INTRODUCTION

TOP ABSTRACT INTRODUCTION STRUCTURE OF CARDIAC AND... PKG REGULATION OF CA2+... PKA REGULATION OF CA2+... PKC REGULATION OF CAV1.2... G PROTEIN SUBUNIT REGULATION... REGULATION OF CAV1.2 CHANNELS... REFERENCES

CA_V1.2 channels are members of the superfamily of voltage-dependent Ca²⁺ channels that includes low voltage-activated, rapidly inactivating channels and high voltage-activated channels. The nomenclature for voltage-dependent Ca²⁺ channels has recently changed from alphabetic descriptors, i.e., L-, N-, P/Q-, and R-type, to names based on a system previously developed to describe K⁺ channels. This system uses numerals to define families and subfamilies of channels based on similarities in amino acid sequences. Thus the Ca_V1 family includes Ca²⁺ channel ₁-subunits encoded by four separate genes that give rise to Ca_V1.1, Ca_V1.2, Ca_V1.3, and Ca_V1.4 (formerly _1S, _1C, _1D, and _1F, respectively) (see Ref. 43). This review focuses specifically on signaling pathways involved in the regulation of cardiac and smooth muscle Ca_V1.2 channels. Ca_V1.2 channels in both cardiac and smooth muscle contribute to contraction by delivering extracellular Ca²⁺ to the cytoplasm and as a trigger for Ca²⁺-induced Ca²⁺ release. A number of excellent reviews have been published in recent years on Ca²⁺ channels (7, 37, 40, 55, 73, 74, 85, 114, 162, 163). The specific focus of this review, therefore, is on the most recent advances in our understanding of Ca_V1.2a and Ca_V1.2b channel regulation in cardiac and smooth muscle by cGMP-dependent protein kinase (PKG), cAMP-dependent protein kinase (PKA), and protein kinase C (PKC).

	STRUCTURE OF CARDIAC AND SMOOTH MUSCLE CA2+ CHANNELS

TOP ABSTRACT INTRODUCTION STRUCTURE OF CARDIAC AND... PKG REGULATION OF CA2+... PKA REGULATION OF CA2+... PKC REGULATION OF CAV1.2... G PROTEIN SUBUNIT REGULATION... REGULATION OF CAV1.2 CHANNELS... REFERENCES

The Ca²⁺ channels examined in this review are multisubunit protein complexes composed of a pore-forming ₁-subunit and several auxiliary subunits including an intracellularly located -subunit and an extracellularly located, disulfide-linked ₂/-subunit (Fig. 1). The cardiac and smooth muscle ₁-subunits are splice variants of the same gene and are designated Ca_V1.2a and Ca_V1.2b, respectively. Ca_V1.2a and Ca_V1.2b exhibit 93% homology in amino acid sequence. The molecular mass of each is ~240 kDa, and each ₁-subunit contains ~2,170 amino acids (11, 37, 73, 81, 90, 92, 119, 160, 185). The ₁-subunit defines the ionic pore of the channel and contains four repeats, each with six transmembrane segments. Cardiac and smooth muscle ₁-subunits are distinguished by a minor difference in the NH₂ terminus, in the hydrophobic segments IS6 and IVS3, and by an insertion in the I-II cytoplasmic linker in the smooth muscle subunit (Fig. 1) (9, 37, 92, 119). Splice variants of Ca_V1.2 have been reported to exist within a single tissue (10, 44). An interesting new twist in the story are recent reports suggesting that the COOH-terminal end of Ca_V1.2a is cleaved in vivo but remains colocalized with the "body" of the ₁-subunit. A proline-rich domain between residues 1973 and 2001 of the ₁-subunit has been identified as the mediator of the COOH-terminal membrane association (49, 51). Because the COOH-terminal region suppresses Ca_V1.2 channel activity (182), the channel could be regulated by changes in the association of the COOH-terminal fragment with the body of the ₁-subunit.

