PHYSIOLOGY IN MEDICINE In collaboration with The American Physiological Society, Thomas E. Andreoli, MD, Editor

Calcium Currents and Arrhythmias: Insights from Molecular Biology Stephen R. Shorofsky, MD, PhD, and C. William Balke, MD Calcium channels are critical to normal cardiac function. They are involved in the generation and conduction of the action potential and in contraction. Three surface membrane channels have been identified. The L-type Ca channel is most abundant and is responsible for Ca entry into the cell that triggers contraction. T-type Ca channels are most prevalent in the conduction system and are probably involved in automaticity. A newly described TTX-sensitive calcium current may be important in “boosting” or enhancing conduction and contraction. The main intracellular Ca channel resides in the sarcoplasmic retic-

ulum and is responsible for the release of the Ca that activates contraction. Oscillatory behavior of this channel influences the sarcolemmal membrane, causing delayed aftercontractions and arrhythmias such as those seen in digoxin toxicity. The on-going molecular characterization of these channels will enhance our knowledge of their normal function and dysfunction in disease states, leading to the development of new therapeutic agents to treat arrhythmias and contractile dysfunction. Am J Med. 2001;110:127–140. 䉷2001 by Excerpta Medica, Inc.

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cent data concerning the molecular structure, function, and regulation of these channels and identifies areas where modulation of Ca channel function may have an important role in the generation of arrhythmias and in the progression and treatment of cardiac diseases.

alcium channels are wonderfully complex proteins with vitally important roles in cardiac function. The opening of calcium (Ca) channels is involved in pacemaker depolarization, supports conduction through the atrioventricular (AV) node, and maintains the distinctive plateau of the cardiac action potential. In addition, they serve as the primary gatekeepers for Ca entry into cells, thus transducing the electrical signals at the surface of the myocyte into the biochemical and mechanical events that result in a contraction. It is no wonder, therefore, that Ca channels have attracted intense interest over the last three decades. Ion channels in the cardiac cell membrane are responsible for the generation and control of the action potential. Thus, alterations in channel function often lead to abnormal electrical activity and arrhythmias. In fact, genetic defects in cardiac sodium and potassium channels have been shown to cause the congenital long QT syndrome and its resultant arrhythmia, torsades de pointes (1). To date, there has not been a specific arrhythmia that results from an identified Ca channel defect in humans, possibly because Ca channels have such a crucial role in cardiac function that significant abnormalities in channel function are lethal. This review, however, focuses on re-

From the Departments of Physiology and Medicine, University of Maryland School of Medicine, Baltimore, Maryland. Requests for reprints should be addressed to C. William Balke, Department of Physiology, University of Maryland School of Medicine, Howard Hall, Room 525, 660 West Redwood Street, Baltimore, Maryland 21201-1541. 䉷2001 by Excerpta Medica, Inc. All rights reserved.

NOMENCLATURE Heart cells actually contain at least five types of Ca currents, three of which (L-type, T-type, and the channels responsible for the newly described current (ICaTTX) are expressed on the surface membrane, and two (the sarcoplasmic reticulum Ca release channel and the IP3 receptor) in internal membranes. The Table summarizes the nomenclature, localization, and salient physiological roles of each Ca channel type. When clinicians speak of the Ca channel in the heart, they usually mean the L-type Ca channel. This is not only because of the fact that it exists in large quantities in myocytes and was first recorded in 1967 in cardiac Purkinje fibers (2), but also because these channels function as the receptors for a number of clinically useful drugs, including nifedipine, verapamil, and diltiazem. This review concentrates on the L- and T-type Ca channels, because they play the most obvious and prominent role in cardiac arrhythmogenesis. The sarcoplasmic reticulum (SR) Ca release channel, the IP3 receptor, and the recently reported Ca-permeable Na channel (ICaTTX) are reviewed briefly. 0002-9343/01/$–see front matter 127 PII S0002-9343(00)00586-6

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Table 1. Cardiovascular Calcium Channels Calcium Channel Type

Location

Ligands

L-type calcium channel

Sarcolemma

Dihydropyridines Benzothiazepines Phenylalkylamines

T-type calcium channel

Sarcolemma

Mibefradil

ICa(TTX) channel

Sarcolemma

Tetrodotoxin

Calcium release channel (ryanodine receptor) IP3 receptor

SR

Ryanodine

SR

Inositol triphosphate

Physiological Roles Atrioventricular nodal conduction Slow response upstroke Excitation–contraction coupling Plateau depolarization Pacemaker depolarization Cell growth and differentiation? Augmenting depolarization Excitation–contraction coupling? SR calcium release Modulation of SR Ca release ? Other functions

SR ⫽ sarcoplasmic reticulum.

