Skip Navigation

Cardiovascular Research 2005 65(1):28-39; doi:10.1016/j.cardiores.2004.09.028
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by van der Heyden, M. A.G.
Right arrow Articles by Opthof, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by van der Heyden, M. A.G.
Right arrow Articles by Opthof, T.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2004, European Society of Cardiology

Molecular aspects of adrenergic modulation of cardiac L-type Ca2+ channels

Marcel A.G. van der Heyden*, Tessa J.M. Wijnhoven and Tobias Opthof

Department of Medical Physiology, University Medical Center Utrecht, P.O. Box 85060, 3508 AB Utrecht, The Netherlands

* Corresponding author. Tel.: +31 30 2538900; fax: +31 30 2539036. Email address: m.a.g.vanderheyden{at}med.uu.nl

Received 28 July 2004; revised 22 September 2004; accepted 29 September 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Structure
 3. Function
 4. Molecular regulation and...
 5. Potential phosphorylation...
 6. Conclusions
 References
 
L-type Ca2+ channels are predominantly regulated by β-adrenergic stimulation, enhancing L-type Ca2+ current by increasing the mean channel open time and/or the opening probability of functional Ca2+ channels. Stimulation of β-adrenergic receptors (ARs) results in an increased cyclic adenosine monophosphate (cAMP) production by adenylate cyclase (AC) and consequently activation of protein kinase (PK) A and phosphorylation of L-type Ca2+ channels by this enzyme. β1-Adrenergic receptors couple exclusively to the G protein Gs, producing a widespread increase in cAMP levels in the cell, whereas β2-adrenergic receptors couple to both Gs and Gi, producing a more localized activation of L-type Ca2+ channels. Other signaling intermediates (protein kinase C, protein kinase G or protein tyrosine kinase (PTK)) either have negative effects on L-type Ca2+ current, or they interact with the stimulatory effect of the protein kinase A pathway.

KEYWORDS Adrenergic (ant)agonists; Ca-channel; Ca (cellular); G-proteins; Protein kinase A; Protein kinase C; Protein kinase G; Protein phosphatase; Protein phosphorylation; Second messengers; Tyrosine protein kinase

Abbreviations: AR, adrenergic receptor • AC, adenylate cyclase • AKAP, A kinase anchoring protein • ATP, adenosine triphosphate • Ca2+, calcium • cAMP, cyclic adenosine monophosphate • cGMP, cyclic guanosine monophosphate • DAG, 1,2-diacylglycerol • GC, guanylate cyclase • G protein, guanosine 5'-triphosphate (GTP)-binding protein • GSNO, nitrosoglutathione • ICa-L, L-type Ca2+ current • InsP3, inositol 1,4,5-triphosphate • NO, nitric oxide • PIP2, phosphatidylinositol 4,5-biphosphate • PK, protein kinase • PLC, phospholipase C • PTK, protein tyrosine kinase • Ser, serine • Thr, threonine


   1. Introduction
Top
 Abstract
 1. Introduction
2. Structure
 3. Function
 4. Molecular regulation and...
 5. Potential phosphorylation...
 6. Conclusions
 References
 
When Orkand and Niedergerke [1] described an inward Ca2+ current in Science in 1964, this current was not yet known as the L-type Ca2+ current (ICa-L). The earlier work on the fast inward Na+ current during the years after the Second World War in squid axon by Weidmann and Coraboeuf and many others had attracted more attention. Orkand and Niedergerke [1] observed that the inward Ca2+ current peaked ‘late’ (in fact it was only 20–30 ms) after the upstroke of the action potential. This "second inward current" was interpreted as something that nature had found to help transform the very short action potential of nerve tissue and skeletal muscle into a cardiac action potential with its substantial longer duration. It contributed to insight in one of the unique characteristics of the ventricle. The long duration of its action potentials causes equally long refractoriness, which protects against re-entrant arrhythmias [2], but also prevents tetanic type of contractions, incompatible with the cyclic function of the heart. Furthermore, the role of the cardiac ICa-L was instrumental to the notion that the heart relies almost completely on an intracellular reallocation type of Ca2+ homeostasis, very different from that of skeletal muscle. This might suggest that ICa-L has a relatively late appearance during evolution. This is not the case. In invertebrate species the inward Ca2+ current and the so-called transient outward current are the first membrane currents which appear during early development [3]. Recent studies in mammalian embryonic tissue [4] have confirmed that this also applies to vertebrates and the recent developments in research on embryonic stem cells have corroborated this notion (see Ref. [5] for references). Thus, ICa-L is also an ‘early current’ in several types of cells developing in the cardiovascular direction and derived from embryonic stem cells from mouse [6] and man [7] or from murine carcinoma cells [8].

ICa-L constitutes the dominant factor in mediating positive inotropy in all types of cardiac tissue [9]. It also contributes to physiological frequency regulation in the sinus node [10]. Thirdly, it is an important parameter for the duration of the plateau phase of the action potential and is thereby a major determinant of action potential duration and refractoriness. These three physiological functions are under control of catecholamines of circulating and neurohumoral origin. In this brief review we focus on known and putative sites of adrenergic-induced phosphorylation of the L-type Ca2+ channel.


   2. Structure
Top
 Abstract
 1. Introduction
 2. Structure
3. Function
 4. Molecular regulation and...
 5. Potential phosphorylation...
 6. Conclusions
 References
 
Voltage-gated Ca2+ channels are heteromultimeric protein complexes. The three-dimensional structure of the bovine cardiac L-type calcium channel has recently been resolved [11] (reviewed in Ref. [12]). The largest subunit (~190–240 kDa) is the poreforming {alpha}1 subunit, which is associated with an intracellularly located β subunit (~55 kDa) and a mostly extracellularly located disulfide-linked {alpha}2{delta} subunit (~170 kDa). The transmembrane {alpha}1 subunit contains four homologous domains (I–IV), each of which is composed of six membrane-spanning {alpha} helices (S1–S6) [13–15]. The S5 and S6 segments and the membrane-associated pore loop (P-loop) between them form the central pore through which ions flow down their electrochemical gradient. The P-loop contains four negatively charged glutamate residues that are required for the Ca2+ selectivity of the channel [13–17]. The fourth transmembrane segment (S4) in each homologous domain contains a positively charged residue (arginine or lysine) at every third or fourth position. This segment serves as the voltage sensor for gating. Moving outward and rotating under the influence of the electric field after depolarization of the membrane, the S4 segments initiate a conformational change that opens the central pore. Thus, the S4 segment controls switching between open and closed conformations of the channel and thus determines whether current will flow [14–16]. The S6 segments form the receptor sites for the pore-blocking Ca2+ antagonist drugs specific for L-type Ca2+ channels. These segments, together with a motif in the cytoplasmic linker between domains I and II and a motif in the cytoplasmic C-terminus, also provide voltage-dependent channel inactivation [17]. Several {alpha}1 subunits have been identified and the {alpha}1C isoform is the one that is expressed at high levels in cardiac muscle, but also in smooth muscle and in the brain [16].