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Model of the cardiac and smooth muscle CaV1.2 channel (1) and associated auxiliary subunits. In addition to the pore-forming 1-subunit, there is an intracellularly localized -subunit and an extracellularly located, disulfide-linked 2/-subunit. Sites of divergence between the cardiac and smooth muscle 1-subunits are indicated at the NH2 terminus, in the hydrophobic segments IS6 and IVS3 and by an insertion between I and II cytoplasmic linker. Evidence points to a functional role for cGMP-dependent protein kinase (PKG) phosphorylation of CaV1.2a at serine 533 (82). The same PKG phosphorylation site exists in CaV1.2b (i.e., serine 528). Evidence also suggests a functional role for cAMP-dependent protein kinase (PKA) phosphorylation of CaV1.2a at serine 1928 (34, 50, 131). The same PKA phosphorylation site exists in CaV1.2b (i.e., serine 1923). Additional studies suggest that phosphorylation of serine residues 478 and 479 on the 2a-subunit also are involved in functional regulation of channels by PKA (18). Although ~18 potential protein kinase C (PKC) phosphorylation sites exist on the 1-subunit, no studies to date have clearly identified a role for these sites in channel activation. However, recent studies suggest that threonine-27 and threonine-31 on CaV1.2a are involved in PKC-induced inhibition of the channel (115). These sites are not present on CaV1.2b.

Four different genes encode mammalian voltage-gated Ca²⁺ channel -subunits (₁-₄; Ref. 11). In addition, each of the four gene products can be alternatively spliced, giving rise to two to four splice variants per -subunit. Overall, these -subunits vary from ~53 to 71 kDa (for review, see Refs. 12 and 37). In cardiac muscle the ₂-subunit predominates (49, 59, 77, 134), whereas in smooth muscle at least three different -subunits (i.e., _1b, ₂, and ₃, 54-68 kDa molecular mass) have been identified (10, 31, 54, 77, 176). In contrast to the ₁-subunit, the -subunit does not contain putative transmembrane domains, although there are hydrophobic regions. The -subunit tightly binds to a highly conserved 18-amino acid sequence in the cytoplasmic linker between repeats I and II of the ₁-subunit (136). -Subunits target the ₁-subunit to the plasma membrane (12, 48, 53, 196) and facilitate Ca_V1.2 channel currents (12, 32, 53, 76, 122, 154). The _2a-subunit is unique in that it is posttranslationally palmitoylated by addition of a 16-carbon palmitic acid group to cysteine residues of the -subunit through a labile thioester linkage (26, 27). This palmitoylation confers unique regulatory properties on Ca_V1 channels (138), although it is uncertain whether such palmitoylated subunits actually exist in either cardiac or smooth muscle cells.

The ₂/ complex consists of an extracellularly located ₂-subunit linked via a disulfide bond to a membrane-spanning -subunit. The ₂ and  proteins are encoded by a single gene (35). ₂/-Subunits (~175 kDa) have been identified in both cardiac (37, 49, 183) and smooth muscle (3). This subunit appears to increase Ca²⁺ channel currents (91, 154) but also has been associated with prevention of prepulse facilitation (33).

	PKG REGULATION OF CA2+ CHANNELS

TOP ABSTRACT INTRODUCTION STRUCTURE OF CARDIAC AND... PKG REGULATION OF CA2+... PKA REGULATION OF CA2+... PKC REGULATION OF CAV1.2... G PROTEIN SUBUNIT REGULATION... REGULATION OF CAV1.2 CHANNELS... REFERENCES

PKG catalyzes the phosphorylation of a number of intracellular proteins that modulate muscle contraction. Two main types of PKG are present in eukaryotic cells, type I and type II (104). Type I PKG is a homodimer, and each subunit has a mass of ~78 kDa. Type II PKG also exists as a dimer whose subunit mass is ~86 kDa. The regulatory and catalytic domains of this molecule are both contained within a single polypeptide sequence. PKG type I is widely distributed and is isolated from soluble extracts of tissues, whereas PKG type II is a particulate form of the enzyme and has a limited tissue distribution. PKG type I is present in both cardiac and smooth muscle cells, although the level of PKG is much greater in smooth muscle than in cardiac muscle (106). This difference is attributable to some degree to a developmental decline in PKG levels from newborn to adult heart (95).