L-TYPE CALCIUM CHANNELS Physiological and Pathophysiological Roles Sarcolemmal Ca channels constitute the major pathway for Ca entry into the cell. The existence of a Ca-selective current in heart muscle has been recognized for more than 25 years (2). Two distinct types of channels with high selectivity for Ca ions (L-type and T-type) are now known to coexist in the surface membranes of heart cells (3,4). The two types of channels can be distinguished on the basis of their gating properties (ie, their pattern of opening and its voltage dependence) and their pharmacological sensitivity. We first consider the “traditional” Ca current of heart muscle, which is sensitive to modulation by a variety of drugs. This current flowing through so-called L-type channels (L for large and long lasting) (4) is well characterized with respect to its role in cardiac excitability, arrhythmogenesis, and excitation– contraction coupling. These channels are fairly plentiful, being present in some 30,000 copies per ventricular myocyte (5). The L-type Ca channel is important in normal and abnormal cardiac excitation. Ca current supports excitation in the sinoatrial (SA) and atrioventricular (AV) nodes as well as conduction through the AV node. Interestingly, the AV node is the only site in the body where Ca channels actually conduct an excitatory impulse; this distinctive feature, as well as use-dependent drug binding explains why Ca channel blockers preferentially suppress AV nodal conduction. In working heart muscle (atria and ventricles) and in specialized conducting tissue, the principal excitatory inward current underlying the action potential plateau flows through L-type Ca channels. Everything else remaining equal, an increase in Ca current prolongs depolarization and thereby increases the height and duration of the action potential plateau. Conversely, blockade of L-type channels shortens the action potential in working myocardium and renders nodal tissue inexcitable. 128

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L-type Ca channels also play potentially important roles in arrhythmia generation and their treatment. Ca channels are capable of mediating the inward current that underlies some types of early afterdepolarizations (EADs) that cause triggered arrhythmias and torsades de pointes in animals (6,7). As mentioned earlier, there has not been a demonstration in humans of a Ca channel defect causing torsades de pointes, or any other arrhythmia thought to be the result of EADs. Ca channels can also support slow conduction in reentrant circuits, especially in depolarized tissue, although this mechanism appears to predominate in only a small minority of patients with inducible ventricular tachycardia. By their effects on automaticity and AV node conduction, L-type Ca channels may be involved in the generation of certain automatic arrhythmias [eg, repetitive monomorphic right ventricular outflow tachycardia (RVOT) and some atrial tachycardias], and modulation of these Ca channels is useful in treating many supraventricular tachycardias (eg, AV nodal reentry, atrial fibrillation, and atrial flutter). An appreciation of the role of Ca channels in excitation– contraction coupling is important in understanding the potential side effects of Ca channel blockers. Ringer (8) first realized in 1883 that Ca ions must be present in the extracellular medium for the heart to generate force. Even though the amount of Ca entering the cell during a single action potential through L-type channels appears to be too small to account fully for contractile activation (9), Ca influx through these channels is a necessary prerequisite for the initiation of contraction (10 –12). Several organic L-type channel inhibitors exist. The classic members of this drug family come from three chemical groups: the phenylalkylamines (eg, verapamil), the benzothiazepines (eg, diltiazem), and dihydropyridines (eg, nifedipine, nitrendipine, and amlodipine) (13). Exposure to any of the Ca channel antagonists can abolish

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Figure 1. Schematic model of the transmembrane topology of the ␣1 subunit of the L-type Ca channel. The P loops in each repeat are indicated by the thick lines. [Reproduced with permission from W.B. Saunders Company, Cardiac Electrophysiology, 3rd ed. (147).]

contractile force in isolated myocardial preparations by interrupting the flux of Ca into the cell by means of L-type Ca channels. There is good evidence that the high-affinity binding sites for these compounds are located on the ␣1 subunit of the L-type Ca channel (14), the structure of which is discussed below.

Structure–function Relationship Channels are pore-forming proteins that open and close in response to defined stimuli. The processes whereby channels open and close are collectively known as gating, whereas permeation describes how a given channel conducts ions when it is open. The conceptual distinction between gating on the one hand and permeation on the other predates the molecular biology era. Now that many channels have been cloned, expressed, and mutated, specific and separate structures have been identified that mediate gating and permeation. Among biological voltagedependent ion channels, those selective for potassium (K channels) are the smallest and, not coincidentally, the most thoroughly characterized (15). Despite the fact that channels selective for Na or Ca are larger and more complex than K channels, several prominent features have emerged. L-type Ca channels were first purified from skeletal muscle and consist of five subunits: ␣1 (165 kD), ␣2 (130 kD) linked to the ␦ subunit (28 kD) by a disulfide bond, ␤ (55 kD), and ␥ (32 kD) (16,17). The primary sequence of the ␣1 subunit was then determined from the cDNA sequence encoding the protein in rabbit skeletal muscle (14). Remarkable homology exists between the ␣1 subunit of the Ca channel and the pore-forming (␣) subunit of voltage-dependent Na channels, both having four homologous domains composed of six transmembrane segments each (16). Figure 1 shows a schematic diagram of

the ␣1 subunit of the L-type Ca channel (the presumed pattern of assembly with the other subunits is diagrammed in Figure 2). The overall protein, deduced from the cDNAs of various isoforms cloned to date (including that from heart), is more than 2,000 amino acids long and consists of four internally homologous domains (I to IV). Each domain is believed to span the membrane six times (S1 to S6). The fourth such segment (S4) in each domain is distinguished by the repetition, at every third position, of positively charged amino acids (lysine or arginine) (16,18). Because Na and Ca channels open in response to depolarization, elucidation of the S4 structure immediately suggested a physical correlate for the voltage sensor involved in activation gating. Site-directed mutagenesis to modify the net charge in S4 in Na channels alters the voltage dependence of activation, verifying the prediction that S4 functions as a voltage sensor (19). The ␣1 subunit contains the binding sites for Ca channel blocking drugs, including the dihydropyridines, benzothiazepines, and phenylalkylamines (14). The cloned ␣1 subunit can be expressed in Xenopus oocytes, dysgenic myotubes, or mammalian tissue culture cells and is capable of conducting Ca currents (20 –22). These studies established that the fundamental properties of the channel allowing for selective permeation of Ca across the membrane reside entirely in the ␣1 subunit. Beyond these basic observations, much remains to be learned about the specific sites in the L-type Ca channel that underlie its distinctive gating and permeation properties. Expression of chimeric ␣1 constructs in dysgenic mouse muscle has revealed that the loop between domains II and III is important in isoform-specific excitation– contraction coupling (22), whereas domain I confers features of activation gating (23).

