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Cardiovascular Research 2006 71(3):430-442; doi:10.1016/j.cardiores.2006.04.012
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Copyright © 2006, European Society of Cardiology

Molecular aspects of adrenergic modulation of the transient outward current

Marcel A.G. van der Heydena,*, Tessa J.M. Wijnhovena and Tobias Opthofa,b

aDepartment of Medical Physiology, University Medical Center Utrecht, The Netherlands
bExperimental and Molecular Cardiology Groups, Academic Medical Center, Amsterdam, The Netherlands

* Corresponding author. Department of Medical Physiology, University Medical Center Utrecht, Yalelaan 50, 3584 CM Utrecht, The Netherlands. Tel.: +31 30 2538900; fax: +31 30 2539036. Email address: m.a.g.vanderheyden{at}med.uu.nl

Received 16 September 2005; revised 5 April 2006; accepted 20 April 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Molecular basis of...
 3. Function in the...
 4. Function in the...
 5. Molecular regulation and...
 6. Potential phosphorylation...
 7. Conclusions
 References
 
Transient outward channels have a different impact on action potential configuration in small mammals compared to large mammals. Small mammals depend primarily on Ito1 for repolarization, while in larger animals Ito1 only indirectly determines action potential duration by setting the level of the plateau. Transient outward channel expression and distribution also differ between animal species. Nevertheless, the primary protein sequence of the underlying Kv1.4, Kv4.2 and Kv4.3 {alpha}1-subunits displays remarkably high levels of amino acid identity. Transient outward channels are subject to {alpha}- and β-adrenergic regulation, mainly decreasing Ito1. However, adrenergic stimulation is also an important determinant of transient outward channel downregulation in cardiac disease. Adrenergic stimulation of PKA as well as PKC leads to an inhibition of Ito1, which has been correlated with phosphorylation of the Kv1.4, Kv4.2 and Kv4.3 {alpha}1-subunits. Calmodulin-dependent kinase II, on the other hand, has been shown to be involved in an increase of Ito1. Comparison of Kv1.4, Kv4.2 and Kv4.3 primary amino acid sequences demonstrates a strong conservation of (potential) phosphorylation sites between different species, despite the fact that Ito1 has a different effect on action potential configuration in mammalian species.

KEYWORDS Action potentials; Adrenergic (ant) agonists; G-proteins; Phosphorylation; Protein kinase A; Protein kinase C; Tyrosine protein kinase; Transient outward current

Abbreviations: AKAP, A-kinase anchoring protein • AR, adrenergic receptor • AC, adenylate cyclase • ATP, adenosine trisphosphate • CaMKII, Ca2+ calmodulin-dependent kinase II • cAMP, cyclic adenosine monophosphate • cGMP, cyclic guanosine monophosphate • DAG, 1,2-diacylglycerol • ERK, extracellular-signal-regulated kinase • G protein, guanosine 5'-triphosphate (GTP)-binding protein • ICa-L, L-type Ca2+ current • Ito1, transient outward current • InsP3, inositol 1,4,5-trisphosphate • PIP2, phosphatidylinositol 4,5-bisphosphate • PK, protein kinase • PLC, phospholipase C • PTK, protein tyrosine kinase • Ser, serine • Thr, threonine • Tyr, tyrosine


   1. Introduction
Top
 Abstract
 1. Introduction
2. Molecular basis of...
 3. Function in the...
 4. Function in the...
 5. Molecular regulation and...
 6. Potential phosphorylation...
 7. Conclusions
 References
 
Two membrane currents, the transient inward (calcium) current (ICa-L) and the transient outward (potassium) current (Ito1), may be considered as the oldest membrane currents from an evolutionary point of view. This has been established in invertebrates [1], but also in mammalian embryonic tissue [2]. The respective mRNAs of ICa-L and Ito1 are also ‘early’ appearing gene products in cells derived from embryonic stem cells in mouse [3,4] and man [5]. Here, we review the molecular adrenergic regulation of Ito1.

Although the transient outward current has two components, one carried by K+ and blocked by 4-aminopyridine (Ito1) [6] and a second one which activates after an intracellular rise of Ca2+, which is insensitive to 4-aminopyridine [7] and which is carried by Cl–, only the first component is described in this review and it will be mentioned simply as the transient outward current (Ito1). A further classification can be made on basis of kinetics and results in a fast component Ito1.f and a sustained component Ito1.s.

Ito1 was discovered in snail neurons [8]; in mammalian tissue, it was first described in Purkinje fibers probably by their prominent phase 1 of the action potential [6,9]. There are, however, also older reports on an early plateau current [10,11], which was originally considered a Cl– current [10,12]. Subsequently, Ito1 was also described in rabbit atrium [13] and ventricle [14], in rat ventricle at the single cell level [15], and–at the single channel level–in isolated bovine Purkinje cells [16]. There are, however, important species differences and differences between parts of the heart. In guinea pig atrium and ventricle [17,18] Ito1 is virtually absent. It is present in rabbit AV nodal cells [19], but not in rabbit sinoatrial nodal cells [20]. Even within the ventricles regional differences exist (see below).