Role of PKG in Cardiac Muscle

Three different mechanisms have been suggested to account for cGMP-induced inhibition of Ca_V1.2 channels, i.e., 1) direct phosphorylation of the channel by PKG, 2) PKG-induced activation of a phosphatase, leading to dephosphorylation of the channel, and 3) cGMP stimulation of phosphodiesterase 2 (PDE2), leading to a reduction in cAMP levels. The implication of the second and third possibilities listed is that cGMP reduces the phosphorylation of Ca_V1.2 produced by PKA. Each of these possibilities is discussed below and is illustrated in Fig. 2.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Mechanisms proposed for cGMP-induced inhibition of CaV1.2 channels. In this schematic, guanylyl cyclase (G cyclase) is activated by nitric oxide (NO) to give rise to an increase in cGMP levels. cGMP then activates PKG to directly phosphorylate the 1-subunit of the Ca2+ channel. Another possibility is that PKG activates a protein phosphatase (PP), which then dephosphorylates the channel. Finally, cGMP may stimulate phosphodiesterase 2 (PDE2), which reduces cAMP levels and thus reduces stimulation of the channel by PKA. AKAP, A-kinase anchoring protein; A cyclase, adenylyl cyclase.

Evidence to support the notion that PKG directly phosphorylates Ca²⁺ channels has been derived from functional studies of Ca_V1.2a channels expressed in oocytes (82). In this study, PKG was activated by addition of the membrane-permeant species 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP). This cyclic nucleotide inhibited wild-type Ca²⁺ channel currents. In contrast, when serine residue 533 of the ₁-subunit was replaced with alanine, 8-Br-cGMP no longer had an effect on channel activity, suggesting that PKG produces inhibition by phosphorylating the channel at serine 533 (82). The actions of 8-Br-cGMP appeared to be independent of either the - or the ₂/-subunit of Ca_V1.2a because inhibition still occurred in the absence of these subunits.

Protein phosphorylation represents a balance between kinase and phosphatase activity. Thus second messenger pathways that modify this balance can upregulate or downregulate channel activity to meet physiological demands. An additional mechanism by which PKG, in particular, could inhibit Ca²⁺ channel activity is through stimulation of phosphatase activity, leading to dephosphorylation of the channel. When inhibitors of either protein phosphatase 1 (PP1) or 2A (PP2A) are applied to cardiac myocytes, Ca²⁺ channel stimulation generally has been observed, suggesting that there is both basal phosphorylation and dephosphorylation of the channel (for review, see Ref. 69). The notion that PKG may exert its effect by activating a phosphatase is not new. Smooth muscle myosin phosphatase is a known target of PKG, and activation of this phosphatase contributes to the well-known phenomenon of myofilament "desensitization" (72, 180). In addition, there is evidence suggesting that the actions of cGMP on Ca²⁺-activated K⁺ channels in rat pituitary tumor cells are due to PKG-induced stimulation of PP2A (187). In a more recent study, 8-Br-cGMP was reported to inhibit IBMX-stimulated Ca²⁺ channel currents in guinea pig ventricular myocytes, and this effect was abolished by the type 1 and 2A phosphatase inhibitor okadaic acid, leading to the conclusion that PKG inhibited the channels by activation of a phosphatase (145).