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Figure 2. Schematic representation of the regulatory subunits and their putative relationship to the ␣1 subunit of the L-type Ca channel. [Reproduced with permission from W.B. Saunders Company, Cardiac Electrophysiology, 3rd ed. (147).]

Na and Ca channels enter a long-lived nonconducting state during maintained depolarization, a gating process known as “inactivation” (24,25), which is not altered by changes in S4 (19). Unlike S4, no feature of Na or Ca channels suggests a role in inactivation merely from inspection of the primary sequence. Nevertheless, mutagenesis has revealed that the cytoplasmic linker between domains III and IV, which has been highly conserved during evolution, forms at least part of the inactivation gate of Na channels (19). Thus, two regions of the Na channel are clearly implicated in gating: S4 senses voltage and initiates activation gating, and the III–IV linker figures prominently in inactivation. Mutations in either region do not grossly influence pore properties. The functional roles of S4 and of the III-IV linker in the Ca channel have yet to be investigated, partly because the Ca channel clones have not been available as long as those for the Na channel, but also because Ca channels have proved to be considerably more difficult to express in surrogate cells. Nevertheless, the fact that Na channels are so similar in their overall molecular architecture gives good reason to predict the likely functions of the homologous regions of the Ca channel. 130

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A confluence of biophysical and molecular biological data has defined regions and residues in the Na channel ␣ subunit that are important in forming the permeation pathway. A region of striking sequence conservation occurs between the fifth and sixth membrane-spanning regions (S5 to S6), also called the P loop (P for pore or permeation) (26). There is now solid evidence, from electrophysiological analysis of mutant Na and K channels, that the P loops from each of the four domains come together in the center of the protein to line the pore (15,27). The P loops have been proposed to form ␤-hairpins that form a ␤-barrel structure, but the precise conformation is not yet known. Ca channels are very selective. Typically, the permeability of the channel to Ca is a thousand-fold higher than the permeability to K or Na (28). Yet, a single Ca channel can conduct more than 106 Ca ions per second across the cell membrane (29). To explain how a Ca channel reconciles the conflicting demands of maintaining a large conductance in the face of exquisite selectivity, models have been proposed in which Ca binds with high affinity at two or more sites down the permeation pathway (30,31). High-affinity binding makes the channel selective, while

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the presence of multiple binding sites enables a high throughput rate: simultaneous occupancy by two or more Ca ions accelerates flux resulting from electrostatic ion–ion repulsion. The model handily explains the ability of Ca channels to conduct large fluxes of monovalent cations when divalent cations are absent (30,31). Remarkably, this model and others (32) were proposed without any knowledge of the actual protein sequence of any voltage-dependent ion channels. The P loops from Na and Ca channels are largely conserved but differ in the presence of acidic residues in repeats III and IV. Substituting acidic residues in these positions of the Na channel produces the essential features of permeation in Ca channels (monovalent permeability at very low [Ca], Ca selectivity at higher [Ca]) without grossly changing other properties of the channel, such as gating kinetics (33). While confirming that the S5–S6 region in each repeat of the Na channel is an integral part of the permeation pathway, these results speak only indirectly to the question of how the Ca channel itself forms the pore. More direct confirmation for the role of S5–S6 in Ca channel permeation comes from recent mutagenesis studies that suggest that a ring of highly conserved glutamate residues in the P loops confers Ca selectivity (34 –36). Dihydropyridine (DHP) Ca channel ligands bind specifically to L-type Ca channels and, like local anesthetics (LA) acting on Na channels, do so in a voltage-dependent manner. Bean (37) demonstrated that voltage protocols that favor Ca channel inactivation markedly enhance the potency of channel block by nitrendipine; he invoked the modulated receptor hypothesis to explain the results, postulating a 3,000-fold higher affinity for the inactivated state than for the resting state. In agreement with this interpretation, Kamp and Miller (38,39) showed that radiolabeled DHPs bind much more avidly during surface membrane depolarization, either in intact cells or in membrane vesicles. Numerous other studies have pointed out the importance of the gating-related channel state in the binding not only of DHPs but also of benzothiazepines and phenylalkylamines to L-type Ca channels (40,41). Virtually nothing is known about the structural basis for the interaction; particularly intriguing is the fact that photoaffinity-labeling experiments place the DHP binding site at the external face of the channel (in contact with III-S5–S6 and with the external end of IV-S6), far from any putative intracellular inactivation gate (14).

