Regulacion Canales CA Cardiaco y Liso

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281:1743-1756, 2001.Am J Physiol Cell PhysiolKathleen D. Keef, Joseph R. Hume and Juming Zhongchannels (CaV1.2a,b) by protein kinasesRegulation of cardiac and smooth muscle Ca2+

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invited review

Regulation of cardiac and smooth muscle Ca2

channels(CaV1.2a,b) by protein kinases

KATHLEEN D. KEEF,1 JOSEPH R. HUME,1,2 AND JUMING ZHONG21Department of Physiology and Cell Biology and 2Department of Pharmacology,

University of Nevada School of Medicine, Reno, Nevada 89557

Keef, Kathleen D., Joseph R. Hume, and Juming Zhong. Regu-lation of cardiac and smooth muscle Ca2 channels (CaV1.2a,b) byprotein kinases. Am J Physiol Cell Physiol 281: C1743C1756,2001.High voltage-activated Ca2 channels of the CaV1.2 class (L-

type) are crucial for excitation-contraction coupling in both cardiac andsmooth muscle. These channels are regulated by a variety of secondmessenger pathways that ultimately serve to modulate the level ofcontractile force in the tissue. The specific focus of this review is on themost recent advances in our understanding of how cardiac CaV1.2a andsmooth muscle CaV1.2b channels are regulated by different kinases,including cGMP-dependent protein kinase, cAMP-dependent protein ki-nase, and protein kinase C. This review also discusses recent evidenceregarding the regulation of these channels by protein tyrosine kinase,calmodulin-dependent kinase, purified G protein subunits, and identifi-cation of possible amino acid residues of the channel responsible forkinase regulation.

cGMP-dependent protein kinase; cAMP-dependent protein kinase; pro-tein kinase C; G proteins

CAV1.2 CHANNELS are members of the superfamily of volt-age-dependent Ca2 channels that includes low volt-age-activated, rapidly inactivating channels and highvoltage-activated channels. The nomenclature for volt-age-dependent Ca2 channels has recently changedfrom alphabetic descriptors, i.e., L-, N-, P/Q-, andR-type, to names based on a system previously devel-oped to describe K channels. This system uses numer-als to define families and subfamilies of channels basedon similarities in amino acid sequences. Thus the CaV1family includes Ca2 channel 1-subunits encoded byfour separate genes that give rise to CaV1.1, CaV1.2,CaV1.3, and CaV1.4 (formerly 1S, 1C, 1D, and 1F,respectively) (see Ref. 43). This review focuses specifi-cally on signaling pathways involved in the regulationof cardiac and smooth muscle CaV1.2 channels. CaV1.2channels in both cardiac and smooth muscle contributeto contraction by delivering extracellular Ca2 to thecytoplasm 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, 5573, 74, 85, 114, 162, 163). The specific focus of thisreview, therefore, is on the most recent advances in ourunderstanding of CaV1.2a and CaV1.2b channel regulation in cardiac and smooth muscle by cGMP-dependent protein kinase (PKG), cAMP-dependent proteinkinase (PKA), and protein kinase C (PKC).

STRUCTURE OF CARDIAC AND SMOOTH MUSCLE

CA2 CHANNELS

The Ca2

channels examined in this review are multisubunit protein complexes composed of a pore-forming1-subunit and several auxiliary subunits including an intracellularly located -subunit and anextracellularly located, disulfide-linked 2/-subuni(Fig. 1). The cardiac and smooth muscle 1-subunitsare splice variants of the same gene and are designatedCaV1.2a and CaV1.2b, respectively. CaV1.2a andCaV1.2b exhibit 93% homology in amino acid sequenceThe molecular mass of each is 240 kDa, and each1-subunit contains 2,170 amino acids (11, 37, 73, 8190, 92, 119, 160, 185). The 1-subunit defines the ionic

Address for reprint requests and other correspondence: K. Keef,Dept. of Physiology and Cell Biology/MS 352, Univ. of Nevada Schoolof Medicine, Reno, NV 89557 (E-mail: [email protected]).

Am J Physiol Cell Physio281: C1743C1756, 2001

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pore of the channel and contains four repeats, eachwith six transmembrane segments. Cardiac andsmooth muscle 1-subunits are distinguished by a mi-nor difference in the NH2 terminus, in the hydrophobicsegments IS6 and IVS3, and by an insertion in the IIIcytoplasmic linker in the smooth muscle subunit (Fig.1) (9, 37, 92, 119). Splice variants of CaV1.2 have beenreported to exist within a single tissue (10, 44). Aninteresting new twist in the story are recent reportssuggesting that the COOH-terminal end of CaV1.2a iscleaved in vivo but remains colocalized with the bodyof the 1-subunit. A proline-rich domain between resi-dues 1973 and 2001 of the 1-subunit has been identi-

fied as the mediator of the COOH-terminal membraneassociation (49, 51). Because the COOH-terminal re-gion suppresses CaV1.2 channel activity (182), thechannel could be regulated by changes in the associa-tion of the COOH-terminal fragment with the body ofthe 1-subunit.