Another means by which Ca²⁺ channel phosphorylation could be reduced is to increase the rate of breakdown of cAMP by PDEs. A number of different isoenzymes of PDEs exist in cells, and these have been subdivided into seven families. At least four of these families (i.e., PDE1-4) are present in cardiac muscle (177). PDE1 and PDE2 hydrolyze both cAMP and cGMP, whereas PDE3 and PDE4 hydrolyze cAMP. PDE2 is stimulated by cGMP, whereas PDE3 is inhibited by cGMP and PDE4 is insensitive to cGMP (135). In some tissues, cGMP may stimulate Ca_V1.2a channel currents via inhibition of PDE3 (88, 128), whereas in others, cGMP may inhibit Ca_V1.2a channel current by stimulating PDE2 (45, 117). This opposing effect of cGMP on PDE2 vs. PDE3 serves to underscore the complexity of interpreting studies in which cGMP is applied to cells.

Role of PKG in Smooth Muscle

The notion that PKG inhibits Ca_V1.2b via the activation of a phosphatase is an intriguing one. Application of inhibitors that block both PP1 and PP2A (e.g., okadaic acid and calyculin A) have generally increased Ca²⁺ channel activity (46, 56, 100, 123, 124), suggesting that basal channel activity is determined by a balance of kinase and phosphatase activity. Some studies in smooth muscle have directly applied activated phosphatases to isolated patches or to the whole cell via cell dialysis. These studies have generally revealed inhibition of channel activity, i.e., the opposite effect [e.g., PP2A application (56, 70) or PP2B application (148)]. To date, there have been no studies directly linking activation of a phosphatase to the PKG-induced inhibition of Ca_V1.2b channels observed in smooth muscle.

Five of the seven PDE isoenzyme families have been identified in vascular smooth muscle, including PDE1-4 and PDE5, which is a cGMP-hydrolyzing PDE (for review, see Ref. 135). PDE3 accounts for approximately half of the cAMP-hydrolyzing activity in the pulmonary artery and aorta (140, 141). To date, there are no studies investigating the possible role of cGMP activation of PDE2 to account for the inhibitory effect of cGMP on Ca_V1.2b.

	PKA REGULATION OF CA2+ CHANNELS

TOP ABSTRACT INTRODUCTION STRUCTURE OF CARDIAC AND... PKG REGULATION OF CA2+... PKA REGULATION OF CA2+... PKC REGULATION OF CAV1.2... G PROTEIN SUBUNIT REGULATION... REGULATION OF CAV1.2 CHANNELS... REFERENCES

PKA is a tetramer consisting of two catalytic subunits bound to a regulatory subunit dimer. Like PKG, PKA is ubiquitously expressed in smooth muscle and cardiac muscle. Initially, two different forms of the regulatory subunit were defined, i.e., R-I and R-II, where the PKA-RI complex is essentially cytosolic and the PKA-RII complex almost exclusively membrane bound. However, recent molecular cloning techniques have revealed significant heterogeneity in both the catalytic and regulatory subunits of PKA (165).

Role of PKA in Cardiac Muscle

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Mechanisms proposed for -adrenergic stimulation of CaV1.2 channels. -Adrenergic receptors (-AR) are stimulated with agonists such as isoproterenol. The -receptor is coupled to the G protein Gs, which leads to activation of the G protein subunits Gs and G. Gs stimulates A cyclase, leading to enhanced levels of cAMP that activate PKA localized to the region of CaV1.2 via an AKAP. PKA is then proposed to activate the Ca2+ channel via phosphorylation of one or more of the Ca2+ channel subunits. In contrast, G is proposed to lead to stimulation of PKC, possibly via phosphatidylinositol 3-kinase, leading to phosphorylation of 1 or more of the Ca2+ channel subunits. Finally, evidence exists for direct activation of CaV1.2 via a direct membrane-delimited action of Gs on the channel.