Regulation Beta-adrenergic stimulation. Sympathetic stimulation causes an increase in heart rate and augmented contractility. L-type Ca channels figure prominently in mediating these effects of ␤-adrenergic stimulation (42– 44). When an agonist binds to the cardiac ␤ receptors, a series of events takes place. The occupied ␤ receptor activates a

second membrane protein called G protein (for its ability to bind and to split guanosine triphosphate [GTP]). The activated ␣ subunit of the G protein in turn stimulates the membrane-bound enzyme adenylate cyclase. Biological membranes contain several types of G proteins (45,46). The type that mediates stimulation of adenylate cyclase (Gs) carries the subscript s to distinguish it from others of inhibitory (Gi) or unrelated (Go) function. The enzyme adenylate cyclase catalyzes the formation of cyclic adenosine 3⬘,5⬘-monophosphate (cAMP) from ATP, tending to increase the cellular content of cAMP (43). Cyclic AMP regulates a number of cellular functions by promoting selective protein phosphorylation, initiated by the binding of cAMP to the regulatory subunit of cAMP-dependent protein kinase (protein kinase A). The binding of cAMP liberates the catalytic subunit of the enzyme, which then phosphorylates a variety of cellular proteins (47). The cardiac L-type Ca channel appears to be a particularly important substrate for phosphorylation by protein kinase A, based on evidence primarily from functional studies (44,48,49). Ca channels, when phosphorylated by ␤-adrenergic stimulation, are considerably more active than in the nonphosphorylated state, thus producing an increase in the Ca current during each action potential (44,48,50). An increase in Ca current can occur by several different means when one examines the underlying single-channel events. Macroscopic current (I) is the product of the number of channels in the membrane available for opening (N), the probability that a channel will open (Po), and the current carried by a single, open channel at that voltage (i), or I ⫽ N 䡠 Po 䡠 i. Activation of the cAMP-dependent phosphorylation pathway results in an increase in channel availability (seen as an increase in the number of openings during a depolarizing pulse) and an increase in the probability of opening. The latter is manifested both as an increased frequency of openings during a depolarizing pulse (51) and as an augmentation of the number of ultralong openings (52). Under control conditions, single-channel openings are quite brief and often do not occur at all during depolarizations to various test potentials. In contrast, exposure to 1 ␮M isoproterenol (a ␤-adrenergic agonist) increases channel availability (as evidenced by a reduction in the number of blank sweeps) and increases the occurrence of a long-opening mode of the channel. Thus, a complex chain of molecular events produces an increase in L-type Ca current and ultimately enhanced Ca delivery to the cytoplasm. The increase in Ca current increases conduction through the AV node and increases the firing rate of normal and subsidiary pacemakers. Abnormal slow conduction will be accelerated to the extent that it is mediated by Ca channels, thus altering the likelihood of reentry. Recently it has become clear that G proteins can di-

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rectly influence the activity of ion channels, even in the absence of second messengers, such as cAMP. Exposure to a purified preparation of activated Gs ␣ subunits increases the likelihood that L-type Ca channels will open in bilayers (53). Although such cell-free experiments are consistent with the idea that part of the ␤-adrenergic stimulation of Ca channels is mediated directly by G proteins, the fact that cAMP and its derivatives mimic the effects of ␤ agonists on Ca currents in intact cells implies that G proteins are not required. Nevertheless, the recognition of direct ion channel gating by G proteins opens the novel possibility of drugs that might act on this G protein link, rather than on the more conventional second-messenger–mediated cascade. Regulation by intracellular magnesium and Mg-ATP. Although often taken for granted, the importance of magnesium (Mg) in the control of cell physiology cannot be overemphasized. Whether the cell is using energy substrates, pumping up or dissipating ion gradients across a membrane, contracting, or synthesizing nucleic acids and proteins, Mg participates in the process. The vital requirement for Mg and Mg-nucleotide complexes in supporting enzyme activity has long been recognized. More recently, a growing body of work has elucidated a number of mechanisms whereby ion channels can sense and respond to Mg and Mg nucleotides. L-type Ca channels are subject to several modulatory actions of Mg. Direct channel blocking effects of both extracellular and intracellular Mg have been observed (54). Furthermore, the rate of inactivation of inward Ba or Na currents through L-type Ca channels has been reported to increase, and the amplitude of the currents to decrease, when intracellular Mg was raised from 0.3 to 3 mM in frog cardiomyocytes (55). When Ca is the charge carrier, raising intracellular Mg also reduces the amplitude of Ca current in guinea pig cardiomyocytes (56). In the frog, this effect is accentuated by prior channel phosphorylation, perhaps by altering the affinity of the Mg binding site (55). Although the role of Mg-ATP in the cAMP-mediated phosphorylation of Ca channels has been recognized, relatively little attention has been paid to investigating the effects of Mg-ATP itself on L-type Ca channels. Previous studies have shown that the inclusion of Mg-ATP in sufficient quantities in intracellular solutions can retard the rate of “rundown” of Ca current (57). In addition, a positive correlation between intracellular Mg-ATP and the amplitude of cardiac L-type Ca currents has been reported (58). Both these effects have been assumed to be related to the importance of Mg-ATP as a substrate for phosphorylation. Exploiting the Mg binding properties of the photosensitive compounds [DM-nitrophen (59) and caged Mg], O’Rourke et al (60) compared the effects of various intra132