Four different genes encode mammalian voltage-gated Ca2 channel -subunits (14; Ref. 11). Inaddition, each of the four gene products can be alter-natively spliced, giving rise to two to four splice vari-ants per -subunit. Overall, these -subunits varyfrom 53 to 71 kDa (for review, see Refs. 12 and 37). In

cardiac muscle the 2-subunit predominates (49, 5977, 134), whereas in smooth muscle at least threedifferent -subunits (i.e., 1b, 2, and 3, 5468 kDamolecular mass) have been identified (10, 31, 54, 77176). In contrast to the 1-subunit, the -subunit doesnot contain putative transmembrane domains, although there are hydrophobic regions. The -subunitightly binds to a highly conserved 18-amino acid se

quence in the cytoplasmic linker between repeats I andII of the 1-subunit (136). -Subunits target the 1subunit to the plasma membrane (12, 48, 53, 196) andfacilitate CaV1.2 channel currents (12, 32, 53, 76, 122154). The 2a-subunit is unique in that it is posttranslationally palmitoylated by addition of a 16-carbonpalmitic acid group to cysteine residues of the -subunit through a labile thioester linkage (26, 27). Thispalmitoylation confers unique regulatory properties onCaV1 channels (138), although it is uncertain whethersuch palmitoylated subunits actually exist in eithercardiac or smooth muscle cells.

The 2/ complex consists of an extracellularlylocated 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 (3749, 183) and smooth muscle (3). This subunit appearsto increase Ca2 channel currents (91, 154) but alsohas been associated with prevention of prepulse facilitation (33).

PKG REGULATION OF CA2 CHANNELS

PKG catalyzes the phosphorylation of a number ofintracellular proteins that modulate muscle contraction. Two main types of PKG are present in eukaryoticcells, type I and type II (104). Type I PKG is a ho

modimer, and each subunit has a mass of 78 kDaType II PKG also exists as a dimer whose subunit massis 86 kDa. The regulatory and catalytic domains othis molecule are both contained within a singlepolypeptide sequence. PKG type I is widely distributedand is isolated from soluble extracts of tissues, whereasPKG type II is a particulate form of the enzyme andhas a limited tissue distribution. PKG type I is presentin both cardiac and smooth muscle cells, although thelevel of PKG is much greater in smooth muscle than incardiac muscle (106). This difference is attributable tosome degree to a developmental decline in PKG levelsfrom newborn to adult heart (95).

Role of PKG in Cardiac Muscle

In cardiac muscle there is continued controversyconcerning the role of the cGMP/PKG pathway in theregulation of myocardial function (94). Although anumber of studies have reported cGMP/PKG-mediatedinhibition of CaV1.2a channels (13, 61, 64, 82, 103, 116145, 169, 170, 179), others have reported the oppositeeffect, particularly when cAMP levels are elevated (6288, 96, 128, 145). However, in most cases where stimulation has been observed, the proposed mechanismwas not direct PKG-mediated activation of CaV1.2a

Fig. 1. Model of the cardiac and smooth muscle CaV1.2 channel (1)and associated auxiliary subunits. In addition to the pore-forming1-subunit, there is an intracellularly localized -subunit and anextracellularly located, disulfide-linked 2/-subunit. Sites of diver-gence between the cardiac and smooth muscle 1-subunits are indi-cated at the NH2 terminus, in the hydrophobic segments IS6 andIVS3 and by an insertion between I and II cytoplasmic linker.

Evidence points to a functional role for cGMP-dependent proteinkinase (PKG) phosphorylation of CaV1.2a at serine 533 (82). Thesame PKG phosphorylation site exists in CaV1.2b (i.e., serine 528).Evidence also suggests a functional role for cAMP-dependent proteinkinase (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 ofserine residues 478 and 479 on the 2a-subunit also are involved infunctional regulation of channels by PKA (18). Although 18 poten-tial protein kinase C (PKC) phosphorylation sites exist on the 1-subunit, no studies to date have clearly identified a role for thesesites in channel activation. However, recent studies suggest thatthreonine-27 and threonine-31 on CaV1.2a are involved in PKC-induced inhibition of the channel (115). These sites are not presenton CaV1.2b.

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but, rather, indirect mechanisms. For example, cGMPinhibits phosphodiesterase 3 (PDE3) (177). A reductionin PDE3 activity could lead to enhanced cAMP levelsand, consequently, Ca2 channel stimulation (88, 128).However, in the case of newborn rabbit ventricularcells, the stimulatory effect of cGMP on CaV1.2a ap-peared to be directly related to PKG, because it wasabolished by the PKG inhibitor KT-5823 but not by the

PKA inhibitor 522 or the PDE inhibitor IBMX (96).Three different mechanisms have been suggested to

account for cGMP-induced inhibition of CaV1.2 chan-nels, i.e., 1) direct phosphorylation of the channel byPKG, 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 implicationof the second and third possibilities listed is that cGMPreduces the phosphorylation of CaV1.2 produced byPKA. Each of these possibilities is discussed below andis illustrated in Fig. 2.

Evidence to support the notion that PKG directlyphosphorylates Ca2 channels has been derived from

functional studies of CaV1.2a channels expressed inoocytes (82). In this study, PKG was activated by ad-dition of the membrane-permeant species 8-bromo-guanosine 3,5-cyclic monophosphate (8-Br-cGMP).This cyclic nucleotide inhibited wild-type Ca2 channelcurrents. In contrast, when serine residue 533 of the1-subunit was replaced with alanine, 8-Br-cGMP nolonger had an effect on channel activity, suggestingthat PKG produces inhibition by phosphorylating thechannel at serine 533 (82). The actions of 8-Br-cGMPappeared to be independent of either the - or the2/-subunit of CaV1.2a because inhibition still oc-curred in the absence of these subunits.