The {alpha}1C subunit interacts with accessory subunits and especially the β subunit is required to form fully functional Ca2+ channels and/or to alter certain channel properties. Accessory subunits determine the activation and inactivation kinetics of the channels. The β subunit also controls targeting of the {alpha}1C subunit to the membrane [17,18]. The cytoplasmically located β subunit is strongly hydrophilic. A highly conserved 18-amino acid sequence in the cytoplasmic loop connecting domains I and II has been identified as the interaction domain of the {alpha}1 subunit for the β subunit [13–15,17].

The {alpha}2{delta} complex, which is less tightly associated with the {alpha}1 subunit, consists of an extracellularly located {alpha}2 subunit linked to a hydrophobic membrane-spanning {delta} subunit. The {alpha}2 subunit is very hydrophilic and has many glycosylation sites. The {alpha}2 and {delta} subunits are encoded by a single gene. The mature forms of these subunits are derived by post-translational proteolytic processing, but they remain associated through a disulfide bond [13,14,17,18]. The extracellular {alpha}2 subunit interacts with the S5–S6 linker in domain III of the {alpha}1 subunit [17].


   3. Function
Top
 Abstract
 1. Introduction
 2. Structure
 3. Function
4. Molecular regulation and...
 5. Potential phosphorylation...
 6. Conclusions
 References
 
3.1. Basic function
From all cardiac ion currents the ICa-L is the most extensively studied. Excellent and extensive reviews on its basic kinetics and interaction with several types of ligands are available [19,20]. ICa-L links membrane depolarization to contraction of the heart by the fact that the Ca2+ ions that enter the cell during the depolarization (see below) give rise to subsequent far more massive Ca2+ release from the sarcoplasmic reticulum into the cytosol. The channels are closed at the resting potential, but activate upon depolarization. L-type Ca2+ channels are activated at relatively positive voltages, with a threshold at about –30 mV [19,20]. These features are even present at early embryonic stage ([21] and references therein). L-type Ca2+ channels are further distinguished by a large single channel conductance, a slow voltage-dependent inactivation, marked regulation by protein kinase (PK)A-dependent pathways, and a specific high affinity for Ca2+ channel blockers. These Ca2+ currents have been designed L-type, as they conduct large, relatively long-lasting currents [14,16,19,20]. Interestingly, and unlike in neurons, inactivation also occurs when Ba2+ has taken the place of Ca2+ as charge carrier [22]. Therefore, inactivation cannot solely be due to a rise in intracellular Ca2+.

The density of ICa-L increases fivefold in the first 7 days after birth in primary cultures of newborn rat ventricular myocytes bridging the gap in density between freshly isolated newborn cells (1.6 pA/pF) and freshly isolated adult cells (8.1 pA/pF) [23]. Such an increase in density has also been demonstrated in rabbit ventricular cells [24]. Interestingly, this increase is not homogeneous over the sarcolemma: the developing T-tubule system strongly expresses L-type Ca2+ channels, leading to a threefold higher density in T-tubules compared to the rest of the sarcolemma at least in the rat [25]. The amount of functional L-type Ca2+ channels, and maybe also expression, decreases again with aging [26].

3.2. Adrenergic receptors
Adrenergic receptors (ARs) are G protein-coupled receptors, which contain seven hydrophobic membrane-spanning {alpha}-helical domains. Highest amino acid conservation is present in the transmembrane regions, which determine the specificity of ligand binding. The cytoplasmic regions, which interact with other cellular proteins to mediate various signaling events, have more variability [27].

In the human heart nine AR subtypes exist, which mediate a variety of cellular functions. They are encoded by distinct genes. The most abundant types are the β-ARs. There are three subtypes: β1, β2 and β3. The β1-AR and β2-AR couple to Gs proteins to activate adenylate cyclase (AC), which mediates the conversion of adenosine triphosphate (ATP) into cyclic adenosine monophosphate (cAMP). This leads to the activation of PKA, which in turn phosphorylates several substrates, including L-type Ca2+ channels. The β2-ARs also couple to Gi proteins, which counteract the Gs coupled activation of AC, resulting in a reduction of cAMP levels [27–30]. The physiological impact as well as the mechanism of action of β3-ARs is less clear, although a more prominent role in heart failure has been suggested. Because β3-ARs have been reported to produce negative inotropy in human ventricle, a future therapeutic modality might be their blockade in the setting of heart failure (see Ref. [31] 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 and is coupled via a Gq protein to the activation of phospholipase C (PLC), which causes formation of InsP3 and DAG. The latter mediates the activation of PKC, which phosphorylates many substrates, including L-type Ca2+ channels. Also, three {alpha}2-ARs ({alpha}2A, {alpha}2B and {alpha}2C) exist in the human heart.

Interestingly, L-type Ca2+ channel mRNA levels are increased by β-adrenergic signaling, while {alpha}-adrenergic signaling produces the reciprocal effect [32].

3.3. Response to adrenergic stimulation
Phosphorylation of L-type Ca2+ channels promotes Ca2+ influx and thus enhances myocyte contraction. L-type Ca2+ channels are regulated by different kinases, including PKA, PKC, PKG and protein tyrosine kinase (PTK) (see Sections 4.1–4.4). They are also regulated by G protein subunits in vitro. Both the cardiac {alpha}1C and β2a subunits of L-type Ca2+ channels have been demonstrated to be direct targets of phosphorylation. Multiple modes of gating have been observed at the single channel level: mode 0 in which channels do not open or open very rarely in response to depolarization, mode 1 in which the probability of opening is low with brief open times, and mode 2 in which the probability of opening is much higher and the openings are long-lasting and the closings are brief [14,33]. The increase in Ca2+ currents observed after the activation of PKA are due to an increase in the open state probability of the channel, resulting from a shift in gating mode [17,34].

There is an enormous literature on the effects of catecholamines on ICa-L which can be subdivided between data obtained in multicellular preparations and in isolated cells/single channels. Also, a subdivision can be made between the effects of {alpha}-adrenergic and β-adrenergic effects. Within the context of this paper it is impossible to review this literature in detail. We wish to underscore here that serum has been reported to inhibit basal ICa-L [35] and discrepancies between older literature (often on multicellular preparations) and more recent data (often on (sub)cellular preparations) is in part caused by this confounding factor.

In summary, data obtained in multicellular preparations [36–38] and in isolated cells [20] point to an increase of ICa-L by β1-adrenergic stimulation (Section 3.5). The peak inward current increases primarily by a decrease of the closed time of the channels. {alpha}-Adrenergic stimulation is not as effective as β-adrenergic stimulation in multicellular preparations [36–39]. In isolated cells the direct effects are also controversial [20,40,41] (see Section 3.4). It should be noted, however, that methodological aspects are involved because perforated patch-clamp recordings have demonstrated a clear-cut increase in ICa-L after stimulation of the {alpha}1-AR [42].