Additional complications have arisen in attempts to demonstrate a functional correlate of PKA-induced phosphorylation in expression systems. Although some studies have reported PKA-induced stimulation of cardiac Ca_V1.2a currents (50, 90, 150, 199, 202), others have failed to show any effect of PKA (33, 118, 209). A variety of explanations have been offered for the negative results, including the importance of auxiliary subunits, particularly the -subunit (90) and, more recently, the need for channel targeting of PKA by AKAP (50). One report suggested that an additional, as yet unidentified, component of the pathway is important for PKA-induced regulation of the Ca²⁺ channel, because PKA failed to stimulate Ca_V1.2a channels unless oocytes were injected with mRNA from cardiac cells (23). Yet another study suggested that PKA leads to phosphorylation of a 700-kDa protein that couples to the cardiac -subunit (60). Despite studies that suggest an obligatory role for additional regulatory subunits, several studies have reported PKA-induced enhancement of expressed ₁-subunits of Ca_V1.2a in the absence of other regulatory proteins (150, 202). In summary, evidence exists in both native cells and expression systems to suggest that PKA can stimulate cardiac Ca_V1.2a channels via direct phosphorylation of serine 1928 on the ₁-subunit. However, additional sites appear to contribute to this process. The variable results reported from expression systems may be related to differences in the cell types used, differences in the regulatory proteins coexpressed with the ₁-subunit, and the activity of other enzymes in the cells (e.g., PDEs, phosphatases) that modify the basal levels of phosphorylation of the channel. Indeed, in some studies, although activated PKA was reported to be without effect on channel activity, inhibition of endogenous PKA decreased channel activity, suggesting that the Ca_V1.2a channels were maximally phosphorylated under basal conditions (131, 132, 159).

Role of PKA in Smooth Muscle

The stimulatory effect of PKA on cardiac Ca_V1.2a channels corresponds well with the known positive inotropic effect of -adrenoceptor stimulation in this muscle. However, in smooth muscle, agonists that activate adenylyl cyclase typically cause relaxation, making it more difficult to reconcile a stimulatory effect of PKA on Ca_V1.2b currents. It is possible that the Ca²⁺ entry associated with PKA stimulation is specifically targeted to events that are unrelated to global cytoplasmic Ca²⁺ concentration; e.g., an increase in the subsarcolemmal Ca²⁺ concentration may be coupled to enhancement of Ca²⁺-activated K⁺ channel activity (57). Alternatively, the enhanced Ca²⁺ entry may serve to fill stores in the sarcoplasmic reticulum (SR).

Some of the controversy surrounding the actions of cAMP on Ca_V1.2b channels may be related to the higher levels of PKG present in smooth muscle compared with cardiac muscle (106). Neither cAMP nor cGMP display absolute specificity for their respective kinases, i.e., the equilibrium dissociation constant for activation of PKG by cAMP is ~10-fold greater than that for PKA (see, for example, Ref. 39). Thus some of the actions of cAMP that have been attributed to PKA on Ca_V1.2b channels may, in fact, be due to PKG. In support of this hypothesis are studies showing that higher concentrations of 8-Br-cAMP and forskolin lead to inhibition of Ca_V1.2b channel currents and that this effect is blocked by PKG inhibitors (80, 144). These data suggest that Ca_V1.2b channels can be inhibited by "cross-over" activation of PKG by cAMP. Cross-over activation of PKG by cAMP also has been proposed by others to contribute to the effects of cAMP (see, for example, Refs. 105 and 186). Interestingly, in the presence of PKG inhibitors the reverse cross-over activation can be demonstrated as well, i.e., cGMP activation of PKA, leading to stimulation of Ca_V1.2b channel current (144). A similar action of cGMP on PKA has been proposed to underlie the NO-induced relaxation in PKG-I-deficient mice (146). This "cross talk" between cyclic nucleotides and protein kinases is illustrated in Fig. 4.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Mechanisms proposed for cGMP stimulation and cAMP inhibition of CaV1.2 channels (red arrows). Cross talk between the cyclic nucleotides may lead to cAMP-induced activation of PKG to inhibit the channel or cGMP-induced activation of PKA to stimulate the channel. Cross talk is more likely to occur when higher levels of cyclic nucleotide are achieved in the cell. Finally, cGMP also has been reported to inhibit PDE3, which leads to an increase in cAMP levels and, hence, stimulation of the channel via PKA.