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cellular concentrations of Mg-ATP on L-type Ca currents with the predominant mechanism for regulating Ca channels, ␤-adrenergic receptor activation, and cAMPdependent phosphorylation. In a series of elegant studies, they demonstrated that a direct relationship exists between intracellular Mg-ATP levels and L-type Ca current amplitude, which was independent of phosphorylation. Elucidation of the phosphorylation-independent pathway does not diminish the importance of cAMP-dependent phosphorylation as the primary mechanism for increasing Ca channel activity in the heart. However, it does stimulate speculation as to the supplementary role of Mg-ATP in linking metabolism to Ca influx. As pointed out by Goldhaber et al (61), cytosolic Ca transients are reduced in ventricular myocytes during metabolic inhibition as a result of both a reduction in L-type Ca current and impaired coupling between the Ca current and SR Ca release, even though the caffeine-releasable pool of Ca is actually greater than in the control condition. Similarly, Taniguchi et al (62) reported that Ca current depression induced by metabolic inhibition could be reversed by injection of ATP. During times of metabolic stress, the ability of channels to sense Mg-ATP levels may serve as a protective mechanism to reduce the metabolic demand associated with Ca removal by cutting back on the trigger for Ca release. This type of stopgap regulation would be carried out at no energy cost to the cell and would function even when the phosphorylation pathway is in high gear. One situation in which this may occur is during ischemia, when cytosolic Mg increases, Mg-ATP decreases (63), and the sympathetic nerves are in overdrive. The overall picture of ion channel activity at this time may include inhibition of the SR Ca release channels (64,65), activation of ATP-sensitive K channels, and perhaps a reduction in Ca current as a result of both the decrease in Mg-ATP and the increase in Mg.

T-TYPE CALCIUM CHANNELS Physiological and Pathophysiological Roles T-type Ca channels are found in cardiac and vascular smooth muscle. They can be distinguished from L-type channels on the basis of their distinctive biophysical characteristics (gating and permeation properties), relative distribution, differential pharmacology, and structure (66). Compared with L-type channels, T-type channels open at significantly more negative membrane potentials that are near the resting potential, rapidly inactivate (hence, T for transient), slowly deactivate, and have a low conductance (hence, T for tiny) (3,4,66). In the heart, T-type Ca channels are expressed in highdensity in SA and AV nodal tissue (67) and Purkinje cells (68), and are relatively sparse or absent in most atrial and ventricular cells. This distribution is consistent with their

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putative role in pacemaker impulse formation. T-type channels probably do not contribute to the action potential upstroke of cardiac ventricular cells, because their relatively small and transient current component is dominated by the coincident and much larger classical Na current (69). However, T-type channels may play a role in action potential generation, particularly in cell types that lack a high density of Na channels, such as vascular smooth muscle (70). T-type channels have been implicated in cell growth under both physiological and pathophysiological conditions. T-type channels are abundant in the heart during fetal development (71) and during rapid growth in the early postnatal period (72). In addition, cardiac T-type Ca channel density is increased under several conditions that are associated with myocyte growth, including rats with growth-hormone–secreting tumors (73), ventricular cells from failing cardiomyopathic hamsters (74), and endothelin-1 induced hypertrophy in neonatal rat ventricular cells (75). Experimentally induced pressure overload hypertrophy in cats induced a re-expression of Ttype Ca channels in ventricular cells that normally do not have any demonstrable T-type Ca current (76). Furthermore, pretreatment with a selective inhibitor of T-type channels, mibefradil, reduces intimal proliferation after experimentally induced vascular injury in rats (77). Of interest, long-term treatment with mibefradil in a rat model of chronic heart failure was associated with an increase in survival (78). Although the precise mechanism for this response is as yet unknown, the reappearance of the fetal phenotype may be a marker for the transition of cardiac hypertrophy to heart failure and may contribute to the increased incidence of sudden death and pump failure characteristic of the failing heart. Compared with the well-studied L-type Ca channel, the pharmacology of the T-type channels is less well characterized. Several drugs and inorganic compounds affect T-type channels, including amiloride (79) and its derivative 3,4-dichlorobenzamil (80), verapamil, diltiazem (81), flunarizine (81), and tetradrine. Nickel and cadmium also block T-type channels. However, all of these agents also act on L-type Ca channels and, therefore, are not useful for dissecting out the effects of selective T-type channel modulation on cardiac electrical or mechanical activity. Recently, a novel Ca channel antagonist, mibefradil, has been shown to inhibit both L- and T-type channels with a marked selectivity for the T-type channel (82– 85). It preferentially affects the coronary vasculature relative to the myocardium. In contrast to other Ca channel antagonists, its vasodilatory effects are not associated with negative inotropic or conduction abnormalities, but it did cause sinus slowing. Although its clinical utility has been compromised by serious drug interactions (86), it remains potentially useful as a research tool for a detailed characterization of the contribution that T-type Ca chan-

nels may make to cardiac electrical and mechanical function.