3.4. Response to {alpha}-adrenergic stimulation
Activation of {alpha}1-ARs in adult rat ventricular cells does not affect ICa-L, but in neonatal rat ventricular myocytes the {alpha}1-adrenergic agonist phenylephrine concentration-dependently increases ICa-L [43]. This stimulating effect of phenylephrine is reversed by the nonselective {alpha}1-AR antagonist prazosin. Clonidine, an {alpha}2-AR agonist, has no effect on ICa-L. The {alpha}2-AR antagonist yohimbine and the β-AR antagonist propanolol do not inhibit the effect of phenylephrine on ICa-L, whereas an {alpha}1A-AR antagonist, but not an {alpha}1B-AR antagonist, abolishes the effect of phenylephrine. In the presence of propranolol, the nonselective adrenergic agent norepinephrine also increases ICa-L in neonatal rat [43]. These results suggest that the increase in ICa-L in neonatal rat ventricular cells is mediated via {alpha}1A-ARs, although an inhibition of ICa-L in neonatal rat ventricular myocytes in response to phenylephrine has been reported as well [44].

3.5. Response to β-adrenergic stimulation
The effects of stimulation of β-ARs on ICa-L are predominant over those of {alpha}-ARs. Although three types of β-ARs exist in the human heart [31], the effects of stimulation of β1-ARs and β2-ARs are more important in the mammalian heart and concern an increase in contractility, heart rate, and amplitude of the cardiac action potential. The increase in ICa-L by β-adrenergic stimulation is not caused by a change in single channel conductance or in the number of functional channels, but by an increase in the mean channel open time and the probability of channel opening. Activation of β-ARs results in a shift of gating mode 0 to gating modes 1 and 2 [14,34]. Thereby β-adrenergic stimulation results in an increased number of channels being open at a time, which can explain the increase in ICa-L.

The non-selective β-AR agonist isoproterenol increases ICa-L by augmenting cAMP levels [45,46]. However, the increase in Ca2+ influx via L-type Ca2+ channels in response to β-AR stimulation also acts as a negative feedback on the AC activity. L-type Ca2+ channels are probably already phosphorylated under basal conditions, because the decrease of ICa-L by the PKA inhibitor H-89 can be reversed with either forskolin or the PP inhibitor okadiac acid.

The stimulatory effect of the β2-AR agonist zinterol on ICa-L in frog ventricular myocytes is maximal and not additive to the stimulatory effects of isoproterenol. The PKA inhibitor PKI reverses this effect of zinterol. Therefore, the increase in ICa-L via β2-ARs probably results from stimulation of AC and phosphorylation of the Ca2+ channels by PKA [47].

The β1-AR activates Gs proteins, but dual coupling of β2-ARs to Gs and Gi proteins in rat ventricular myocytes has been demonstrated [48]. After treatment with the Gi inhibitor pertussis toxin, the β2-AR-stimulated increase of ICa-L is enhanced, while the effect of β1-AR stimulation on these Ca2+ currents is unaffected. This indicates that a coupling occurs between β2-ARs and Gi proteins, exerting negative feedback on the cellular responses to β2-AR stimulation [30,48,49] (but see also Ref. [50]).

There is evidence that β-AR stimulation is also involved in myocyte apoptosis [51]. β-Adrenergic modulation of ICa-L via Gs proteins is gradually established during development. In myocytes at early developmental stage, forskolin has a weak stimulatory effect on ICa-L, whereas isoproterenol has no effect at all. However, within a couple of days these substances become effective both in developing cardiomyocytes derived from embryonic tissues and in the embryos themselves [52,53]. The reduced β-adrenergic response in very early cells is, at least partially, due to the high intrinsic activity of protein phosphatases and phosphodiesterases [52].


   4. Molecular regulation and intracellular pathways
Top
 Abstract
 1. Introduction
 2. Structure
 3. Function
 4. Molecular regulation and...
5. Potential phosphorylation...
 6. Conclusions
 References
 
4.1. Regulation of ICa-L by protein kinase A
Activation of β-ARs results in the activation of ICa-L (see Section 3.5) along many pathways (see Sections 4.2–4.4). The pathway via PKA, which will ultimately lead to phosphorylation of residues of the channel itself, causes an increase in ICa-L. Activation of G{alpha}s subunits by β-AR agonists (i) stimulates AC (ii), the enzyme that mediates the conversion of ATP into cAMP (iii). Binding of cAMP to the regulatory subunits of PKA (iv) results in the liberation of the catalytic subunits (v), which phosphorylate specific serine and threonine residues of the L-type Ca2+ channel (vi) [13,34]. The localization of AC is close to the L-type Ca2+ channels in the T-tubules [54]. There is evidence that the β-AR colocalizes with caveolin3, a component of caveolae [55] and the same has been demonstrated for AC [56]. This needs not be in conflict, because it is possible that caveolae and T-tubules are associated as well [57].

Two forms (of different size) of the main subunit ({alpha}1C) of the L-type Ca2+ channel have been detected: a full-length form of ~240–250 kDa and a C-terminally truncated form of ~190–210 kDa. The full-length rabbit {alpha}1C subunit is phosphorylated both in vitro and in vivo by PKA in response to elevated cAMP concentrations, but the truncated channel subunit is not [34,53–61]. In intact cardiac myocytes, the majority of {alpha}1C subunits are full-length. The truncated form of the {alpha}1C subunit is generated by post-translational proteolytic processing [53]. The C-terminal fragment of 30–50 kDa contains a proline-rich domain, which mediates membrane association. Deletion of either the proline-rich domain or truncation of the C-terminus results in an increase of ICa-L, which suggests that a region in the C-terminal domain has an inhibitory effect on the function of L-type Ca2+ channels [62–64].

According to previous literature, the full-length rabbit cardiac {alpha}1 subunit contains six potential PKA phosphorylation sites: Ser 124 in the N-terminal part, and five others in the C-terminal part at positions 1575, 1627, 1700, 1848, and 1928. Mutation of Ser 1928 to alanine results in complete loss of cAMP-mediated phosphorylation and in reduction of ICa-L [34,65]. The C-terminally truncated {alpha}1C subunit lacks Ser 1928 and, thereby, is no longer a substrate for PKA, confirming that, despite the presence of six putative sites, Ser 1928 is the only site, which is in fact phosphorylated by PKA in the {alpha}1C subunit [34,59–61]. A previous report on the phosphorylation of the {alpha}1C subunit by PKA at Ser 1627 and possibly Ser 1700 [18], has not been confirmed.

Besides phosphorylation, dephosphorylation is also a strictly regulated process. The protein phosphatase inhibitors okadaic acid, microcystin and calyculin A inhibit dephosphorylation of the {alpha}1C subunit, albeit in different ways [33,66,67]. Protein phosphatase 2A binds to the 557 amino acids of the C-terminal of the {alpha}1C subunit and reverses phosphorylation of Ser 1928 of the L-type Ca2+ channel by PKA [33].

None of the important sites phosphorylated by PKA in skeletal muscle are conserved in the cardiac isoform of the channel, and in reverse, the cardiac phosphorylation site (Ser 1928) is not conserved in skeletal muscle {alpha}1s [14,18,65].