Only one study to date has demonstrated enhancement of Ca_V1.2b channel currents with raised cAMP in an expression system (90). In this study, the ₁-subunit of Ca_V1.2b was coexpressed with the skeletal muscle _1a-subunit. cAMP produced current stimulation only in the presence of the -subunit. The authors suggested that the kind of -subunit present in a tissue may underlie tissue-specific effects of cAMP/PKA. However, other -subunits (e.g., ₂ or ₃) were not tested in this study. A recent study of A7r5 cells also suggests that cAMP-dependent stimulation of the channel requires a functional -subunit (87). Two other studies have failed to show enhancement of the Ca_V1.2b ₁-subunit by cAMP/PKA in an expression system (89, 209). In one case the ₁-subunit was expressed with ₂- and ₂/-subunits in HEK-293 cells (209), and in the other case the ₁-subunit was expressed alone in Chinese hamster ovary cells (89). This variability between studies of expressed smooth muscle Ca_V1.2b channels is similar to the variability reported for the actions of PKA on expressed cardiac Ca_V1.2a channels.

	PKC REGULATION OF CAV1.2 CHANNELS

TOP ABSTRACT INTRODUCTION STRUCTURE OF CARDIAC AND... PKG REGULATION OF CA2+... PKA REGULATION OF CA2+... PKC REGULATION OF CAV1.2... G PROTEIN SUBUNIT REGULATION... REGULATION OF CAV1.2 CHANNELS... REFERENCES

Eleven isoforms of PKC have been identified in mammalian tissues, and these are divided into three groups: 1) classic or conventional PKCs (cPKC) that are activated by diacylglycerol or phorbol ester and are Ca²⁺ sensitive (including , I, II, ), 2) novel or new PKCs (nPKC) that are activated by diacylglycerol or phorbol ester but are not Ca²⁺ dependent (, , , , L, µ), and 3) atypical PKCs (aPKC) that are not activated by diacylglycerol, phorbol ester, or Ca²⁺ (/, ) (for review, see Ref. 75).

Role of PKC in Cardiac Muscle

Role of PKC in Smooth Muscle

	G PROTEIN SUBUNIT REGULATION OF CAV1.2 CHANNELS

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The PKA and PKC pathways discussed above are generally linked to agonist-induced activation of G protein subunits. Activated G protein subunits can regulate different ionic channels not only through intracellular second messenger pathways but also through a direct membrane-delimited gating of channels. For example, neuronal Ca_V2.2 channels (formerly N-type or _1B) are inhibited by G protein-activated PKC and by the direct binding of G to the ₁-subunit (78). Direct G binding takes place in the intracellular loop between repeat I and II as well as in a second downstream sequence (38). However, the requisite binding motif for this interaction is absent in Ca_V1.2 channels. In the case of Ca_V1.2 channels, evidence has been presented to suggest a direct membrane-delimited activation of Ca_V1.2a and Ca_V1.2b channels by the G protein subunit G_s; however, these effects are controversial (29, 170).

Role of G Protein Subunits in Cardiac Muscle

Recent studies have provided further evidence for a membrane-delimited gating of Ca_V1.2a by G_s subunits. The whole cell Ca²⁺ channel current in cardiac myocytes from transgenic mice overexpressing cardiac G_s is 490% higher than in cells from wild-type control animals (98). Furthermore, in Xenopus oocytes, antisense knockdown of endogenous G_s reduced currents of expressed Ca_V1.2a channels, whereas coexpression of G_s with Ca_V1.2a enhanced currents (15). Interestingly, PKA inhibitors did not have any detectable action on the effects of G_s in either the transgenic mice or the oocyte model. In fact, a small cAMP-dependent decrease of current was observed in oocytes coexpressed with Ca_V1.2a and the ₂-adrenergic receptor (15). The authors concluded that coexpression of G_s, but not its acute activation via -adrenergic receptors, enhances Ca_V1.2a currents via a PKA-independent pathway. A recent study of Ca_V1.2a and ₂-subunits expressed in Chinese hamster fibroblasts with -adrenoceptors also suggested the presence of an additional cAMP-independent mechanism of channel activation (198). Thus controversy continues to surround the issue of direct membrane-delimited effects of G_s on Ca_V1.2a, although a high-affinity binding site for G_s on Ca_V1.2a has yet to be identified.