Structure-function Relationship Until recently, information regarding the structure of Ttype Ca channels was inferential and indirect. Augmentation of T-type current by exposure to neuraminidase (87) suggested that the molecules are glycosylated and that such glycosylation might modulate function. Other structural features were implied from the comparison with L-type channels. Despite important differences in single-channel conductance (T-type channels are about one third to one half as large as L-type channels) (88), their selectivity for Ca over monovalents is comparable, and both types of channel can conduct monovalent cations well when divalent cations are absent (88). On the other hand, T-type channels do not exhibit differences in inactivation depending on the ion that carries the charge, unlike L-type channels, which display prominent Ca-mediated inactivation (4,88). Finally, an important structural difference in the outer channel mouth regions is suggested by the fact that T-type channels do not bind dihydropyridines (3,4). Perez-Reyes et al (89) recently cloned the first member (␣1G) of a new family of Ca channels in rat, mouse, and human neuronal tissue that is distinct from all previously cloned Ca channels. They localized the gene encoding human ␣1G to human chromosome 17q22 and the mouse locus to distal mouse chromosome 11. Functional expression studies in Xenopus oocytes provided compelling evidence that ␣1G was, indeed, a T-type Ca channel on the basis of its negative voltage range of activation, fast inactivation, slow deactivation, and tiny unitary conductance of approximately 7.5 pS. On the heels of this landmark report, Perez-Reyes et al cloned a second member of this family of T-type Ca channels (␣1H) from human heart (90). The cDNA of ␣1H was consistent with the general structure of a voltage-gated Ca channel, and there was a high degree of homology with human and mouse ␣1G. Specifically, both ␣1H and ␣1G are predicted to have a four-domain structure with conserved pore loops and voltage sensors. In addition, ␣1H lacks both an identifiable motif for ␤ subunit binding and a Ca binding domain that would confer Ca-dependent inactivation. In contrast to human ␣1G, human ␣1H was mapped to a different chromosome, human chromosome 16p13.3. Both ␣1G and ␣1H were found in brain and heart. However, ␣1G was most abundant in brain, and ␣1H was most abundant in heart. Importantly, heterologous expression of ␣1H in HEK cells produced Ca currents with the biophysical hallmarks of native T-type channels including a single-channel conductance for barium of approximately 5.3 pS and a sensitivity to mibefradil at the concentration reported for native T-type Ca channels. The identification of multiple genes encoding T-type Ca

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channels represents a major advance in the study of voltage-gated ion channels. The availability of recombinant T-type channels provides the tools necessary to adequately separate the contributions of T- and L-type Ca channels to cardiac function and to identify T-type Ca channel–related pathophysiology.

Regulation T-type Ca channels are not prominently regulated by ␤-adrenergic mechanisms (91) but do exhibit a prominent up-regulation with norepinephrine (92), phenylephrine (an ␣-adrenergic agonist) (93), and extracellular ATP (94). Endothelin has also been found to augment Ca entry through T-type channels in heart cells (75). These agents only modestly alter L-type currents. Developmental changes in the relative densities of L-type and T-type channel expression have been deduced (94,95) and may turn out to be clinically important in distinguishing mechanisms of pediatric versus adult automaticity. In the cultured chick myocyte model, developmentally young cells exhibit large T-type currents (94). The mammalian correlates of these changes remain to be fully defined. Interestingly, Nuss and Houser (76) have observed that T-type currents are enhanced in hypertrophic ventricular cells from the cat, and this may represent an electrophysiological example of the general truism that cardiac hypertrophy turns back the developmental clock by activating a “program” of fetal gene expression.

TETRODOTOXIN-SENSITIVE CALCIUM CHANNELS (ICa(TTX)) Recently, a new and functionally distinct inward current component has been identified in rat ventricular cells (96). The channels that carry this current are permeable to both Na and Ca and are blocked by tetrodotoxin (TTX), hence their designation as ICa(TTX). This new component displays different kinetics, different voltage ranges for both activation and inactivation, and different permeability properties from classical sodium channels and L- and T-type Ca channels. Specifically, ICa(TTX) activates over a more negative voltage range than classical Na and T-type channels. Therefore, it may act to amplify the depolarization delivered by Purkinje fibers or adjacent ventricular cells and so provide the immediate trigger for the generation of the cardiac action potential. To the extent that ICa(TTX) triggers the cardiac action potential, it may be important for cardiac arrhythmias and their control. In addition, pharmacological modulation of ICa(TTX) would be predicted to affect the timing and conduction of the cardiac action potential without compromising the action potential itself (ie, Vmax or overshoot potential). Consequently, this pharmacological strategy would be expected to have fewer and less severe side effects compared with traditional antiarrhythmic 134

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agents that are targeted to the classical cardiac Na channel. Because ICa(TTX) is permeable to Ca as well as Na, it may also play a role in providing the Ca trigger for SR Ca release by one of two possible mechanisms. 1) Na permeation of ICa(TTX) could elevate the concentration of intracellular Na in a “restricted space” near the sarcolemmal membrane and thereby activate Ca influx by means of the Na/Ca exchanger (ie, “reverse mode”) to a level that contributes to the trigger for SR Ca release. 2) Ca permeation of ICa(TTX) could contribute directly to the trigger for SR Ca release. Apart from this initial characterization and the demonstration that the current is augmented by ␤-adrenergic stimulation (96,97), no information is yet available regarding the relative permeabilities, structure, and regulation of ICa(TTX). Importantly, ICa(TTX) may represent a distinct new Na (or Ca) channel isoform or a differentially regulated or otherwise posttranslationally modified Na (or Ca) channel.