Besides the {alpha}1C subunit, also the β2 subunit is a second important target of PKA [68]. PKA still increases ICa-L generated by channels with a truncated {alpha}1C subunit, when they are associated with a wild type β2a subunit [61]. Although the rat β2a subunit contains two strong consensus sites for PKA-mediated phosphorylation at Thr 164 and Ser 591, the actual sites of PKA-mediated phosphorylation are at other residues, because mutants that lack both of the consensus sites remain good substrates for phosphorylation by PKA [69]. Phosphopeptide mapping and β2a truncation demonstrated that the major sites of PKA-mediated phosphorylation occur at three loose consensus sites for PKA: Ser 459, Ser 478 and Ser 479. Mutation of Ser 459 to alanine results in a reduced rate and degree of phosphorylation of the β2a subunit by PKA [69], without altering the basic functional properties of the regulatory β2a subunit [61]. Mutation of Ser 478 and Ser 479 to alanine, however, completely abolishes the PKA-induced phosphorylation [69] and prevents PKA-induced ICa-L [34,61]. Phosphorylation of the β2a subunit at Ser 478 and Ser 479 is pivotal for the regulation of the cardiac L-type Ca2+ channel in response to PKA. Phosphorylation of the other associated subunit, the {alpha}2{delta} complex, which is less tightly associated with the {alpha}1 subunit and consists of an extracellular subunit, has not been detected [34].

For the regulation of the L-type Ca2+ channel by PKA, localization of the enzyme to the Ca2+ channel is required. PKA is often anchored to specific subcellular compartments by PKA anchoring proteins (AKAPs). These proteins contain a targeting domain that directs the AKAP to a specific cellular site, and a kinase anchoring domain that binds the regulatory subunits of PKA [14,61]. Targeting PKA in close proximity to the L-type Ca2+ channel by an AKAP may facilitate phosphorylation of the channel. Anchoring of PKA to the membrane through association with AKAP79 indeed facilitates PKA-mediated phosphorylation of Ser 1928 in the rabbit {alpha}1C subunit. AKAP15 directly interacts with {alpha}1C through a leucine zipper motif present in the C-terminal tail of the subunit [70]. Phosphorylation of the β2a subunit however does not require an AKAP [53,60,61]. Thus, for appropriate PKA-dependent phosphorylation and stimulation of L-type Ca2+ channels the enzyme has to be anchored to the membrane by an AKAP. Another important giant sarcolemmal protein (AHNAK) with comparable function has been described as well [71,72].

4.2. Regulation of ICa-L by protein kinase C
Activation of Gq subunits by {alpha}-ARs (i) stimulates PLC (ii), which hydrolyzes phosphatidylinositol 4,5-biphosphate (PIP2) to inositol 1,4,5-triphosphate (InsP3) and diacylglycerol (DAG) (iii). The latter activates PKC (iv), which in turn phosphorylates many substrates, including L-type Ca2+ channels (v) [34].

It has been shown recently that the {alpha}1C subunit of the L-type Ca2+ channel contains two alternative first exons, exon1a and 1b, which display tissue specific expression in human and rat mediated by alternative promoter usage [73–76]. Exon1a is specifically expressed in cardiac tissue, and codes for a 46 amino acid region of the N-terminus in contrast to the 16 amino acid short N-terminal version coded for by exon1b. The activation of PKC results in a decrease or in a transient increase followed by a decrease of cardiac ICa-L. Deletion of the initial 46 amino acids of the long version N-terminus of the rabbit {alpha}1C subunit increases Ca2+ currents [77,78] by increasing single channel open probability with an order of magnitude [77]. Similar findings were observed when comparing the long and short N-terminus form of the human {alpha}1C channel [73]. Therefore, the first 46 amino acids of the N-terminus of the {alpha}1C subunit have a long-term negative effect on channel gating.

Co-expression of the β2a subunit increases ICa-L, but less in the N-terminal deletion mutant channel than in channels with the full-length {alpha}1C subunit. The β2a subunit also counteracts the inhibitory effect of PKC. It is proposed that there is an interaction between the β2a subunit and the N-terminus of the {alpha}1C subunit, resulting in an allosteric competition with the N-terminus to exert its inhibiting effect on gating. The first 5 amino acids of the N-terminus have been identified as very important and the first 20 amino acids as crucial for the inhibitory effect on gating of the {alpha}1C subunit of the L-type Ca2+ channel gating and for the interaction between the {alpha}1C subunit and the β2a subunit [79]. Interestingly, none of the first 5 amino acids of the {alpha}1C subunit are Ser or Thr. Thus, PKC cannot directly phosphorylate this segment. The N-terminus of the rabbit cardiac {alpha}1C subunit contains two putative PKC phosphorylation sites at Thr 27 and Thr 31, but there are conflicting data on the question whether phosphorylation of these sites in fact occurs [78,79] and, if they occur, whether they are relevant for function [34,79]. Conflicting findings have also been observed in studies with direct activators of PKC. It has been suggested that distinct isoforms of PKC may have opposing effects on L-type Ca2+ channels [13,34].

4.3. Regulation of ICa-L by protein kinase G
It is difficult to present a clear outline of the effect of the PKG pathway on the regulation of ICa-L, because it is not clear whether the effect of PKG induces direct phosphorylation of the L-type Ca2+ channel or whether the inhibitory effect of PKG on the PKA pathway, resulting in a decreased cAMP formation, is predominant. Moreover, the cyclic guanosine monophosphate (cGMP)/PKG pathway affects the response to adrenergic stimulation despite the fact that the pathway itself is not directly activated by ARs. Thus, the primary activator of the pathway is not an adrenergic agonist but NO (i), which increases the formation of cGMP from GTP mediated by the cytoplasmic GC (ii). cGMP exerts both stimulatory and inhibitory effects on ICa-L. This second messenger activates PKG (iii), which either directly phosphorylates the L-type Ca2+ channel (iv) or activates a protein phosphatase (v) that dephosphorylates the L-type Ca2+ channel (vi). It also stimulates phosphodiesterase 2 (vii), which reduces cAMP levels (viii) and thus inhibits stimulation of the L-type Ca2+ channel by PKA [13].

According to previous literature, the rabbit {alpha}1C subunit contains two potential PKG phosphorylation sites at Ser 533 and Ser 1371. PKG inhibits rabbit ICa-L by phosphorylating the {alpha}1C subunit of the channel at Ser 533 [15]. Ser 1371 is located in the fourth transmembrane segment of domain IV of the {alpha}1C subunit. So it is not possible that this residue is phosphorylated by PKG in vivo, because only intracellularly located residues are potential targets.

In some cell preparations, a PKG-mediated effect can only be observed after prior activation of the L-type Ca2+ channel by PKA [15,80]. The cGMP analogue 8-BrcGMP has no effect on basal single channel gating in mice, but diminishes the PKA-induced activation of L-type Ca2+ channels. It still has to be investigated whether cGMP exerts this effect via a direct interaction with PKA or by the activation of PKG [80]. PKG can also activate a sarcolemma bound-protein phosphatase, which dephosphorylates L-type Ca2+ channels that were previously phosphorylated by PKA [13]. Finally, there is also evidence that cGMP exerts its inhibitory effect via cGMP-stimulated phosphodiesterase activity, which results in the breakdown of cAMP and subsequent reduction of PKA-mediated increase in ICa-L [13,15,81].