In the mammalian heart, the transient outward current is responsible for early repolarization directly following the upstroke of the action potential (phase 1). In small mammals like mice or rats, this current directly determines action potential duration in the absence of relevant membrane current during the plateau phase of the action potential. This explains the short action potential duration (and short QT intervals) in rats and mice [21]. In larger mammals, including man, Ito1 primarily affects the level of the plateau phase and, therefore, only indirectly determines action potential duration. Thus, inhibition of Ito1 will lead to a more positive (depolarized) level of the plateau phase, whereas an increase of Ito1 will lead to a more negative (hyperpolarized) level of the plateau phase. This will affect the amplitude of several membrane currents, among which ICa-L and both delayed rectifier currents, IKr and IKs. The effect of a decrease or increase in Ito1 may therefore seem quite paradoxical in the heart of large mammals, although the basic conductance of Ito1 is critical [22–24]. Thus, a decrease in Ito1, which is after all an outward, repolarizing current, may cause shortening of the action potential rather than the expected prolongation and vice versa as has been demonstrated in model studies of human atrium [23] and canine ventricle [23,24], based on extensive experimental data.

Ito1 is regionally distributed over the ventricle in dog [25], cat [26], rabbit [27] and man [25]. In general, the current density is higher at the subepicardium than at the subendocardium. In the non-diseased human ventricle, the current density is approximately 50% higher in subepicardial cells than in subendocardial cells [28].

By its early peak just after the upstroke of the action potential Ito1 has only indirect effects on inotropy and on (dispersion in) action potential duration. From its strong frequency dependence, analyzed in detail by Boyett [29], it logically follows that also all these indirect effects strongly depend on heart rate [30]. These physiological functions are under control of catecholamines of circulating and neurohormonal origin. In this review, we focus on known and putative sites of adrenergic-induced phosphorylation of transient outward channels.


   2. Molecular basis of Ito1
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 Abstract
 1. Introduction
 2. Molecular basis of...
3. Function in the...
 4. Function in the...
 5. Molecular regulation and...
 6. Potential phosphorylation...
 7. Conclusions
 References
 
2.1 Molecular structure
A comprehensive discussion on the {alpha}1-subunit structure, expression in various tissues, and interacting β-subunits and other proteins can be found in several excellent reviews [31–34]. Kv4.2 and Kv4.3 are the voltage-gated K+ channels reported to contribute to cardiac Ito1 [35,36], while Kv1.4 is responsible for Ito1.s [37,38]. Transient outward channels and delayed rectifier channels are structurally related [12]. The {alpha}1-subunit of all these voltage-dependent K+ channels has a similar overall structure as the {alpha}1c-subunit of voltage-gated Ca2+ channels [39–41], with the only difference that it is composed of four separate subunits, which are assembled into a tetrameric structure with the N- and C-terminal ends located intracellularly. Assembly of four {alpha}1-subunits into a tetrameric structure is required to create a functional K+ channel [42]. Linkage of subunits from different channel families into one heterotetrameric structure is responsible for the larger variability of K+ channels compared to Na+ or Ca2+ channels. Each of the four subunits comprises six transmembranous hydrophobic segments (S1 to S6), which are connected by extracellular loops S1–S2, S3–S4 and S5–S6 and intracellular loops S2–S3 and S4–S5. The S4 segment serves as a voltage sensor. The S5–S6 loop constitutes the lining of the pore of the channel and renders ion selectivity. A β-subunit is reported to interact with highly conserved domains immediately preceding the S1 segment of the {alpha}1-subunit [42]. However, no reports demonstrate the involvement of an {alpha}2{delta} complex as in the L-type Ca2+ channel [41]. The Kv4.3 channel exists in two isoforms: a full-length variant and a shorter one, which has a 19-amino acid deletion in the C-terminus.

2.2 Developmental expression, species specificity and regional distribution of {alpha}1-subunits
During mouse and rat embryonic development, Kv1.4 is the main contributant to Ito1. Postnatal, Kv1.4 levels decrease, while Kv4.x levels increase. In the adult rat, cardiomyocyte Kv4.2 is the main determinant of Ito1 [43–45]. Therefore, Kv1.4 is considered as a "fetal" gene. Whether similar temporal expression patterns are occurring in humans also has thus far not been established.