Protein phosphorylation represents a balance be-tween kinase and phosphatase activity. Thus secondmessenger pathways that modify this balance can up-

regulate or downregulate channel activity to meetphysiological demands. An additional mechanism bywhich PKG, in particular, could inhibit Ca2 channeactivity is through stimulation of phosphatase activityleading to dephosphorylation of the channel. Wheninhibitors of either protein phosphatase 1 (PP1) or 2A(PP2A) are applied to cardiac myocytes, Ca2 channestimulation generally has been observed, suggesting

that there is both basal phosphorylation and dephosphorylation of the channel (for review, see Ref. 69). Thenotion that PKG may exert its effect by activating aphosphatase is not new. Smooth muscle myosin phosphatase is a known target of PKG, and activation othis phosphatase contributes to the well-known phenomenon of myofilament desensitization (72, 180). Inaddition, there is evidence suggesting that the actionsof cGMP on Ca2-activated K channels in rat pituitary tumor cells are due to PKG-induced stimulationof PP2A (187). In a more recent study, 8-Br-cGMP wasreported to inhibit IBMX-stimulated Ca2 channel currents in guinea pig ventricular myocytes, and thiseffect was abolished by the type 1 and 2A phosphataseinhibitor okadaic acid, leading to the conclusion thatPKG 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 beensubdivided into seven families. At least four of thesefamilies (i.e., PDE14) are present in cardiac muscle(177). PDE1 and PDE2 hydrolyze both cAMP andcGMP, whereas PDE3 and PDE4 hydrolyze cAMPPDE2 is stimulated by cGMP, whereas PDE3 is inhibited by cGMP and PDE4 is insensitive to cGMP (135)

In some tissues, cGMP may stimulate CaV1.2a channecurrents via inhibition of PDE3 (88, 128), whereas inothers, cGMP may inhibit CaV1.2a channel current bystimulating PDE2 (45, 117). This opposing effect ocGMP on PDE2 vs. PDE3 serves to underscore thecomplexity of interpreting studies in which cGMP isapplied to cells.

Role of PKG in Smooth Muscle

In contrast to its role in cardiac muscle, the role ofPKG as an important mediator of vascular relaxationis well established (for review, see Ref. 19), particularlywith regard to the actions of the endothelium-relaxing

factor nitric oxide (NO) and nitrovasodilators such assodium nitroprusside. PKG reduces cytoplasmic Ca2

concentration via several mechanisms, and a numberof studies suggest that one of these mechanisms is theinhibition of CaV1.2b channels. For example, CaV1.2bcurrents are reduced by NO and by NO donors such assodium nitroprusside and by the membrane-permeableanalog 8-Br-cGMP (1, 14, 80, 93, 109, 111, 139, 144167, 195). Current inhibition is reversed with PKGblockers (144, 167). However, the mechanism by whichPKG reduces CaV1.2b channel activity is still unknown. Mutation experiments such as those described

Fig. 2. Mechanisms proposed for cGMP-induced inhibition of CaV1.2channels. In this schematic, guanylyl cyclase (G cyclase) is activatedby nitric oxide (NO) to give rise to an increase in cGMP levels. cGMPthen activates PKG to directly phosphorylate the 1-subunit of theCa2 channel. Another possibility is that PKG activates a proteinphosphatase (PP), which then dephosphorylates the channel. Fi-nally, cGMP may stimulate phosphodiesterase 2 (PDE2), whichreduces cAMP levels and thus reduces stimulation of the channel byPKA. AKAP, A-kinase anchoring protein; A cyclase, adenylyl cyclase.

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for the 1-subunit of CaV1.2a (82) have yet to be re-peated for CaV1.2b.

The notion that PKG inhibits CaV1.2b via the acti-vation of a phosphatase is an intriguing one. Applica-tion of inhibitors that block both PP1 and PP2A (e.g.,okadaic acid and calyculin A) have generally increasedCa2 channel activity (46, 56, 100, 123, 124), suggest-ing that basal channel activity is determined by a

balance of kinase and phosphatase activity. Some stud-ies in smooth muscle have directly applied activatedphosphatases to isolated patches or to the whole cellvia cell dialysis. These studies have generally revealedinhibition 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 directlylinking activation of a phosphatase to the PKG-in-duced inhibition of CaV1.2b channels observed insmooth muscle.

Five of the seven PDE isoenzyme families have beenidentified in vascular smooth muscle, including PDE14 and PDE5, which is a cGMP-hydrolyzing PDE (forreview, see Ref. 135). PDE3 accounts for approximately

half of the cAMP-hydrolyzing activity in the pulmonaryartery and aorta (140, 141). To date, there are nostudies investigating the possible role of cGMP activa-tion of PDE2 to account for the inhibitory effect ofcGMP on CaV1.2b.

PKA REGULATION OF CA2 CHANNELS

PKA is a tetramer consisting of two catalytic sub-units bound to a regulatory subunit dimer. Like PKG,PKA is ubiquitously expressed in smooth muscle andcardiac muscle. Initially, two different forms of theregulatory 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 membranebound. However, recent molecular cloning techniqueshave revealed significant heterogeneity in both thecatalytic and regulatory subunits of PKA (165).