In frog ventricular myocytes the NO donor sodium nitroprusside inhibits stimulation of L-type Ca2+ channel activity by the β-adrenergic agonist isoproterenol or by the AC activator forskolin via activation of cGMP-stimulated phosphodiesterase-2. The effect is reversed by scavenging NO or by the inhibition of phosphodiesterase-2 [82]. Thus in frog myocytes, stimulation of guanylate cyclase (GC) by NO leads to a reduction of cAMP levels near the L-type Ca2+ channels due to activation of phosphodiesterase-2 and thus inhibits stimulation of the L-type Ca2+ channel by PKA.

Developmental aspects seem to be involved, because PKG seems to increase basal ICa-L in newborn rabbit ventricular cells, but not in adult myocytes. Different isoforms of PKG exist and differing ratios of these isoforms in newborn compared to adult rabbit myocytes may be responsible for different roles of cGMP depending of developmental stages [81].

4.4. Regulation of ICa-L by protein tyrosine phosphorylation
There is evidence for a role of tyrosine phosphorylation in regulating myocardial β-adrenergic responses, because β-adrenergic stimulation of L-type Ca2+ channel activity by isoproterenol is antagonized by a number of phosphotyrosine phosphatase inhibitors [83]. However, no clear overall picture has emerged at this moment. Stimulatory as well as inhibitory effects of PTK inhibitors on ICa-L have been reported. For example, the PTK inhibitor genistein increases ICa-L in human atrial myocytes [84], but reduces ICa-L in guinea pig ventricular myocytes [85]. PKC seems to be involved in the mechanism [84]. It has been hypothesized that genistein inhibition of membrane-bound PTK decreases ICa-L, whereas inhibition of cytosolic PTK increases ICa-L [17]. Others report that genistein inhibits ICa-L by a tyrosine kinase independent mechanism [86].


   5. Potential phosphorylation sites of L-type Ca2+ channels
Top
 Abstract
 1. Introduction
 2. Structure
 3. Function
 4. Molecular regulation and...
 5. Potential phosphorylation...
6. Conclusions
 References
 
There may be more phosphorylation sites in the {alpha}1C subunit of L-type Ca2+ channels than the ones found in literature. Amino acid sequences from the {alpha}1C subunits of different species, containing the exon1a coded region, were retrieved from the GenBank database. The alignment of the amino acid sequences was compared with the alignment made by Mikami et al. [87]. The different domains (extracellular, transmembrane and intracellular) were determined, because only the intracellular domains will be potential targets for PKs. Fig. 1 and Table 1 show the potential phosphorylation sites of PKA, PKC and PKG in man (Hs), guinea pig (Cp), mouse (Mm), rabbit (Oc) and rat (Rn) as determined by using NetPhos (http://www.cbs.dtu.dk/services/NetPhos/). The PK consensus sequences are listed in Table 1. Accession codes of the used sequences are listed in the legend of Fig. 1.


Figure 1
Figure 1
Figure 1
View larger version (370K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 (A) Comparative alignment of the amino acid sequences of the L-type Ca2+ channel {alpha}1C subunits. Dashed gaps were introduced to optimize the alignment. The S1–S6 segments of domains I, II, II and IV are indicated. Potential phosphorylation sites (see also Table 1) were labelled as follows. Unique phosphorylation sites: ({downarrow}) PKA; ({diamondsuit}) PKC; ({blacktriangledown}): PKG. Common phosphorylation sites: ({circ}) PKA and PKC; (bullet) PKA and PKG; (*) PKA, PKC and PKG. Identical residues for all species are indicated by white lettering over black shading. Abbreviations and accesion numbers: Hs: Homo sapiens, AAA17030 and AC005342; Cp: Cavia porcellus, AB016287; Mm: Mus musculus, NM_009781 and genomic information; Oc: Oryctolagus cuniculus, X15539; Rn: Rattus norvegicus, AAL47073. (B) Schematic representation of {alpha}1C. 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.

 

View this table:
[in this window]
[in a new window]

  		Table 1 Potential and proven phosphorylation sites of the {alpha}1C subunit in different species

 
There are (potential) phosphorylation sites, conserved and non-conserved, in literature (see for example Ref. [88]), that were not detected by NetPhos. Thus, there may be more potential phosphorylation sites than the ones presented by us in Table 1. In general, it can be concluded that the potential phosphorylation sites in different species are highly conserved. Remarkably, the established rabbit PKC phosphorylation site Thr 27 [78] is not conserved in other species, while rabbit phosphorylation site PKC Thr 31 [78] is conserved in guinea pig and human, but not in mouse and rat. Instead, positively charged amino-acids are present at these sites, while negatively charged amino-acids would allow PKC mediated inhibition according to McHugh et al. [78].


   6. Conclusions
Top
 Abstract
 1. Introduction
 2. Structure
 3. Function
 4. Molecular regulation and...
 5. Potential phosphorylation...
 6. Conclusions
References
 
L-type Ca2+ channels are predominantly regulated by β-adrenergic stimulation, enhancing ICa-L by increasing the mean channel open time and/or the opening probability of functional Ca2+ channels. Stimulation of β-ARs results primarily in an increased cAMP production by AC and consequently activation of PKA and phosphorylation of L-type Ca2+ channels by this enzyme. β1-ARs couple exclusively to the G protein Gs, producing a widespread increase in cAMP levels in the cell, whereas β2-ARs couple to both Gs and Gi, producing a more localized activation of L-type Ca2+ channels. In neonatal rat ventricular myocytes, ICa-L is also regulated by {alpha}-adrenergic stimulation, but it still is not clear whether activation of {alpha}1-ARs results in activation or in a reduction of ICa-L. In adult rat ventricular myocytes activation of {alpha}1-ARs increases ICa-L, but only in experiments with the perforated patch-clamp technique. Thus methodological issues at present obscure the physiological significance. The effects of adrenergic stimulation are exerted by phosphorylation of the L-type Ca2+ channel subunits by PKA, PKC and PKG.

6.1. PKA pathway
Activation of G{alpha}s stimulates AC, which mediates the conversion of ATP into cAMP. This second messenger activates PKA, which increases ICa-L via phosphorylation of one or more subunits of the L-type Ca2+ channel. In rabbit ventricular myocytes, phosphorylation of Ser 1928 in the {alpha}1C subunit is of functional importance for the stimulation of the L-type Ca2+ channel in response to PKA. The rat β2a subunit is also phosphorylated by PKA at Ser 478 and Ser 479. Phosphorylation of both residues is required for stimulation of the cardiac L-type Ca2+ channel. For appropriate phosphorylation of the {alpha}1C subunit, PKA has to be anchored to the membrane in close proximity to the L-type Ca2+ channel by an AKAP, whereas PKA-dependent phosphorylation of the β2a subunit does not require an AKAP.

6.2. PKC pathway
Activation of G{alpha}q stimulates PLC, which hydrolyzes PIP2 to InsP3 and DAG. The latter mediates the activation of PKC, which phosphorylates L-type Ca2+ channels, but decreases ICa-L. The first 46 amino acids of the N-terminus of the {alpha}1C subunit have a negative effect on channel gating. Phosphorylation of both Thr 27 and Thr 31 of this subunit by PKC inhibits L-type Ca2+ channel activity.