In ferret, Kv4.2 is localized primarily in left ventricular epicardial myocytes and is uniformly expressed over the entire right ventricular free wall, whereas Kv4.3 is more uniformly expressed in the left ventricular wall and is sparse to absent in right ventricular tissue [35]. There are also significant differences in the pattern of Kv4 expression between different species. For example, Kv4.2 can be detected in rat myocytes, but is not expressed at significant levels in canine or human myocytes where Kv4.3 appears as the main Ito1 determinant. Furthermore,–and in contrast with rats and mice, see top of this section–, rabbit atrial and ventricular myocytes display Ito1 currents almost exclusively generated by Kv1.4, which is the most important Ito1 {alpha}1-subunit in this species. For a more detailed description of regional and species-specific expression patterns, we refer to the review of Oudit et al. [31] and Patel and Campbell [34]. These differences are thought to be an adaptation to the different requirements for cardiac function in mammals of different sizes [36].


   3. Function in the normal heart
Top
 Abstract
 1. Introduction
 2. Molecular basis of...
 3. Function in the...
4. Function in the...
 5. Molecular regulation and...
 6. Potential phosphorylation...
 7. Conclusions
 References
 
3.1 Basic function
In rabbit atrial cells, transient outward channels have a unitary conductance of 14 pS and there are about 1600 channels per myocyte (density 3–4/µm2) in normal Tyrode's solution [46]. As stated in Section 1, the amplitude of Ito1 directly affects phase 1 of the action potential, but only indirectly influences action potential duration in ventricular myocytes of larger mammals. The latter effect depends on the potential range of phase 2 of the action potential (plateau), which affects activation and inactivation of ICa-L and of the delayed rectifier currents. With so many parameters, it is hard to predict the effect of changes in Ito1 on action potential duration. The higher density of Ito1 in the subepicardium is considered to explain the shorter action potential duration and refractoriness in these cells, although this seems not the case in the human ventricle [47,48].

Ito1 appears as the first outward current in relatively early embryonic stages as was derived from a study in cardiomyocytes differentiated in vitro from mouse embryonic stem cells [49]. In neonatal rabbit ventricular cells, the density of transient outward channels is half of that observed in adult cells [50]. It is not clear whether a similar increase causes the substantial postnatal action potential shortening, which is so characteristic for rat ventricle [51]. In senescent rats, Ito1 appears subject to further change, but some studies report an increase [52], whereas others report a decrease [53]. Action potential duration does not change very much as long as intracellular Ca2+ is buffered [53]. The changes in Ito1 due to aging per se appear small compared to the pathophysiological changes during the development of hypertrophy when a decrease in Ito1 and an increase in action potential duration seem causally correlated [54]. The very slow recovery from inactivation may underlie the remarkable and unique action potential shortening at extremely long cycle lengths in adult rabbit ventricular myocytes [55].

Ito1 is strongly rate-dependent [29]. Abrupt changes in rate and pauses have important consequences for the early repolarization of the membrane [30]. Ito1 fails to recover from previous inactivation at very high heart rates [29,30], but species differences are very relevant here. When one compares frequency changes of 0.1 Hz to 4.0 Hz, peak Ito1 is reduced by 11% and 97% in human and rabbit atrial myocytes, respectively [55].

3.2 Adrenergic receptors
Adrenergic receptors (ARs) are coupled to membrane-bound G proteins. The ARs comprise seven hydrophobic membrane-spanning {alpha}-helical domains. Amino acid conservation is highest in the transmembrane regions, which determine the specificity of ligand binding. The cytoplasmic regions, which communicate with other cellular signaling proteins, show more variability [56].

There are nine AR subtypes in the human heart. They mediate a plethora of cellular functions. The nine AR subtypes are encoded by distinct genes. The most abundant types are the β-ARs with three subtypes: β1, β2 and β3. The β1-AR and β2-AR activate AC after coupling to Gs proteins. AC mediates the conversion of ATP into cAMP. cAMP activates PKA, which subsequently phosphorylates several substrates. The β2-ARs couple also to Gi proteins, which counteract the Gs-coupled activation of AC, resulting in a reduction of cAMP levels [56–59]. The physiological impact of β3-ARs is less clear, although they may be upregulated in heart failure. β3-ARs have been reported to produce negative inotropy in human ventricle. Therefore, a future therapeutic modality might be their blockade in the setting of heart failure (see [60] for references).

Three subtypes of the {alpha}1-AR have been identified: {alpha}1A, {alpha}1B and {alpha}1D. The {alpha}1A-AR is the most abundant in the human heart. It activates PLC after coupling to a Gq protein. This subsequently causes formation of InsP3 and DAG. The latter mediates the activation of PKC, which phosphorylates many substrates. Also three {alpha}2-ARs ({alpha}2A, {alpha}2B and {alpha}2C) exist in the human heart.

With respect to Ito1 regulation, the abovementioned pathways of {alpha}- and β-adrenergic regulation require some refining. Gallego et al. [61] recently indicated that {alpha}1-adrenergic regulation, i.e. decrease of Ito1 in rat cardiomyocytes is mediated by PKA rather than PKC.