Role of PKA in Cardiac Muscle

The cardiac CaV1.2a channel has long been recog-nized as a target of the adenylyl cyclase/PKA path-way. Stimulation of -adrenoceptors leads to en-hanced single-channel activity as well as a three- tosevenfold increase in whole cell current in cardiaccells (114, 150, 171, 173) (see Fig. 3). Recent reportssuggest that 1-adrenergic receptors, which couple

exclusively to the G protein Gs, produce a morewidespread increase in cAMP levels in the cell (i.e.,diffuse activation), whereas 2-adrenergic receptors,which can be coupled to both Gs and Gi, produce amore localized activation of CaV1.2a channels (24).Early studies directed toward determining whetherPKA phosphorylates CaV1.2 channels were compli-cated by the fact that the majority of biochemicallyisolated 1-subunits undergo a proteolytic cleavagethat removes a substantial portion of the COOH-terminal tail (22, 36, 68, 99, 202). In 1992, Yoshidaand coworkers (202) showed that PKA-induced phos-

phorylation occurred in the 250-kDa form of the1-subunit but not in the truncated 200-kDa formLater studies confirmed the importance of theCOOH-terminal tail and identified serine 1928 asthe site of PKA-induced phosphorylation (34, 50, 68120, 131). Interestingly, although the COOH-terminal of the 1-subunit is cleaved, Hosey and colleagues (49, 51) have provided evidence that thecleaved portion of the 1-subunit remains associatedwith the body and thus may still be involved in

PKA-induced regulation of the channel. Other studies have shown that the cardiac -subunit also isphosphorylated during activation of the adenylyl cyclase/PKA pathway (50, 58, 137), and the sites ofphosphorylation appear to be three loose consensussites for PKA in the COOH terminus rather than thetwo strong consensus sites identified for PKA (52). Ina later study, two of these three sites (i.e., serine-478and serine-479) were shown to be critical for PKAinduced regulation of the 1-subunit (18). The relative contribution of the 1- vs. 2-subunit phosphorylation to PKA regulation of CaV1.2a in vivo has yetto be determined and, indeed, may change with development and/or other conditions present in the

cell. There are reports that PKA-induced phosphorylation of the 1-subunit is dependent on localizingPKA to the vicinity of the channel via A-kinaseanchoring protein (AKAP) (Fig. 3), whereas phosphorylation of the 2a-subunit does not requireAKAP (50). However, another study failed to reproduce a PKA-dependent increase in current amplitudein HEK-293 cells expressing the cardiac 1- and2a-subunits, even when these subunits were coexpressed with AKAP79 (33).

Additional complications have arisen in attempts todemonstrate a functional correlate of PKA-induced

Fig. 3. Mechanisms proposed for -adrenergic stimulation of CaV1.2channels. -Adrenergic receptors (-AR) are stimulated with agonists such as isoproterenol. The -receptor is coupled to the G proteinGs, which leads to activation of the G protein subunits Gs and GGs stimulates A cyclase, leading to enhanced levels of cAMP thaactivate PKA localized to the region of CaV1.2 via an AKAP. PKA ithen proposed to activate the Ca2 channel via phosphorylation oone or more of the Ca2 channel subunits. In contrast, G iproposed to lead to stimulation of PKC, possibly via phosphatidylinositol 3-kinase, leading to phosphorylation of 1 or more of the Ca 2

channel subunits. Finally, evidence exists for direct activation oCaV1.2 via a direct membrane-delimited action of Gs on thechannel.




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phosphorylation in expression systems. Although somestudies have reported PKA-induced stimulation of car-diac CaV1.2a currents (50, 90, 150, 199, 202), othershave failed to show any effect of PKA (33, 118, 209). Avariety of explanations have been offered for the neg-ative results, including the importance of auxiliarysubunits, particularly the -subunit (90) and, morerecently, the need for channel targeting of PKA by

AKAP (50). One report suggested that an additional, asyet unidentified, component of the pathway is impor-tant for PKA-induced regulation of the Ca2 channel,because PKA failed to stimulate CaV1.2a channels un-less oocytes were injected with mRNA from cardiaccells (23). Yet another study suggested that PKA leadsto phosphorylation of a 700-kDa protein that couples tothe cardiac-subunit (60). Despite studies that suggestan obligatory role for additional regulatory subunits,several studies have reported PKA-induced enhance-ment of expressed 1-subunits of CaV1.2a in the ab-sence of other regulatory proteins (150, 202). In sum-mary, evidence exists in both native cells andexpression systems to suggest that PKA can stimulatecardiac CaV1.2a channels via direct phosphorylation ofserine 1928 on the 1-subunit. However, additionalsites appear to contribute to this process. The variableresults reported from expression systems may be re-lated to differences in the cell types used, differences inthe regulatory proteins coexpressed with the 1-sub-unit, and the activity of other enzymes in the cells (e.g.,PDEs, phosphatases) that modify the basal levels ofphosphorylation of the channel. Indeed, in some stud-ies, although activated PKA was reported to be withouteffect on channel activity, inhibition of endogenousPKA decreased channel activity, suggesting that theCaV1.2a channels were maximally phosphorylated un-

der basal conditions (131, 132, 159).