SARCOPLASMIC RETICULUM CALCIUM RELEASE CHANNELS (RYANODINE RECEPTORS) Physiological and Pathophysiological Roles The Ca release channels of the sarcoplasmic reticulum (SR) are entirely intracellular; they mediate the rapid efflux of Ca from the SR lumen into the cytosol during each cardiac cycle. In the scientific literature, SR Ca release channels are often called ryanodine receptors because they bind the plant-derived toxin ryanodine with nanomolar affinity. The SR has been generally accepted as the predominant source of activator Ca for activation of the myofilaments and contraction in both skeletal and cardiac muscle (98,99). However, ultrastructural comparison with skeletal muscle reveals that cardiac SR occupies a much smaller fraction of the intracellular volume (100). Furthermore, the mechanism of signal transduction coupling the cell membrane and the terminal cisternae of the SR is still incompletely understood. As complicating factors, substantial differences have been reported regarding the contribution of SR Ca in atrial as opposed to ventricular tissue, between mammalian and lower vertebrate hearts, and even among ventricles from different mammalian species (101).

Structure–function Relationship Ca release channel proteins are the largest biological channels yet described; they are so large that the shadows of their cytoplasmic protrusions can be visualized in routine electron micrographs as the “foot” processes that span the gap between the SR and surface membranes. In contrast to membrane structures embedded in the sarco-

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lemma, functional proteins located in the SR are often studied under experimental conditions where the integrity of the cell has to be disrupted by “skinning” myocardial cells or by preparing vesicular membrane fractions from the SR. At the intact cellular level, several agents that inhibit SR Ca release markedly reduce contractility, and drugs that facilitate SR Ca release activate contraction. Functional studies (10,11) suggest that in the intact cell, Ca release from the SR is triggered by the increase of intracellular free Ca concentration ([Ca]) initially produced by the L-type Ca current. Such observations support the mechanism of Ca-induced Ca release from the SR, first described in skinned preparations (102). Several recent studies have advanced our understanding of the quantitative relationship (or the gain) between the trigger Ca provided by the L-type Ca current and activator Ca provided by the SR Ca release channel (103,104). They have shown that SR Ca release is controlled by events localized to the region of the L-type Ca channels and SR Ca release channels (ryanodine receptors), and these events may be very different from those seen macroscopically in the whole cell. Several recent experimental studies provide support for this local coupling of the L-type Ca channel and the SR Ca release channel. 1) Immunocytochemical and ultrastructural studies demonstrate the close physical proximity and clustering of L-type Ca channels and ryanodine receptors. In ventricular cells, L-type Ca channels were closely associated with ryanodine receptors at the T tubules (105). In atrial cells, which lack T tubules, L-type Ca channels and ryanodine receptors also co-localized to the sarcolemma. 2) Spontaneous, spatially localized nonpropagating transient elevations in [Ca] (ie, Ca sparks) have been observed in resting single cardiac cells (106) and in intact cardiac muscle (107). 3) Lo´pez-Lo´pez et al (108,109) and others (110,111) have shown that SR Ca release was controlled locally (ie, Ca sparks) by L-type Ca current in voltage-clamped rat ventricular cells. Ca sparks evoked by membrane depolarization behave in a stochastic manner (108 –110) and have a voltage and time dependence similar to that of the L-type Ca channel (109). 4) Ca sparks do not appear to be elicited by Ca entry by means of the reverse mode of the Na/Ca exchanger under certain experimental conditions (109). 5) Consistent with the immunocytochemical and ultrastructural studies, Ca sparks do not occur randomly throughout the cell but localize to the region of the T tubule and the junctional SR (112) where L-type Ca channels and SR Ca release channels co-localize (105). Thus, there is a preponderance of evidence that Ca entry by means of L-type Ca channels is the major trigger of SR Ca release and that this trigger Ca “grades” the release of SR Ca from a single or cluster of ryanodine receptors in close proximity. Finally, other sources of trigger Ca including Na/Ca exchange and voltage per se have not been shown to be sufficient quan-

titatively to account for the Ca transients recorded in mammalian muscle. Thus, several lines of evidence point to the SR as the primary site of origin for activator Ca in mammalian heart muscle (113). Interestingly, the SR seems to lose its capability to regulate diastolic Ca tightly when the cell is “Ca overloaded.” Under these conditions, the SR releases Ca spontaneously and asynchronously (114,115). The resultant Ca oscillations produce delayed afterdepolarizations (DADs) observed when the Ca loading of the cell is excessive, as in digitalis toxicity, and may result in lethal arrhythmias (116). At the molecular level, biochemical techniques and recent single channel studies have provided more direct evidence to improve our understanding of SR function and pharmacology. The Ca release channel serves as the pathway for the sudden systolic delivery of Ca from the SR to the cytosol, triggered by the increment in cytosolic Ca itself, and facilitated by the presence of ATP. This molecule has been purified from skeletal and cardiac muscle SR (117–119) by several laboratories using procedures that exploit the high affinity of this channel to the plant alkaloid, ryanodine. The ryanodine-binding protein has been characterized electrophysiologically as a channel passing Ca ions and, with a several-fold lower permeability, potassium ions. It can be blocked by the dye, ruthenium red. Ryanodine changes the properties of the channel in a complex manner, producing long-lasting openings of low conductance at nanomolar ryanodine concentrations. Thus, it causes a continuous leakage of Ca out of the SR, which consequently loses its ability to store and release Ca phasically in the intact cell. The SR Ca release channel has been cloned from both skeletal and cardiac muscle (120). The cardiac cDNA is 16,532 base pairs in length and encodes a protein of 4,969 amino acids that is 66% identical with the skeletal muscle gene product. Four of these identical subunits are believed to assemble to form the functional Ca release channel spanning the SR membrane. The molecule is dominated by a huge N-terminal domain that appears to form the “foot processes” that span the cytosolic gap between the SR and the transverse tubules in skeletal muscle. The putative channel resides entirely within the carboxy terminal end of the molecule. One interesting difference between the skeletal and cardiac muscle isoforms arises from a difference in phosphorylation sites (121); the cardiac receptor has a multifunctional Ca-calmodulin– dependent protein kinase site that is absent in the skeletal muscle isoform, hinting at possible differences in regulatory mechanisms (see below). Because ryanodine acts effectively to dissociate the SR from excitation– contraction coupling (122,123), it has been widely used as an experimental tool (101,124,125). A homologous mode of action has been proposed for

