agonist, isoprenaline, nor to its counteracting agent,carbachol. These actions on ICaL are specific for the Thr-containing ahnak peptide, while the Ile-containing (wild-type)ahnak peptide does essentially not modify basal ICaL nor does itinterfere with the isoprenaline response of the channel [59].

Fig. 1. Scheme illustrating the role of ahnak in beta-adrenergic modulation of cardiac Cav1.2 channel activity. A) Under basal condition, Cav1.2 conductance isreprimed by strong interaction between dephosphorylated ahnak and dephosphorylated β2 subunit. B) PKA phosphorylation of ahnak-C1 and/or β2-subunitrelieves the inhibitory effects. C) Relief from ahnak repression is also achieved by intracellular perfusion with mutated ahnak peptides leading to increased ICaL[59]. Inset, superimposed original traces of ICaL evoked by conditions demonstrated in A, B, and C, respectively.

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Thus, individuals with Thr5236 ahnak may exhibit defects inchannel gating. Though Ile5236Thr ahnak is not considered tobe a disease-causing mutation, it can substantially contribute toindividual differences in the response to beta-adrenergicchallenges or beta-blocker therapy.

A second ahnak polymorphism results in a Thr or Met atamino acid 5549, respectively. This mutation does notinfluence the specific features of recombinant ahnak-C2, i.e.strong actin binding and high-affinity β2 subunit interaction[61]. The consequence of this mutation on ICaL is not yetknown. Likewise, information is lacking whether theadditional ahnak polymorphisms reported are functional.

3.4. Tuning the ahnak-C1/β2 subunit interaction

As the Thr5236-ahnak peptide specifically mimics theisoprenaline actions on ICaL, we elucidated whether the

proximal C-terminal ahnak region, ahnak-C1 (aa 4646-5288), plays a role in the signal transduction pathway betweenbeta-adrenergic receptor and Cav1.2 channel. Binding assaysreveal the ahnak-C1/β2 subunit binding is indeed phosphor-ylation-dependent. The binding affinity decreases by ∼50%upon PKA phosphorylation of both protein partners. Surpris-ingly, the ahnak-C1/β2 subunit interaction is similarlyattenuated by Thr5236-ahnak peptide, but not by therespective wild-type ahnak peptide [59]. The specific actionselicited by PKA phosphorylation and Thr5236-ahnak peptideare explained by the assumption that the ahnak-C1 domainoperates as a phosphorylation-dependent suppressor of ICaL.Consequently, relief from ahnak-C1 suppression makes theβ2subunit more available for the ion-conducting Cav1.2 subunitleading to enhanced current density. Hence, a model isproposed in which Cav1.2 activity is reversibly regulated bytuning the ahnak-C1/β2 subunit interaction. Overall, the

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actions exerted by PKA-mediated phosphorylation andThr5236-ahnak peptide are ascribed to a functional uncouplingof the β2 subunit from the inhibitory ahnak-C1 domainleading to robust up-regulation of ICaL as seen in nativecardiomyocytes upon sympathetic stimulation. (Fig. 1).

In general, functional uncoupling of inhibitory regulatoryproteins upon sympathetic stimulation is in line with un-coupling of phospholamban (PKA phosphorylated) fromSERCA 2a [62] or uncoupling of FKBP12.6 (PKAphosphorylated) from RyR2 [63]. Mice with targetedablation of either phospholamban or FKBP12.6 are viable,fertile, and exhibit hyper-contractile cardiac phenotypes.Phospholamban-deficient mice show increased Ca2+ cyclingcapacity with no signs of Ca2+ overload or hypertrophy [64],while mice with targeted ablation of FKBP12.6 developcardiac hypertrophy at higher age in a sex-specific mannerdue to Ca2+ overload in males [65]. Interestingly, phospho-lamban-deficient cardiomyocytes exhibit accelerated ICaLdecay kinetics which is believed to protect the cell from Ca2+

overload [66]. This model demonstrates the close interrelatedroles of ICaL and SERCA2a/ phospholamban in cardiomyo-cyte Ca2+ cycling.

3.5. Targeting the ahnak-C2/β2 subunit interaction

The functional role of the distal C-terminal ahnak domain,ahnak-C2, in Cav1.2 channel gating has been first examinedin rat ventricular cardiomyocytes by Alvarez et al. [58].Intracellular perfusion with ahnak-C2-derived fragments,which efficiently and reversibly bound to the β2 subunit,augmented ICaL predominantly through slowing the channelinactivation. Importantly, the effects of isoprenaline werefully retained and were additive to those of ahnak-C2fragments. This implies an inhibitory role of ahnak-C2 onICaL independent of the sympathetic signalling pathway [58].

4. Perspectives

Ahnak-derived peptides have emerged as potent mod-ulators of ICaL in the adult mammalian heart [58,59] offeringnew therapeutic strategies for the treatment of cardiovasculardiseases. For instance, the Thr5236-mutated ahnak peptide isable to enhance ICaL down-stream of PKA. This interventionseems beneficial for contractile support in heart failure inwhich the beta-adrenergic responsiveness is blunted [67,68].By contrast, ahnak peptides derived from the distal C-terminus may impair cardiac function since they slow ICaLinactivation which can induce intracellular Ca2+ overloadand can even cause lethal arrhythmias [69]. We described ourcurrent working hypothesis that ICaL modulation by ahnakpeptides is due to alterations in ahnak/Cavβ interaction, butthe contribution of Cav1.2 remains to be explored.Heterologous expression of different combinations of theCav1.2 channel subunits with different ahnak domains willbe crucial to understand the underlying molecular events.Furthermore, studies on ahnak-deficient mice are required to

shed light upon ahnak's role in ICaL regulation and cardiacfunction in physiological and pathological conditions.

5. Conclusion

Beta-adrenergic stimulation of the cardiac Cav1.2 current(ICaL) is transmitted by PKA and is central to physiologic andpathologic events in the heart. However, a fundamentalquestion of Cav1.2 channel physiology, the mechanismsresponsible for beta-adrenergic enhancement of ICaL remainopen since attempts to reconstitute the PKA modulation inheterologous expression systems are inconclusive. Experi-mental data, reviewed herein, implicate ahnak as new playerin beta-adrenergic control of ICaL. Ahnak is abundantlyexpressed in cardiomyocytes, undergoes PKA phosphoryla-tion, and associates to the intracellular regulatory β2 subunitof Cav1.2 channels. Our model proposes that ahnak bindingcontrols the availability of the β2 subunit to increase ICaL.This paradigm implies that targeting the ahnak/β2 subunitinteraction may be a novel therapeutic strategy to improvecontractile function in the failing heart.

Acknowledgments

I am grateful to Dr. Julio Alvarez for providing the ICaLtraces shown in Fig. 1. Special thanks are due to Dr. IngoMorano for his continuous support and valuable suggestions.Drs. Guy Vassort and Peter Karczewki are acknowledged forcritical reading the manuscript and Steffen Lutter for tech-nical assistance and art work. The original studies referred towere supported by Deutsche Forschungsgemeinschaft andStiftung für Pathobiochemie.

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