Role of PKA in Smooth Muscle

In contrast to the general agreement concerning theactions of the adenylyl cyclase/PKA pathway on car-diac CaV1.2a channels, the details of how PKA modu-lates CaV1.2b channels in smooth muscle, and, indeed,even the direction of this modulation has remainedsomewhat controversial. Over the past 12 years, theadenylyl cyclase/PKA pathway has been reported toinhibit (109, 161, 195), to enhance (47, 80, 84, 93, 113,144, 151, 164, 166, 206), or to have no effect (121, 127,184) on smooth muscle CaV1.2b channels. However, a

preponderance of recent evidence has gradually accu-mulated to suggest that PKA can modulate smoothmuscle CaV1.2b channels in a similar manner to thatdescribed for cardiac CaV1.2a channels. Specifically,studies of various smooth muscle cells have shown that1) the membrane-permeable analog 8-Br-cAMP en-hances CaV1.2b channel currents (80, 144), an effectblocked by PKA inhibitors (87, 206); 2) the adenylylcyclase activator forskolin enhances CaV1.2b channelcurrents (80, 151), an effect blocked by PKA inhibitors(200); 3) activation of adenylyl cyclase with isoproter-enol enhances CaV1.2b channel currents (80, 93, 151,

161, 194), an effect blocked by PKA inhibitors (206); 4activation of adenylyl cyclase with the G protein sub-unit Gs enhances CaV1.2b currents (193, 205), aneffect blocked by PKA inhibitors (205); 5) the catalyticsubunit of PKA enhances whole cell CaV1.2b currentsan effect blocked by PKA inhibitors (144); and 6) application of the catalytic subunit of PKA to the cytosolicsurface of inside-out patches increases the open prob-

ability of CaV1.2b channels (166). In addition, thesmooth muscle 1-subunit shares 93% homology withthe cardiac 1-subunit (92, 185) and contains the sameconsensus PKA phosphorylation site (73). Also, as incardiac muscle, there is evidence that the actions ofPKA on CaV1.2b channels requires targeting via anAKAP (206). All of these observations strongly suggestthat PKA is likely to have a similar action on bothCaV1.2b and CaV1.2a channels (Fig. 3). However, theresponse to PKA differs in two respects. First, moststudies to date have not reported a significant effect ofcAMP on the shape of the current-voltage (I-V) relationship of CaV1.2b channels in smooth muscle cells(80, 151). In contrast, a number of studies of cardiac

muscle have noted a shift to the left of the I-Vrelationship with cAMP (see, for example, Ref. 6). Second, thecAMP pathway in cardiac muscle leads to a 3- to 7-foldincrease in CaV1.2 channel currents, whereas insmooth muscle the stimulation is more modest, i.e.ranging between a 0.5- and 2-fold increase.

The stimulatory effect of PKA on cardiac CaV1.2achannels corresponds well with the known positiveinotropic effect of -adrenoceptor stimulation in thismuscle. However, in smooth muscle, agonists that activate adenylyl cyclase typically cause relaxation, making it more difficult to reconcile a stimulatory effect ofPKA on CaV1.2b currents. It is possible that the Ca2

entry associated with PKA stimulation is specificallytargeted to events that are unrelated to global cytoplasmic Ca2 concentration; e.g., an increase in thesubsarcolemmal Ca2 concentration may be coupled toenhancement of Ca2-activated K channel activity(57). Alternatively, the enhanced Ca2 entry may serveto fill stores in the sarcoplasmic reticulum (SR).

Some of the controversy surrounding the actions ocAMP on CaV1.2b channels may be related to thehigher levels of PKG present in smooth muscle com-pared with cardiac muscle (106). Neither cAMP norcGMP display absolute specificity for their respectivekinases, i.e., the equilibrium dissociation constant foractivation of PKG by cAMP is 10-fold greater than

that for PKA (see, for example, Ref. 39). Thus some othe actions of cAMP that have been attributed to PKAon CaV1.2b channels may, in fact, be due to PKG. Insupport of this hypothesis are studies showing thathigher concentrations of 8-Br-cAMP and forskolin leadto inhibition of CaV1.2b channel currents and that thiseffect is blocked by PKG inhibitors (80, 144). Thesedata suggest that CaV1.2b channels can be inhibited bycross-over activation of PKG by cAMP. Cross-overactivation of PKG by cAMP also has been proposed byothers to contribute to the effects of cAMP (see, forexample, Refs. 105 and 186). Interestingly, in the pres

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ence of PKG inhibitors the reverse cross-over activa-tion can be demonstrated as well, i.e., cGMP activationof PKA, leading to stimulation of CaV1.2b channelcurrent (144). A similar action of cGMP on PKA hasbeen proposed to underlie the NO-induced relaxationin PKG-I-deficient mice (146). This cross talk be-tween cyclic nucleotides and protein kinases is illus-trated in Fig. 4.

Only one study to date has demonstrated enhance-ment of CaV1.2b channel currents with raised cAMP inan expression system (90). In this study, the 1-subunitof CaV1.2b was coexpressed with the skeletal muscle1a-subunit. cAMP produced current stimulation onlyin the presence of the -subunit. The authors sug-gested that the kind of -subunit present in a tissuemay underlie tissue-specific effects of cAMP/PKA.However, other -subunits (e.g., 2 or 3) were nottested in this study. A recent study of A7r5 cells alsosuggests that cAMP-dependent stimulation of thechannel requires a functional -subunit (87). Two otherstudies have failed to show enhancement of theCaV1.2b 1-subunit by cAMP/PKA in an expression

system (89, 209). In one case the 1-subunit was ex-pressed with 2- and 2/-subunits in HEK-293 cells(209), and in the other case the 1-subunit was ex-pressed alone in Chinese hamster ovary cells (89). Thisvariability between studies of expressed smooth mus-cle CaV1.2b channels is similar to the variability re-ported for the actions of PKA on expressed cardiacCaV1.2a channels.