3.3 Response to adrenergic stimulation
Several problems prevent that there is a clear overall picture of the physiological effect of adrenergic stimulation on Ito1. Even when only Ito1 is considered (and not the Ca2+-sensitive and 4-aminopyridine-insensitive transient outward current [7]), one may still define the current amplitude of Ito1 in patch-clamp experiments as either the difference between the peak current after a depolarizing clamp step and the holding current or as the difference between the peak current after a depolarizing clamp step and the steady state current at the end of that same depolarizing clamp step. This causes problems when both components increase in response to whatever stimulation. Moreover, efforts to elucidate submechanisms like responses to {alpha}-adrenergic or β-adrenergic stimulation, f.e. by testing the effect of phenylephrine ({alpha}-agonist) in combination with propranolol (β-antagonist) or the effect of isoproterenol (β-agonist) in combination with prazosin ({alpha}-antagonist), obscure the responses to the natural (neuro)humoral stimulators norepinephrine and epinephrine. The next sections will make clear that there is more information about the inhibiting effect of {alpha}-adrenergic stimulation on Ito1 and that there is more sparse information about the effect of β-adrenergic stimulation on Ito1, but that we in fact do not know the overall effect (see also Refs. [62,63]).

3.4 Response to {alpha}-adrenergic stimulation
The most straightforward way to study the relevance of the {alpha}-adrenergic regulation of Ito1 is to test the effect of norepinephrine in the presence of β-adrenergic blockade (f.e. by propranolol). A decrease in peak current in response to {alpha}-adrenergic agonists (phenylephrine or methoxamine) or norepinephrine in combination with propranolol has been observed in rat ventricular myocytes [64,65] and in rabbit atrial [66] and ventricular myocytes [67]. Because this effect is sensitive to blockade by prazosin, it is considered an {alpha}1-AR-mediated effect [68,69]. Changes in action potential duration–and also cell shortening–are abolished in the presence of blockers of the L-type Ca2+ current, indicating that changes in Ito1 affect action potential duration only indirectly at least in species other than rats and mice (see also Sections 1 and 3.1). In man Ito1, encoded by the long isoform of human Kv4.3, but not by the short isoform, is inhibited by phenylephrine as well [70].

The heart harbors both {alpha}1A- and {alpha}1B-ARs. In rat and canine ventricular myocytes, stimulation of {alpha}1A-ARs decreases Ito1, because this inhibition is abolished by the {alpha}1A-AR antagonist WB4101 in rat [71] and by the {alpha}1A-AR antagonist niguldipine in dog [72]. The effect of stimulation of {alpha}1B-ARs is controversial [73,74]. Stimulation of {alpha}1A-ARs reduces not only Ito1 itself, but also the transcription of Kv4.2 and Kv4.3, resulting in chronically decreased Ito1 [75].

3.5 Response to β-adrenergic stimulation
Almost nothing is known of the overall (combined {alpha}- and β-adrenergic-mediated) effect of norepinephrine on Ito1. To our knowledge, we have to rely completely on experiments on single canine Purkinje cells of Nakayama and Fozzard almost two decades ago [76]. Norepinephrine and isoproterenol both dramatically increase peak and steady state current during depolarizing clamp steps. These effects are mimicked by forskolin and abolished by β-AR blockade with sotalol [76]. However, the β-AR-mediated increase in Ito1 is very small or absent if it is defined as the difference between peak current and steady state current at the end of the depolarizing clamp step (see Section 3.3). The nature of this time-independent component has received more attention since publication of the work of Nakayama and Fozzard in 1988 [76] and it has been labeled as Isus, but it remains questionable whether the β-adrenergic stimulation of Ito1 as claimed by Nakayama and Fozzard [76] in fact should be considered as either stimulation of this sustained component or even of the slow component of the delayed rectifier current (IKs). A role of β-ARs in the regulation of Ito1 has further been suggested in rabbit ventricular myocytes. Carvedilol, a nonselective β-AR antagonist, causes a concentration-dependent prolongation of action potential duration and a concentration-dependent inhibition of Ito1 [77]. However, it should be noted that blockade of basic Ito1 by a β-AR antagonist (carvedilol) is not the same as the abolishment of an increase in Ito1 caused by a β-AR agonist. It does not exclude direct action of carvedilol on the transient outward channel-independent from the β-AR.

By and large, it seems improbable that the clear reduction of Ito1 in response to {alpha}-AR stimulation (Section 3.4) can be overcome by β-AR-mediated effects under normal conditions.