6.3. PKG pathway
Activation of soluble GC results in the conversion of GTP into cGMP. This second messenger activates PKG, which phosphorylates the rabbit {alpha}1C subunit of the L-type Ca2+ channel at Ser 533, resulting in an inhibition of L-type Ca2+ channel activity. Besides direct phosphorylation of the L-type Ca2+ channel, it is also possible that PKG activates a protein phosphatase, which dephosphorylates the channel, or that cGMP activates phosphodiesterase 2, which reduces cAMP levels. Thus stimulation of ICa-L by PKA will be inhibited. However, besides an inhibition of ICa-L, also stimulatory effects of the PKG pathway have been shown.

Using Netphos, the potential phosphorylation sites of the {alpha}1C subunit were determined for PKA, PKC, and PKG. The {alpha}1C subunits of different species were compared and it can be concluded that the potential phosphorylation sites in different species are highly conserved.


   Acknowledgements
 
This study was supported by ZonMW grant MKG.5942 (MvdH).


   Notes
 
Time for primary review 20 days


   References
Top
 Abstract
 1. Introduction
 2. Structure
 3. Function
 4. Molecular regulation and...
 5. Potential phosphorylation...
 6. Conclusions
 References
 

  1. Orkand R.K., Niedergerke R. Heart action potential dependance on external calcium and sodium ions. Science (1964) 146:1176–1177.[Abstract/Free Full Text]
  2. Janse M.J., Wit A.L. Electrophysiological mechanisms of ventricular arrhythmias resulting from myocardial ischemia and infarction. Physiol. Rev. (1989) 69:1040–1169.
  3. Salkoff L.B., Tanouye M.A. Genetics of ion channels. Physiol. Rev. (1986) 66:301–329.[Free Full Text]
  4. Davies M.P., An R.H., Doevendans P., Kubalak S., Chien K.R., Kass R.S. Developmental changes in ionic channel activity in the embryonic murine heart. Circ. Res. (1996) 78:15–25.[Abstract/Free Full Text]
  5. Van der Heyden M.A.G., Hescheler J., Mummery C.L. Spotlight on stem cells–makes old hearts fresh. Cardiovasc. Res. (2003) 58:241–245.[Free Full Text]
  6. Hescheler J., Fleischman B.K., Lentini S., Maltsev V.A., Rohwedel J., Wobus A.M., et al. Embryonic stem cells: a model to study structural and functional properties in cardiomyogenesis. Cardiovasc. Res. (1997) 36:149–162.[Free Full Text]
  7. Mummery C., Ward-Van Oostwaard D., Doevendans P., Spijker R., Van den Brink S., Hassink R., et al. Differentiation of human embryonic stem cells to cardiomyocytes. Role of coculture with visceral endoderm-like cells. Circulation (2003) 107:2733–2740.[Abstract/Free Full Text]
  8. Van der Heyden M.A.G., Van Kempen M.J.A., Tsuji Y., Rook M.B., Jongsma H.J., Opthof T. P19 embryonal carcinoma cells: a suitable model system for cardiac electrophysiological differentiation at the molecular and functional level. Cardiovasc. Res. (2003) 58:410–422.[Abstract/Free Full Text]
  9. Reuter H. Calcium channel modulation by neurotransmitters, enzymes and drugs. Nature (1983) 301:569–574.[CrossRef][Medline]
  10. Irisawa H., Brown H.F., Giles W.R. Cardiac pacemaking in the sinoatrial node. Physiol. Rev. (1993) 73:197–227.[Free Full Text]
  11. Wang M.-C., Collins R.F., Ford R.C., Berrow N.S., Dolphin A.C., Kitmitto A. The three-dimensional structure of the cardiac L-type voltage-gated calcium channel. J. Biol. Chem. (2004) 279:7159–7168.[Abstract/Free Full Text]
  12. Wang M.-C., Dolphin A., Kitmitto A. L-type voltage-gated calcium channels: understanding function through structure. FEBS Lett. (2004) 564:245–250.[CrossRef][ISI][Medline]
  13. Keef K.D., Hume J.R., Zhong J. Regulation of cardiac and smooth muscle Ca2+ channels (Cav1.2a,b) by protein kinases. Am. J. Physiol. Cell Physiol. (2001) 281:C1743–C1756.[Abstract/Free Full Text]
  14. Catterall W.A. Structure and regulation of voltage-gated Ca2+ channels. Annu. Rev. Dev. Biol. (2000) 16:521–555.[CrossRef][ISI][Medline]
  15. Jiang L.H., Gawler D.J., Hodson N., Milligan C.J., Pearson H.A., Porter V., et al. Regulation of cloned cardiac L-type calcium channels by cGMP-dependent protein kinase. J. Biol. Chem. (2000) 275:6135–6143.[Abstract/Free Full Text]
  16. Hosey M.M., Chien A.J., Puri T.S. Structure and regulation of L-type calcium channels: a current assessment of the properties and roles of channel subunits. Trends Cardiovasc. Med. (1996) 6:265–273.[CrossRef][ISI]
  17. Striessnig J. Pharmacology, structure and function of cardiac L-type Ca2+ channels. Cell Physiol. Biochem. (1999) 9:242–269.[ISI][Medline]
  18. Norman R.I., Leach R.N. Subunit structure and phosphorylation of the cardiac L-type calcium channel. Biochem. Soc. Trans. (1994) 22:492–495.[ISI][Medline]
  19. Pelzer D., Pelzer S., McDonald T.F. Properties and regulation of calcium channels in muscle cells. Rev. Physiol. Biochem. Pharmacol. (1990) 114:107–207.[ISI][Medline]
  20. McDonald T.F., Pelzer S., Trautwein W., Pelzer D.J. Regulation and modulation of calcium channels in cardiac, skeletal, and smooth muscle cells. Physiol. Rev. (1994) 74:365–507.[Free Full Text]
  21. Maltsev V.A., Wobus A.M., Rohwedel J., Bader M., Hescheler J. Cardiomyocytes differentiated in vitro from embryonic stem cells developmentally express cardiac-specific genes and ionic currents. Circ. Res. (1994) 75:233–244.[Abstract/Free Full Text]
  22. Hartzell H.C., Duchatelle-Gourdon I. Structure and neural modulation of cardiac calcium channels. J. Cardiovasc. Electrophysiol. (1992) 3:567–578.[ISI]
  23. Gomez J.P., Potreau D., Branka J.E., Raymond G. Developmental changes in Ca2+ currents from newborn rat cardiomyocytes in primary culture. Pflügers Arch. (1994) 428:241–249.[CrossRef][ISI][Medline]
  24. Osaka T., Joyner R.W. Developmental changes in calcium currents of rabbit ventricular cells. Circ. Res. (1991) 68:788–796.[Abstract/Free Full Text]