PKC REGULATION OF CAV1.2 CHANNELS

Eleven isoforms of PKC have been identified in mam-malian tissues, and these are divided into threegroups: 1) classic or conventional PKCs (cPKC) that

are activated by diacylglycerol or phorbol ester and areCa2 sensitive (including, I, II, ), 2) novel or newPKCs (nPKC) that are activated by diacylglycerol orphorbol ester but are not Ca2 dependent (, , , , L,

), and 3) atypical PKCs (aPKC) that are not activatedby diacylglycerol, phorbol ester, or Ca2 (/, ) (forreview, see Ref. 75).

Role of PKC in Cardiac Muscle

In cardiac muscle PKC phosphorylates a variety ofdifferent proteins that contribute to myocardial excit

ability and contraction. Among these is the CaV1.2achannel. Both the 1- and 2-subunits of CaV1.2a arephosphorylated by PKC in vitro with a stoichiometry o23 moles of phosphate per mole of1-subunit and 12moles of phosphate per mole of2a-subunit (137). Thefirst 46 amino acid residues at the NH2-terminal end othe cardiac 1-subunit have been shown to inhibitchannel activity, and it has been suggested that PKCmay enhance CaV1.2a channel activity by relievingNH2-terminal inhibition (153). In this scheme, the siteof PKC phosphorylation is left undefined except toexclude the NH2-terminal region per se from phosphorylation (155). PKC generally has been reported to ac

tivate CaV1.2a channels in heterologous systems (17153, 158), but there are exceptions (115). Some studiesin native cells have reported PKC-mediated stimulation of CaV1.2a (41, 67, 110), whereas others reportinhibition (204). Current stimulation with phorbol esters sometimes has been reported to be transient andfollowed by later current inhibition (97, 172), with thelater effect being PKC independent (5, 17, 97, 172)Furthermore, there is preliminary evidence to suggestthat different PKC isoforms may produce opposingactions on CaV1.2a, with cPKCs producing inhibition oCaV1.2a but nPKCs enhancing channel current (63). Arecent study by McHugh et al. (115) of Ca2 channels

expressed in TSA-201 cells suggests that inhibition ofchannel activity by PKC occurs through phosphorylation of threonine-27 and threonine-31 at the NH2terminal region of the 1-subunit. These data underscore the need for additional studies to clarify thepossibly complex nature of PKC-induced effects onCaV1.2a.

Role of PKC in Smooth Muscle

PKC has been reported to enhance CaV1.2b channecurrents in various smooth muscle preparations(28, 101, 102, 125, 147, 152, 175, 178, 205). As withCaV1.2a, stimulation of CaV1.2b is sometimes followedby current inhibition (147). A number of different agonists, including norepinephrine, endothelin, angiotensin II, and serotonin, have been suggested to stimulateCaV1.2b channels via a PKC-dependent mechanism(for review, see Ref. 55). Because agonists that activatePKC often produce inositol 1,4,5-trisphosphate-induced release of Ca2 from the SR, the PKC-inducedchannel stimulation can be masked by Ca2-inducedinactivation of channels (71, 86). Little is known aboutthe mechanisms underlying CaV1.2b channel modulation by PKC.

Fig. 4. Mechanisms proposed for cGMP stimulation and cAMP inhi-bition of CaV1.2 channels (red arrows). Cross talk between the cyclicnucleotides may lead to cAMP-induced activation of PKG to inhibitthe channel or cGMP-induced activation of PKA to stimulate thechannel. Cross talk is more likely to occur when higher levels of cyclicnucleotide are achieved in the cell. Finally, cGMP also has beenreported to inhibit PDE3, which leads to an increase in cAMP levelsand, hence, stimulation of the channel via PKA.




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G PROTEIN SUBUNIT REGULATION OF

CAV1.2 CHANNELS

The PKA and PKC pathways discussed above aregenerally linked to agonist-induced activation of Gprotein subunits. Activated G protein subunits canregulate different ionic channels not only through in-tracellular second messenger pathways but alsothrough a direct membrane-delimited gating of chan-nels. For example, neuronal CaV2.2 channels (formerlyN-type or 1B) are inhibited by G protein-activatedPKC and by the direct binding of G to the 1-subunit(78). Direct G binding takes place in the intracellu-lar loop between repeat I and II as well as in a seconddownstream sequence (38). However, the requisitebinding motif for this interaction is absent in CaV1.2channels. In the case of CaV1.2 channels, evidence hasbeen presented to suggest a direct membrane-delim-ited activation of CaV1.2a and CaV1.2b channels by theG protein subunit Gs; however, these effects are con-troversial (29, 170).