In our recent studies of G protein activation of native guinea pig cardiac Ca_V1.2a channel currents, we did not obtain any evidence to suggest the presence of a direct membrane-delimited pathway for either G_s or G activation of the channel (208). Rather, the pathways involved appeared to be very similar in nature to those described below for G protein regulation of smooth muscle Ca_V1.2b channels (see also Ref. 205 as well as Fig. 3), leaving open the possibility that the PKA-independent effect of -adrenergic stimulation in cardiac myocytes identified by others may actually be due to G-induced activation of PKC, rather than to a direct membrane-delimited effect of G_s.

Role of G Protein Subunits in Smooth Muscle

	REGULATION OF CAV1.2 CHANNELS BY OTHER KINASES

TOP ABSTRACT INTRODUCTION STRUCTURE OF CARDIAC AND... PKG REGULATION OF CA2+... PKA REGULATION OF CA2+... PKC REGULATION OF CAV1.2... G PROTEIN SUBUNIT REGULATION... REGULATION OF CAV1.2 CHANNELS... REFERENCES

Recent studies suggest that the activity of Ca_V1.2 channels in cardiac and smooth muscle cells also is regulated by other kinases, especially by protein tyrosine kinase (PTK) and calmodulin-dependent kinase (CaMK). Although this review focuses mainly on Ca_V1.2 channel regulation by PKG, PKA, and PKC, a summary of recent reports of channel regulation by PTK and CaMK follows.

Role of PTK

Evidence that vascular Ca_V1.2b channels are regulated by PTK also has been provided. Wijetunge et al. (188) reported that Ca_V1.2b current in rabbit ear artery myocytes was significantly reduced by both genistein and tyrphostin 23 but not by daidzein. Inhibition of tyrosine phosphatases (190) or activation of endogenous c-Src tyrosine kinase (189) increased current in the same cell preparation. PTK inhibitors antagonized these stimulatory effects, suggesting a possible role for endogenous c-Src tyrosine kinase in the modulation of vascular Ca²⁺ channels (189, 191). In rat portal vein myocytes, genistein also reduced whole cell Ca²⁺ channel currents, whereas daidzein was without effect (107). Further studies of these cells revealed that genistein also decreased the mean open time and prolonged closed time of single channels (108).

Although the evidence to date suggests a possible role of PTK in the regulation of Ca_V1.2 channels, no studies have yet extended this work to PTK regulation of expressed cardiovascular Ca_V1.2 channels. A recent study of neuronal Ca_V1.2 channels indicated that potentiation of current by insulin-like growth factor 1 required phosphorylation of the ₁-subunit by PTK (8). It is possible that PTK phosphorylates a similar site on the ₁-subunit of cardiovascular Ca_V1.2 channels.

Role of CaMK

In contrast to CaM-dependent facilitation, some studies have proposed that CaM-dependent inactivation may result from channel dephosphorylation by the CaM-dependent phosphatase calcineurin (4, 21, 148). On the other hand, others have proposed that CaM directly interacts with the Ca²⁺ channel ₁-subunit and that channel phosphorylation is not involved (see, for example, Refs. 129, 133, 142, and 211). Some studies have gone so far as to suggest that direct binding of CaM to the ₁-subunit is a key step in both Ca²⁺-dependent inactivation and facilitation of Ca_V1.2 channels. Recently, it has been shown that mutations in a consensus CaM-binding IQ motif in the COOH-terminal tail of ₁ eliminates both forms of Ca²⁺-dependent automodulation of Ca²⁺ channels (210, 211). Whether this direct interaction between CaM and the channel ₁-subunits is sufficient to induce both Ca²⁺-dependent inactivation and facilitation requires further study, as does the question of the role of CaMK in the process of autoregulation.