  25. Wibo M., Bravo G., Godfraind T. Postnatal maturation of excitation–contraction coupling in rat ventricle in relation to the subcellular localization and surface density of 1,4 dihydropyridine and ryanodine receptors. Circ. Res. (1991) 68:662–673.[Abstract/Free Full Text]
  26. Liu S.J., Wyeth R.P., Melchert R.B., Kennedy R.H. Aging-associated changes in whole cell K+ and L-type Ca2+ currents in rat ventricular myocytes. Am. J. Physiol. Heart Circ. Physiol. (2000) 279:H889–H900.[Abstract/Free Full Text]
  27. Lefkowitz R.J., Rockman H.A., Koch W.J. Catecholamines, cardiac β-adrenergic receptors, and heart failure. Circulation (2000) 101:1634–1637.[Free Full Text]
  28. Brodde O.E., Michel M.C. Adrenergic and muscarinic receptors in the human heart. Pharmacol. Rev. (1999) 51:651–690.[Abstract/Free Full Text]
  29. Chen-Izu Y., Xiao R.P., Izu L.T., Cheng H., Kuschel M., Spurgeon H., et al. Gi-dependent localization of β2-adrenergic receptor signaling to L-type Ca2+ channels. Biophys. J. (2000) 79:2547–2556.[Abstract/Free Full Text]
  30. Xiao R.P. Cell logic for dual coupling of a single class of receptors to Gs and Gi proteins. Circ. Res. (2000) 87:635–637.[Free Full Text]
  31. Conrath C.E., Opthof T. β3-Adrenoceptors in the heart. Cardiovasc. Res. (2002) 56:353–356.[Free Full Text]
  32. Maki T., Gruver E.J., Davidoff A.J., Izzo N., Toupin D., Colucci W., et al. Regulation of calcium channel expression in neonatal myocytes by catecholamines. J. Clin. Invest. (1996) 97:656–663.[ISI][Medline]
  33. Davare M.A., Horne M.C., Hell J.W. Protein phosphatase 2A is associated with class C L-type calcium channels (Cav1.2) and antagonizes channel phosphorylation by cAMP-dependent protein kinase. J. Biol. Chem. (2000) 275:39710–39717.[Abstract/Free Full Text]
  34. Kamp T.J., Hell J.W. Regulation of cardiac L-type calcium channels by protein kinase A and protein kinase C. Circ. Res. (2000) 87:1095–1102.[Abstract/Free Full Text]
  35. Banach K., Bünemann M., Hüser J., Pott L. Serum contains a potent factor that decreases β-adrenergic receptor-stimulated L-type Ca2+ current in cardiac myocytes. Pflügers Arch. (1993) 423:245–250.[CrossRef][ISI][Medline]
  36. Reuter H, Effects of neurotransmitters on the slow inward current. In: DP Zipes, JC Bailey, V Elharrar, editors. The slow inward current and cardiac arrhythmias. The Hague/Boston/London: Martinus Nijhoff Publishers; 1980. p. 205–19.
  37. Carmeliet E, Electrophysiological effects of catecholamines in the heart. In: RA Riemersma, MF Oliver, editors. Catecholamines in the non-ischemic and ischemic myocardium. Amsterdam/New York/Oxford: Elsevier/North Holland biomedical press; 1982. p.77–86.
  38. Dukes I.D., Vaughan Williams E.M. Effects of selective {alpha}1, {alpha}2, β1 and β2 adrenoceptor stimulation on potentials and contractions in the rabbit heart. J. Physiol. (Lond.) (1984) 355:523–546.[Abstract/Free Full Text]
  39. Brückner R., Mügge A., Scholz H. Existence and functional role of {alpha}1-receptors in the mammalian heart. J. Mol. Cell. Cardiol. (1985) 17:639–645.[CrossRef][ISI][Medline]
  40. Hartzell H.C. Regulation of cardiac ion channels by catecholamines, acetylcholine and second messenger systems. Prog. Biophys. Mol. Biol. (1988) 52:165–247.[CrossRef][ISI][Medline]
  41. Zaza A., Kline R.P., Rosen M.R. Effects of {alpha}-adrenergic stimulation on intracellular sodium activity and automaticity in canine Purkinje fibers. Circ. Res. (1990) 66:416–426.[Abstract/Free Full Text]
  42. Liu S.J., Kennedy R.H. {alpha}1-Adrenergic activation of L-type Ca current in rat ventricular myocytes: perforated patch-clamp recordings. Am. J. Physiol. (1998) 274:H2203–H2207.[ISI][Medline]
  43. Liu Q.-Y., Karpinski E., Pang P.K.T. The L-type calcium channel current is increased by alpha-1 adrenoceptor activation in neonatal rat ventricular cells. J. Pharmacol. Exp. Ther. (1994) 271:935–943.[Abstract/Free Full Text]
  44. Gaughan J.P., Hefner C.A., Houser S.R. Electrophysiological properties of neonatal rat ventricular myocytes with {alpha}1-adrenergic-induced hypertrophy. Am. J. Physiol. Heart Circ. Physiol. (1998) 44:H577–H590.
  45. Yatani A., Codina J., Imoto Y., Reeves J.P., Birnbaumer L., Brown A.M. A G protein directly regulates mammalian cardiac calcium channels. Science (1987) 238:1288–1292.[Abstract/Free Full Text]
  46. Yu H.J., Ma H., Green R.D. Calcium entry via L-type calcium channels acts as a negative regulator of adenylyl cyclase activity and cyclic AMP levels in cardiac myocytes. Mol. Pharmacol. (1993) 44:689–693.[Abstract]
  47. Skeberdis V.A., Jurevicius J., Fischmeister R. Beta-2 adrenergic activation of L-type Ca++ current in cardiac myocytes. J. Pharmacol. Exp. Ther. (1997) 283:452–461.[Abstract/Free Full Text]
  48. Xiao R.P., Ji X., Lakatta E.G. Functional coupling of the β2-adrenoceptor to a pertussis toxin-sensitive G protein in cardiac myocytes. Am. Soc. Pharmacol. Exp. Ther. (1995) 47:322–329.
  49. Zhou Y.Y., Cheng H., Bogdanov K.Y., Hohl C., Altschuld R., Lakatta E.G., et al. Localized cAMP-dependent signalling mediates β2-adrenergic modulation of cardiac excitation–contraction coupling. Am. J. Physiol. Heart Circ. Physiol. (1997) 42:H1611–H1618.
  50. Yatani A., Tajima Y., Green S.A. Coupling of β-adrenergic receptors to cardiac Ltype Ca2+ channels: preferential coupling of the β1 versus β2 receptor subtype and evidence for PKA-independent activation of the channel. Cell Signal. (1999) 11:337–342.[CrossRef][ISI][Medline]
  51. Communal C., Singh K., Pimentel D.R., Colucci W.S. Norepinephrine stimulates apoptosis in adult rat ventricular myocytes by activation of the β-adrenergic pathway. Circulation (1998) 98:1329–1334.[Abstract/Free Full Text]
  52. Maltsev V.A., Ji G.J., Wobus A.M., Fleischmann B.K., Hescheler J. Establishment of β-adrenergic modulation of L-type Ca2+ current in the early stages of cardiomyocyte development. Circ. Res. (1999) 84:136–145.[Abstract/Free Full Text]