Role of G Protein Subunits in Cardiac MuscleThe first evidence suggesting a direct membrane-

delimited action of Gs on CaV1.2a channels was fromstudies showing that the GTP analog guanosine 5-O-(3-thiotriphosphate) (GTPS) prolonged the survival ofexcised Ca2 channels and that Ca2 channels incor-porated into a lipid bilayer could be activated indepen-dently of cAMP, ATP, PKA, and the PKC activatorphorbol ester. Preactivated Gs protein and the Gssubunit also were shown to mimic the effects of GTPSon channels in excised patches or in lipid bilayers (79,197). Additional studies have reported that -adrener-gic stimulation of CaV1.2a channels could not be ex-

plained solely by a PKA-dependent pathway, i.e., iso-proterenol still increased CaV1.2a current by 3580%in the presence of inhibitors of the adenylyl cyclase/PKA pathway in guinea pig ventricular myocytes (20,130, 156), whereas forskolin, which is not G proteinlinked, was blocked entirely by the same maneuvers.In contrast, other studies have reported that the stim-ulatory effect of isoproterenol on CaV1.2a is blockedentirely by PKA inhibitors in several different animalspecies (29, 65, 66, 83).

Recent studies have provided further evidence for amembrane-delimited gating of CaV1.2a by Gs sub-units. The whole cell Ca2 channel current in cardiacmyocytes from transgenic mice overexpressing cardiac

Gs is 490% higher than in cells from wild-type controlanimals (98). Furthermore, in Xenopus oocytes, anti-sense knockdown of endogenous Gs reduced currentsof expressed CaV1.2a channels, whereas coexpressionof Gs with CaV1.2a enhanced currents (15). Interest-ingly, PKA inhibitors did not have any detectable ac-tion on the effects of Gs in either the transgenic miceor the oocyte model. In fact, a small cAMP-dependentdecrease of current was observed in oocytes coex-pressed with CaV1.2a and the 2-adrenergic receptor(15). The authors concluded that coexpression of Gs,but not its acute activation via -adrenergic receptors,

enhances CaV1.2a currents via a PKA-independenpathway. A recent study of CaV1.2a and 2-subunitsexpressed in Chinese hamster fibroblasts with -adrenoceptors also suggested the presence of an additionacAMP-independent mechanism of channel activation(198). Thus controversy continues to surround the issue of direct membrane-delimited effects of Gs onCaV1.2a, although a high-affinity binding site for G

on CaV1.2a has yet to be identified.In our recent studies of G protein activation of native

guinea pig cardiac CaV1.2a channel currents, we didnot obtain any evidence to suggest the presence of adirect membrane-delimited pathway for either Gs orG activation of the channel (208). Rather, the pathways involved appeared to be very similar in nature tothose described below for G protein regulation osmooth muscle CaV1.2b channels (see also Ref. 205 aswell as Fig. 3), leaving open the possibility that thePKA-independent effect of-adrenergic stimulation incardiac myocytes identified by others may actually bedue to G-induced activation of PKC, rather than to adirect membrane-delimited effect of Gs.

Role of G Protein Subunits in Smooth Muscle

In 1994, Xiong and colleagues (195) reported thatdialysis of cells with activated Gs gave rise to stimulation of Ca2 channel currents in rabbit portal veinmyocytes. It was suggested that this effect representeda direct membrane-delimited action of Gs on the channel. We directly explored this issue by comparing theeffect of dialyzing cells with either activated Gs orG12. Both Gs and G stimulated CaV1.2b currentsin rabbit portal vein myocytes, whereas inactive subunits [i.e., Gs-guanosine 5-O-(2-thiodiphosphate) andnonprenylated G] were without effect. Of evengreater interest was the observation that different second messenger pathways appeared to be involved inthe actions of the two G protein subunits. Thus Genhanced current via the adenylyl cyclase/PKA pathway, whereas G activated channels via a PKC-dependent pathway (205). Recently, we determined thatboth of these pathways also are present when endogenous Gs and G are stimulated with isoprotereno(Fig. 3) (207). To date, G dimers from four differentsources all have been shown to stimulate CaV1.2bchannels in a PKC-dependent manner. These includeendogenous G13 coupled to G13 in rat portal vein(112), G purified from rat brain Gi/Go (178), G12

purified from Sf9 cells (205), and endogenous Gcoupled to Gs in rabbit portal vein cells (207). Thesedata suggest that G-induced stimulation of CaV1.2bchannels may be a fairly ubiquitous feature of agonistinduced G protein stimulation. Under these circumstances, G protein specificity still could be conferred bythe G subunit coupled to G. The notion that manydifferent G dimers exert similar effects certainly isnot new. This was suggested early on (see, for exampleRef. 174) and has received continued support in morerecent times (30, 143). The question of how G leadsto activation of PKC in this pathway has been only

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recently addressed. Studies of rat portal vein myocytessuggest that at least one mechanism involved is G-induced stimulation of phosphatidylinositol 3-kinase(178). In most of the recent work on G protein subunitregulation, there has been little evidence to support theidea that either Gs or G activates CaV1.2b channelsvia a direct membrane-delimited pathway.

REGULATION OF CAV1.2 CHANNELS BY

OTHER KINASES

Recent studies suggest that the activity of CaV1.2channels in cardiac and smooth muscle cells also isregulated by other kinases, especially by protein ty-rosine kinase (PTK) and calmodulin-dependent kinase(CaMK). Although this review focuses mainly onCaV1.2 channel regulation by PKG, PKA, and PKC, asummary of recent reports of channel regulation byPTK and CaMK follows.