   4. Function in the diseased heart
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 Abstract
 1. Introduction
 2. Molecular basis of...
 3. Function in the...
 4. Function in the...
5. Molecular regulation and...
 6. Potential phosphorylation...
 7. Conclusions
 References
 
4.1 Ischemia and infarction
Although action potential shortening during acute ischemia is a well-known phenomenon, its exact mechanism of action remains incompletely understood. The issue of involvement of ATP-sensitive K+ channels has never been completely resolved, because there is a mismatch between the moment of action potential shortening and the moment of decline in intracellular ATP (for review, see [78]). Moreover, it remains intriguing that the opening of ATP-sensitive K+ channels is not a prerequisite for potassium accumulation during acute ischemia (for review, see [79]). Early {alpha}-AR-mediated reduction of Ito1 may serve as an alternative explanation for action potential shortening during acute ischemia, because the early plateau is set at a more positive potential with concomitant more ICa-L inactivation. There are several indications for changes of the effects of catecholamines on membrane currents and action potential duration during acute ischemia [80]. Little is known on changes in Ito1 during acute ischemia. In murine ventricular cells, anoxia results in a clear-cut reduction of Ito1 [81]. During the subacute phases of ischemia, maintenance of expression of Ito1 may depend on anatomical and/or electrical integrity between the normal and diseased myocardium. In feline [82] and canine cell cultures [83] with absence of intercellular coupling, Ito1 disappears almost completely within days and in surviving subepicardial cells isolated 5 days after coronary occlusion in canine ventricle there is also a prominent decrease in Ito1 [84]. Disappearance of Ito1 leads to action potential shortening for reasons mentioned above (see also Sections 1 and 3.1). Interestingly, β-adrenergic stimulation (norepinephrine or isoproterenol) is able to restore the Ito1 density [83]. This effect is not acute. It occurs within 24 h after addition of either of the agonists [83].

4.2 Hypertrophy and heart failure
Even in a normal heart Ito1 has a regional distribution ((Sections 1, 2.2 and 3.1)). Therefore, it can be expected that cardiovascular diseases, which have a regional nature themselves, either enlarge this regional inhomogeneity or counterbalance it. Ito1 increases during hypertrophy [85–87], albeit that a decrease has also been reported in some animal models (for review, see [86]). The prolongation of the action potential, concomitant with an increase in Ito1, presumably results from the more negative level of the plateau with less ICa-L inactivation and probably less delayed rectifier activation. In contrast to hypertrophy, the final stage of heart failure is associated with a clear reduction in Ito1 [28,88,89]. However, this reduction in Ito1 does not relate to action potential shortening as during subacute ischemia [84], but to action potential prolongation [90–92]), indicating the involvement of other membrane currents. The reduction of Ito1 during end-stage heart failure may be restricted to subendocardial cells [28], thereby changing the endocardial–epicardial gradients in action potential duration and repolarization moments in failing hearts compared to normal hearts.

Interestingly, in several hypertrophy model systems, expression levels of Kv4.2 and Kv4.3 decrease with the progression of hypertrophy. In contrast, a reappearance of the "fetal" Kv1.4 subunit is found. However, expression of the latter is not sufficient to restore the original Ito1 current densities [93,94].

4.3 The diabetic heart
Similar as for some hypertrophy models, also the diabetic ventricle displays a Kv4.2 to Kv1.4 isoform switch [95]. Cardiomyocytes isolated from the diabetic heart display reduced Ito1, which reflects in decreased levels of Kv4.x, and in some studies of Kv1.4 too (reviewed in [96]). Application of insulin restores the Ito1 current densities after a lag time of several hours, indicative for the requirement of transcription and translation and protein trafficking to the sarcolemma. A same type of restoration process was observed with many other agents in diabetic cardiomyocytes [97,98]. It should be realized that the restoration of a pathophysiologically induced downregulation of any current (in this case Ito1 in diabetic rats) by norepinephrine is not the same as an interaction of norepinephrine with the kinetics of a current.

Also, oxidative stress, reflected in an altered redox state is a key determinant of Ito1 downregulation in several pathologies, including diabetic hypertrophy (for reviews on this subject, see [98,99]). Finally, an increased {alpha}1-adrenergic drive also produces reactive oxygen species through the activation of NADPH oxidase. The resulting activated ERK may phosphorylate Ito1 [100,101].

4.4 Other pathologies
Also in diseases other than myocardial ischemia, infarction, hypertrophy, heart failure or diabetes changes in the density of Ito1 occur. In Chagas' disease there is downregulation of Ito1, which is restored by exposure to norepinephrine or isoproterenol [102]. This effect is not mimicked by phenylephrine and, therefore, seems to be based on stimulation of β-ARs [102]. In the hyperthyroid rabbit, there is a prominent upregulation of Ito1 [103].

Finally, Ito1 density is substantially decreased in rats treated with reserpine resulting in a decrease of the tissue norepinephrine content by 97% [104]. This led to a decrease in Ito1 density by 49%. This–again–suggests that the sympathetic nervous system is involved in the regulation of the density of Ito1 [104]. A comparable downregulation of Ito1 is observed in diabetic rats (see previous section). Again, this downregulation is restored by norepinephrine and the effect takes about 24 h [105].