  53. Liu W., Yasui K., Arai A., Kamiya K., Cheng J., Kodama I., et al. β-Adrenergic modulation of L-type Ca2+ channel currents in early-stage embryonic mouse heart. Am. J. Physiol. (1999) 276:H608–H613.[ISI][Medline]
  54. Gao T., Puri T.S., Gerhardstein B.L., Chien A.J., Green R.D., Hosey M.M. Identification and subcellular localization of the subunits of L-type calcium channels and adenylyl cyclase in cardiac myocytes. J. Biol. Chem. (1997) 272:19401–19407.[Abstract/Free Full Text]
  55. Steinberg S.F. β2-Adrenergic receptor signalling complexes in cardiomyocyte caveolae/lipid rafts. J. Mol. Cell. Cardiol. (2004) 37:407–415.[CrossRef][ISI][Medline]
  56. Schwenke C., Yamamoto M., Okumura S., Toya Y., Kim S.-J., Ishikawa Y. Compartmentation of cyclic adenosine 3',5'-monophosphate signalling in caveolae. Mol. Endocrinol. (1999) 13:1061–1070.[Abstract/Free Full Text]
  57. Steinberg S.F., Brunton L.L. Compartmentation of G-protein-coupled signalling pathways in cardiac myocytes. Annu. Rev. Pharmacol. Toxicol. (2001) 41:751–773.[CrossRef][ISI][Medline]
  58. Yoshida A., Takahashi M., Nishimura S., Takeshima H., Kokubun S. Cyclic AMP-dependent phosphorylation and regulation of the cardiac dihydropyridine-sensitive Ca2+ channel. FEBS Lett. (1992) 309:343–349.[CrossRef][ISI][Medline]
  59. De Jongh K.S., Murphy B.J., Colvin A.A., Hell J.W., Takahashi M., Catterall W.A. Specific phosphorylation of a site in the full-length form of the {alpha}1 subunit of the cardiac L-type calcium channel by adenosine 3',5'-cyclic monophosphate-dependent protein kinase. Biochemistry (1996) 35:10392–10402.[CrossRef][ISI][Medline]
  60. Gao T., Yatani A., Dell'Acqua M.L., Sako H., Green S.A., Dascal N., et al. cAMP-dependent regulation of cardiac L-type Ca2+ channels requires membrane targeting of PKA and phosphorylation of channel subunits. Neuron (1997) 19:185–195.[CrossRef][ISI][Medline]
  61. Bünemann M., Gerhardstein B.L., Gao T., Hosey M.M. Functional regulation of L-type calcium channels via protein kinase A-mediated phosphorylation of the β2 subunit. J. Biol. Chem. (1999) 274:33851–33854.[Abstract/Free Full Text]
  62. Gerhardstein B.L., Gao T., Bünemann M., Puri T.S., Adair A., Ma H., et al. Proteolytic processing of the C terminus of the {alpha}1C subunit of L-type calcium channels and the role of a proline-rich domain in membrane tethering of proteolytic fragments. J. Biol. Chem. (2000) 275:8556–8563.[Abstract/Free Full Text]
  63. Gao T., Cuadra A.E., Ma H., Bünemann M., Gerhardstein B.L., Cheng T., et al. C-terminal fragments of the {alpha}1c (Cav1.2) subunit associate with and regulate L-type calcium channels containing C-terminal-truncated {alpha}1c subunits. J. Biol. Chem. (2001) 276:21089–21097.[Abstract/Free Full Text]
  64. Mikala G., Bodi I., Klockner U., Varadi M., Varadi G., Koch S.E., et al. Characterization of auto-regulation of the human {alpha}1 subunit of the L-type calcium channel: importance of the C-terminus. Mol. Cell. Biochem. (2003) 250:81–89.[CrossRef][ISI][Medline]
  65. Perets T., Blumenstein Y., Shistik E., Lotan I., Dascal N. A potential site of functional modulation by protein kinase A in the cardiac Ca2+ channel {alpha}1C subunit. FEBS Lett. (1996) 384:189–192.[CrossRef][ISI][Medline]
  66. Wiechen K., Yue D.T., Herzig S. Two distinct functional effects of protein phosphatase inhibitors on guinea-pig cardiac L-type Ca2+ channels. J. Physiol. (Lond.) (1995) 484:583–592.[Abstract/Free Full Text]
  67. Hirayama Y., Hartzell C. Effects of protein phosphatase and kinase inhibitors on Ca2+ and Cl– currents in guinea pig ventricular myocytes. Mol. Pharmacol. (1997) 52:725–734.[Abstract/Free Full Text]
  68. Haase H., Karczewski P., Beckert R., Krause E.G. Phosphorylation of the L-type calcium channel β subunit is involved in β-adrenergic signal transduction in canine myocardium. FEBS Lett. (1993) 335:217–222.[CrossRef][ISI][Medline]
  69. Gerhardstein B.L., Puri T.S., Chien A.J., Hosey M.M. Identification of the sites phosphorylated by cyclic AMP-dependent protein kinase on the β2 subunit of L-type voltage-dependent calcium channels. Biochemistry (1999) 38:10361–10370.[CrossRef][ISI][Medline]
  70. Hulme J.T., Lin T.W.-C., Westenbroek R.E., Scheuer T., Catterall W.A. β-Adrenergic regulation requires direct anchoring of PKA to cardiac Cav1.2 channels via a leucine zipper interaction with A kinase-anchoring protein 15. Proc. Natl. Acad. Sci. U. S. A. (2003) 100:13093–13098.[Abstract/Free Full Text]
  71. Haase H., Podzuweit T., Lutsch G., Hohaus A., Kostka S., Lindschau C., et al. Signaling from β-adrenoceptor to L-type calcium channel: identification of a novel cardiac protein kinase A target possessing similarities to AHNAK. FASEB J. (1999) 13:2161–2172.[Abstract/Free Full Text]
  72. Alvarez J., Hamplova J., Hohaus A., Morano I., Haase H., Vassort G. Calcium current in rat cardiomyocytes is modulated by the carboxyl-terminal Ahnak domain. J. Biol. Chem. (2004) 279:12456–12461.[Abstract/Free Full Text]
  73. Blumenstein Y., Kanevsky N., Sahar G., Barzilai R., Ivanina T., Dascal N. A novel long N-terminal isoform of human L-type Ca2+ channels is upregulated by Protein Kinase C. J. Biol. Chem. (2002) 277:3419–3423.[Abstract/Free Full Text]
  74. Dai B., Saada N., Echetebu C., Dettbarn C., Palade P. A new promoter for {alpha}1c subunit of human L-type cardiac calcium channel Cav1.2. Biochem. Biophys. Res. Commun. (2002) 296:429–433.[CrossRef][ISI][Medline]
  75. Saada N., Dai B., Echetebu C., Sarna S.K., Palade P. Smooth muscle uses another promoter to express primarily a form of human Cav1.2 L-type calcium channel different from the principle heart form. Biochem. Biophys. Res. Commun. (2003) 302:23–28.[CrossRef]