Role of PTK

Tyrosine phosphorylation is an important regulatorof cell function not only for growth-related responsessuch as gene transcription and cell division but also forrapid cellular responses such as cell adhesion andmuscle contraction. Early studies on possible regula-tion of cardiac Ca2 channels by PTK indicated thatgenistein, a specific PTK inhibitor, dose-dependentlyreduced CaV1.2 channel currents in rat (201) andguinea pig ventricular myocytes (25). However, inthese studies, daidzein, an inactive analog of genistein,exerted the same inhibitory effect on Ca2 channelcurrent as genistein. In contrast, several other studieshave reported inhibition of Ca2 channel currents withPTK blockers that was not mimicked by daidzein (126,181). In feline atrial myocytes, genistein was reportedto have a biphasic effect on Ca2 channel currentswhen examined with the whole cell perforated-patchrecording method (181). Both inhibition and stimula-tion with genistein were not mimicked by daidzein butwere abolished by the tyrosine phosphatase inhibitorvanadate. Interestingly, current stimulation was ab-sent when the conventional whole cell patch recordingmethod was used. The authors concluded thatgenistein inhibits Ca2 channel current by blockingmembrane-bound PTKs and stimulates Ca2 channelcurrent by blocking cytosolic PTKs. Others have re-ported that PTK inhibition leads only to current stim-

ulation and have suggested a PKC-dependent mecha-nism (16). A recent report further proposes that PTKalso may directly antagonize -adrenergic receptors(157). Together, these studies suggest that PTK mayplay a significant role in the regulation of cardiacCaV1.2 channels, but at present even the direction ofthis regulation appears to be controversial. Additionalstudies are required to better understand the possiblycomplicated role(s) that various PTKs play in modulat-ing CaV1.2a activity. Caution is advisable as well wheninterpreting results using PTK antagonists, which mayhave more than one site of action.

Evidence that vascular CaV1.2b channels are regulated by PTK also has been provided. Wijetunge et al(188) reported that CaV1.2b current in rabbit ear artery myocytes was significantly reduced by bothgenistein and tyrphostin 23 but not by daidzein. Inhi-bition of tyrosine phosphatases (190) or activation oendogenous c-Src tyrosine kinase (189) increased current in the same cell preparation. PTK inhibitors an

tagonized these stimulatory effects, suggesting a possible role for endogenous c-Src tyrosine kinase in themodulation of vascular Ca2 channels (189, 191). In ratportal vein myocytes, genistein also reduced whole celCa2 channel currents, whereas daidzein was withouteffect (107). Further studies of these cells revealed thatgenistein also decreased the mean open time and prolonged closed time of single channels (108).

Although the evidence to date suggests a possiblerole of PTK in the regulation of CaV1.2 channels, nostudies have yet extended this work to PTK regulationof expressed cardiovascular CaV1.2 channels. A recentstudy of neuronal CaV1.2 channels indicated that potentiation of current by insulin-like growth factor 1required phosphorylation of the 1-subunit by PTK (8)It is possible that PTK phosphorylates a similar site onthe 1-subunit of cardiovascular CaV1.2 channels.

Role of CaMK

Ca2-dependent inactivation and facilitation oCaV1.2 channels are well-known phenomena, and bothforms of channel autoregulation appear to involve calmodulin (CaM). A number of different studies suggestthat CaMK may be responsible for the positive-feedback, Ca2-dependent facilitation of CaV1.2 channels(42, 168, 192, 203). An early study by Yuan and Bers(203) indicated that repetitive membrane depolarization from 90 to 0 mV in rabbit and ferret ventricularmyocytes induced a staircase increase in Ca2 currentThis effect was completely abolished by dialyzing cellswith either the CaMKII inhibitor CaMKII(290309) orCaMKII(273302). Similar results have been observed inrabbit ventricular myocytes (2). Constitutively activeCaMK also markedly increases the open probability osingle Ca2 channel currents in murine ventricularmyocytes recorded in inside-out patch configuration(42). The stimulatory effect of constitutively activeCaMK required ATP and was not mimicked by CaMalone. It was blocked by the CaMK inhibitory peptideAC3-1 but not by either PKA or PKC inhibitory pep

tides. The authors suggested that CaMK phosphorylates a cell membrane-associated target protein, whichgives rise to frequent, long openings of Ca2 channels

In contrast to CaM-dependent facilitation, somestudies have proposed that CaM-dependent inactivation may result from channel dephosphorylation by theCaM-dependent phosphatase calcineurin (4, 21, 148)On the other hand, others have proposed that CaMdirectly interacts with the Ca2 channel 1-subuniand that channel phosphorylation is not involved (seefor example, Refs. 129, 133, 142, and 211). Some stud-ies have gone so far as to suggest that direct binding o




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CaM to the 1-subunit is a key step in both Ca2-dependent inactivation and facilitation of CaV1.2 chan-nels. Recently, it has been shown that mutations in aconsensus CaM-binding IQ motif in the COOH-termi-nal tail of1 eliminates both forms of Ca2-dependentautomodulation of Ca2 channels (210, 211). Whetherthis direct interaction between CaM and the channel1-subunits is sufficient to induce both Ca2-depen-

dent inactivation and facilitation requires furtherstudy, as does the question of the role of CaMK in theprocess of autoregulation.

We thank Dr. James Kenyon for careful reading of this manu-script and valuable suggestions.

This work was supported by National Heart, Lung, and BloodInstitute Grants HL-40399 (to K. D. Keef and J. R. Hume) andHL-49254 (to J. R. Hume) and by an American Heart AssociationWestern Affiliate Grant-in-Aid (to J. Zhong).

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Regulacion Canales CA Cardiaco y Liso

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