   5. Molecular regulation and intracellular pathways
Top
 Abstract
 1. Introduction
 2. Molecular basis of...
 3. Function in the...
 4. Function in the...
 5. Molecular regulation and...
6. Potential phosphorylation...
 7. Conclusions
 References
 
Kv1.4, Kv4.2 and Kv4.3 contain multiple consensus sites for PKA-, PKC-, ERK-, CamKII- and PKG-mediated phosphorylation. However, little is known about phosphorylation of the cardiac Kv1.4 and Kv4.x channels. In general, PKA-, PKC- and ERK-mediated phosphorylation decreases Ito1, while CamKII-mediated phosphorylation increases Ito1 current.

5.1 Regulation of Ito1 by protein kinase A (PKA)
Only few studies have addressed PKA-mediated regulation of Kv1.4 at the molecular level. Matthias et al. [106] used forskolin to increase PKA activity and thereby reduced Ito1 in Kv1.4 expressing HEK cells. Subsequently, forskolin-induced phosphorylation of Ser229 in HEK cells was found [107]. Remarkably, however, in this latter study, dbcAMP application resulted in enhanced Ito1 densities, which was independent from Ser229 phosphorylation. Moreover, mutation analysis demonstrated that Ser229 is involved in negative regulation of Kv1.4-mediated Ito1 current.

Xiao and McArdle [108] showed that the chemical protein phosphatase BDM concentration-dependently inhibits Ito1 in rat ventricular myocytes. However, 8-Br-cAMP reverses the inhibitory effect of BDM on Ito1. The BDM-induced suppression of Ito1 may be related to dephosphorylation of the Kv4 proteins. The peptide hormone relaxin inhibits Ito1 in rat atrial myocytes, which can be prevented by blockage of PKA activity by 5–24 amide [109]. Thus, relaxin inhibits Ito1 via PKA-dependent phosphorylation. Therefore, PKA seems to play a role in the regulation of Ito1. In constructs of respectively the N- and C-terminal domains of Kv4.2, Thr38 and Ser552 were found to be substrates for PKA in vitro. However, there is a preference for the C-terminal peptide over the N-terminal peptide, which suggests that there may be a preference for Ser552 over Thr38 when Kv4.2 is phosphorylated in vivo. The synthetic peptides were also expressed in COS-7 cells and after the addition of the AC activator forskolin phosphorylated N- and C-terminal peptides were detected [110]. Though phosphorylation studies using the COS-7 overexpression system should be considered with some caution, these results indicate that Thr38 and Ser552 within the Kv4.2 channel can be phosphorylated by endogenous PKA in an intact cell. Although PKA phosphorylates the {alpha}-subunit at position Ser552, the interaction with the ancillary subunit KChIP3 is required to inhibit Ito1 [111]. In contrast, in this setting, Thr38 is not involved in PKA-mediated decrease of Ito1. It was concluded that PKA must act on a complex consisting of pore-forming {alpha}-subunits in combination with ancillary subunits in order to downregulate Ito1. PKA-mediated phosphorylation of several cardiac ion channels has been shown to be dependent of A-kinase anchoring proteins (AKAPs) [112,113]. Specific AKAP isoforms direct PKA to submembrane sites and thereby promote phosphorylation of ion channels, thereby positively influencing efficiency and specificity of phosphorylation. Although no involvement of AKAPs in Ito1 channel subunits has been demonstrated so far, it is conceivable that they play a role.

5.2 Regulation of Ito1 by protein kinase C (PKC)
PKC-dependent inhibition of Kv1.4-based Ito1 has been observed in Xenopus oöcytes after prolonged PMA or endothelin stimulation [114,115]. Furthermore, it has been shown that Kv1.4 {alpha}-subunits become heavily phosphorylated by PKC in vitro. The PKC activator PMA reduces both Kv4.2 and Kv4.3 currents expressed in Xenopus oöcytes as well as native Ito1 in rat ventricular myocytes [64,116]. Pretreatment of the cells with the PKC inhibitors chelerythrine and staurosporine prevents the inhibiting effect of PMA [116]. Ito1 is also inhibited in canine ventricular myocytes after the addition of the PKC activator PDD, whereas the PKC inhibitor bisindolymaleimide has little effect on Ito1 [72]. Thus, activation of PKC is thought to inhibit Ito1.

Activation of the specific PKC{varepsilon} isoform by the DAG analogue DiC8 attenuates Ito1 in rats. This inhibition is found in epicardial cells, not in endocardial cells. This reflects the fact that in endocardial cells there is less PKC{varepsilon} to mediate inhibitory phosphorylation of Kv4 channels than in epicardial cells [117].

The Kv4.3 channel exists in two isoforms: a full-length variant and a shorter one, which has a 19-amino acid deletion in the C-terminus. The long isoform is predominant in rat myocytes [118,119] and in human myocytes [120]. The 19-amino acid sequence in the C-terminus of the long splice variant contains a consensus PKC phosphorylation site (RXXT*XK) after the last membrane-spanning segment. An isoform-specific regulation of Kv4.3 channel activity by PKC-mediated phosphorylation has been demonstrated [70,121]. Reconstitution of the long version in {alpha}1-AR expressing cells leads to a phenylephrine-dependent inhibition of Ito1. Furthermore, heteromeric channels of the long and short splice variants display a similar phenylephrine-mediated inhibition. Mutation of Thr503 in the long Kv4.3 isoform in alanine abolishes inhibition of Ito1 by the {alpha}1-AR agonist phenylephrine or the PKC activator PMA when compared to the wild-type channel [70]. Thus, the threonine at this position is a molecular target for {alpha}1-AR-mediated inhibition.

5.3 Regulation of Ito1 by extracellular-signal-regulated kinase (ERK)
The mitogen-activated protein kinase ERK plays an important role in synaptic plasticity, learning and memory. The amino acid sequence of Kv4.2 contains several consensus phosphorylation sites for this enzyme. Synthetic C-terminal peptides of Kv4.2 are phosphorylated at Thr602, Thr607 and Ser616 by ERK2. The C-terminal constructs are also phosphorylated by the enzyme when expressed in COS-7 cells. Also native Kv4.2 in the rat hippocampus is phosphorylated by ERK2 [122]. Kv4.2 is a substrate for ERK in hippocampal cells, but it still has to be determined whether cardiac Kv4.2 is also phosphorylated by ERK.

5.4 Regulation of Ito1 by protein tyrosine kinase (PTK)
Kv4.2 in rat ventricular myocytes contains a single consensus sequence for phosphorylation by PTK. It has been shown for other channels that PTK-mediated signalling operates downstream of PKC. However, mutation of Tyr 592 to phenylalanine does not result in altered K+ currents when compared to wild-type Kv4.2 proteins upon PKC activation [116]. In addition, no PTK consensus sequence has been determined in Kv4.3.

However, upregulation of Kv4.2 mRNA is prevented by exposing rat cardiac myocytes to the PTK inhibitors genistein and tyrphostin A25. Inhibiting PTK activity has a greater effect on the Kv4.2 expression in low-density than in high-density myocyte cultures [123]. Direct contact between myocytes is suggested to decrease protein tyrosine phosphorylation to regulate the mRNA expression of Kv4.2 channels.

5.5 Regulation of Ito1 by Ca2+ calmodulin-dependent kinase II (CaMKII)
Kv1.4 can be phosphorylated at multiple sites by CaMKII in vitro [124]. From subsequent in vivo studies in HEK cells, it appeared that Ser123 is the physiological relevant CaMKII target site. In response to phosphorylation at this site, a slowing of inactivation kinetics and an acceleration of recovery kinetics was observed, resulting in an increase in Ito1 [124].

Recent studies in human atrial myocytes have shown that inhibition of CaMKII results in a decreased Ito1 [125]. Similar as for Kv1.4, CaMKII activity slows Kv4.3 inactivation and accelerates the rate of recovery from inactivation. Mutation of the CaMKII consensus phosphorylation site Ser550 of Kv4.3 prevents the stimulatory effect of this kinase on Ito1 [126].


   6. Potential phosphorylation sites of transient outward channels based on Kv1.4, Kv4.2 and Kv4.3
Top
 Abstract
 1. Introduction
 2. Molecular basis of...
 3. Function in the...
 4. Function in the...
 5. Molecular regulation and...
 6. Potential phosphorylation...
7. Conclusions
 References
 
As described in Section 1, Ito1 activity and expression varies greatly between the different species. We compared Kv1.4, Kv4.2 and Kv4.3 amino acid sequences of several species with emphasis on phosphorylation sites. Amino acid sequences from Kv1.4, Kv4.2 and Kv4.3 subunits from different species were retrieved from GenBank. The alignments of the amino acid sequences of the Kv1.4, Kv4.2 and Kv4.3 channels are compared with the alignments made by Dixon et al. [36] and Serôdio et al. [127] and the intracellular domains were determined, since these can be a target for intracellular kinases only. The potential phosphorylation sites of PKA, PKC and PKG were determined by using the web-based program Netphos (http://www.cbs.dtu.dk/services/NetPhos/) (Fig. 1 and Table 1 for Kv1.4, Fig. 2 and Table 1 for Kv4.2, and Fig. 3 and Table 1 for Kv4.3). The protein kinase consensus sequences and the accession codes of the sequences that were used are listed in the legend of Table 1 and Figs. 1–3GoGo, respectively. Fig. 4 gives a summary of our analysis for Kv1.4, Kv4.2 and Kv4.3. It becomes obvious from our analyses that, first, Kv1.4, Kv4.2 and Kv4.3 are very well conserved during mammalian evolution. Second, most of the identified–potential–phosphorylation sites are conserved. Third, different roles of Ito1 in action potential formation in different mammalian species are not reflected in differences in the primary sequence of the protein.


Figure 1
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  		Fig. 1 Comparative alignment of the amino acid sequences of the Ito1 Kv1.4 {alpha}-subunit of human, mouse, ferret and rat. The S1–S6 segments are indicated. Potential phosphorylation sites (see also Table 1) were labeled as follows. Unique phosphorylation sites: PKA (arrow), (PKC (diamond). Common phosphorylation sites: PKA and PKC (open circle). Identical residues for all species are indicated by white lettering over black shading. Abbreviations and accession numbers. Hs: Homo sapiens, AAA61275; Mm: Mus musculus, NP_067250; Mp: Mustela putorius, AAB_60261; Rn: Rattus norvegicus, NP_037103.

 

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  		Table 1 Potential and proven phosphorylation sites of the Kv4.2 and Kv4.3 subunit in different species

 

Figure 2
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  		Fig. 2 Comparative alignment of the amino acid sequences of the Ito1 Kv4.2 {alpha}-subunit of human, mouse, ferret, rabbit and rat. The S1–S6 segments are indicated. Potential phosphorylation sites (see also Table 1) were labeled as follows. Unique phosphorylation sites: PKA (arrow), PKC (diamond). Common phosphorylation sites: PKA and PKG (closed circle). Identical residues for all species are indicated by white lettering over black shading. Abbreviations and accession numbers. Hs: Homo sapiens, AJ010969; Mm: Mus musculus, NM_019697; Mp: Mustela putorius, AY147192; Oc: Oryctolagus cuniculus, AF508735; Rn: Rattus norvegicus, S64320.

 

Figure 3
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  		Fig. 3 Comparative alignment of the amino acid sequences of the Ito1 Kv4.3 {alpha}-subunit of human, mouse, rabbit and rat. The S1–S6 segments are indicated. Potential phosphorylation sites (see also Table 1) were labeled as follows. Unique phosphorylation sites: PKA (arrow), PKC (diamond); PKG (triangle). Common phosphorylation sites: PKA and PKC (open circles). Identical residues for all species are indicated by white lettering over black shading. Abbreviations and accession numbers. Hs: Homo sapiens, NM_004980; Mm: Mus musculus, NM_019931; Oc: Oryctolagus cuniculus, AF198445; Rn: Rattus norvegicus, AB003587.

 

Figure 4
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  		Fig. 4 Schematic representation of Kv1.4, Kv4.2 and Kv4.3 (long version). Transmembrane regions are indicated by vertical black thick lines. Phosphorylation sites are indicated as follows: PKA, circles; PKC, triangles; PKG, squares. Open symbols represent potential phosphorylation sites; closed symbols represent phosphorylation sites with experimental evidence.

 

   7. Conclusions
Top
 Abstract
 1. Introduction
 2. Molecular basis of...
 3. Function in the...
 4. Function in the...
 5. Molecular regulation and...
 6. Potential phosphorylation...
 7. Conclusions
References
 
There is a large variability between different mammalian species for the role of Ito1 in action potential formation. In guinea pig, Ito1 is virtually absent in working myocardium. In small mammals, Ito1 is the major determinator of action potential duration, while in larger animals Ito1 sets the level of the plateau phase, and thereby indirectly influences action potential duration. In most animals, Ito1 displays a regional distribution over the ventricle, having a higher density at the subepicardium than at the subendocardium. Nevertheless, the primary sequences of the protein subunits underlying Ito1, Kv1.4, Kv4.2 and Kv4.3, are remarkably well conserved between all mammalian species of which these sequences are available.

Cardiac diseases affect the expression of Ito1, thereby either increasing of decreasing the spatial Ito1 distribution. In general, ischemia, infarction and end-stage heart failure decrease Ito1 densities, while compensatory hypertrophy tends to increase Ito1. Adrenergic effects seem to be involved in at least some of these Ito1 regulating processes during heart disease.

Although far from complete, the current picture of adrenergic regulation of Ito1 shows an important role of {alpha}-adrenergic-mediated inhibition of Ito1. Concomitant β-adrenergic stimulation appears to counteract the {alpha}-adrenergic effect, at least in part. However, it is unclear whether β-adrenergic regulation can stimulate Ito1 in the absence of {alpha}-adrenergic inhibition. The effects of {alpha}- and β-adrenergic stimulation are exerted by phosphorylation of the Ito1 channel subunits by PKA, PKC and probably PKG.

Using Netphos, the potential PKA, PKC and PKG phosphorylation sites of the Kv1.4, Kv4.2 and Kv4.3 subunits of various mammals were determined. It can be concluded that virtually all phosphorylation sites are conserved during evolution.


   Acknowledgements
 
We want to thank Pieter Doevendans for his efforts to enable this work. This study was supported by ZonMW grant MKG.5942 (MvdH).


   Notes
 
Time for primary review 19 days


   References
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 3. Function in the...
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 5. Molecular regulation and...
 6. Potential phosphorylation...
 7. Conclusions
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