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high (millimolar) concentrations of caffeine, which induce a contracture in heart muscle preparations owing to a sudden release of Ca, causing Ca depletion of the SR. The anthracycline cytostatic agent, doxorubicin, has been proposed to affect cardiac contractility by such a mechanism (126,127). In contrast to ryanodine, caffeine and doxorubicin exert a number of additional effects. A specific antiarrhythmic drug has not yet been found to act by interfering with SR Ca release, although this pathway has emerged as a promising target for rational drug design now that the specific molecular components are becoming increasingly well defined.

Regulation Despite a wealth of biophysical data under nonphysiological conditions (with channels reconstituted in lipid bilayers and conducting monovalent cations), little is certain about the physiological factors that regulate SR Ca channel function in living cells. Presumably, Ca activates the channel and initiates Ca-induced Ca release. Another regulatory factor that most surely plays a prominent pathophysiological role in ischemia is ATP; both ATP and, to a lesser extent, Mg-ATP potentiate channel flux by increasing open probability. In ischemia, intracellular ATP depletion would tend to decrease Ca release by means of the ryanodine receptors, perhaps contributing to ischemic contractile failure (which temporally precedes the onset of electrical inexcitability).

fects of ␣-adrenergic stimulation are usually quite small and differ among species (132,133). Angiotensin II receptors are present on cardiac myocytes, and exposure to angiotensin is known to increase cellular IP3 levels (134). Thus, it seems plausible that the postulated arrhythmogenic effects of angiotensin II in heart failure may at least partly be attributable to IP3 elevation, possibly as a result of a feedback effect of elevated intracellular [Ca] on ion channels in the surface membrane. IP3 receptors are found in higher density in specialized cardiac conducting tissue (135) and Purkinje cells (136), consistent with a possible role in cardiac excitability, and in intercalated disks (137), suggesting a potential role in intercellular communication. In addition, IP3 receptors have been implicated in cell growth, differentiation, and apoptosis (138,139).

Structure–function Relationship IP3 receptors are formed of four similar or identical subunits of 315,000 molecular weight (2,749 amino acid residues) arranged in a quatrefoil pattern similar to that described for the ryanodine receptor. Several subunits have been cloned from cerebellar cDNA libraries (140). Hydropathy analysis predicts that 80% of the peptide forms a large cytoplasmic domain, followed by six to eight transmembrane domains per subunit. The membranespanning regions probably form the ion conductance pathway. It is not yet clear whether the cardiac receptor represents the same gene product, a splice variant, or an entirely different gene from the cerebellar isoform(s).

IP3 RECEPTORS

Regulation

Physiological and Pathophysiological Roles

Little is known about the regulation of IP3 receptors aside from the reported effects of angiotensin II and ␣-adrenergic stimulation. IP3 receptors are both autophosphorylated (141) and phosphorylated by several protein kinases, including PKA (142), PKC, and Ca-calmodulindependent kinase II (143). In addition, IP3 has two potential tyrosine phosphorylation sites (144). In T lymphocytes, tyrosine phosphorylation of IP3 appears to modulate intracellular Ca release and may modulate intracellular Ca levels (138,145). However, this putative role has yet to be demonstrated in cardiac cells.

Like ryanodine receptors, inositol triphosphate (IP3) receptors are integral SR membrane proteins that mediate the efflux of Ca from this intracellular store. IP3 receptors are found predominantly in smooth muscle, where they appear to mediate drug-induced receptor-activated SR Ca release. Although IP3 receptors are also found in the myocardium, less is known about their functional role in heart. In contrast to observations made in other tissues (128,129), it appears unlikely that Ca release in cardiac SR is primarily triggered by the second messenger IP3, although a modulatory role cannot be excluded. Although it appears that IP3 is not involved in normal cardiac excitation– contraction coupling, it may have a role in the maintenance of diastolic tension and in the physiological modulation of contractility in response to a variety of drugs and hormones. IP3 is produced by hydrolysis of the membrane phospholipid phosphatidylinositol-biphosphate, catalyzed by the enzyme phospholipase C. A variety of agonists can activate phospholipase C in the heart (130). In particular, the molecular basis of the positive inotropic effect of ␣-adrenergic stimulation might involve this pathway (131). Nevertheless, the inotropic ef136

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CONCLUSIONS Calcium channels are critical to normal cardiac function. They are important in impulse generation, conduction, maintaining the plateau phase of the action potential, and contraction. At least three different surface membrane channels exist and are preferentially distributed in various areas of the heart, giving a clue as to their function. The L-type Ca channel is the most abundant type in all cardiac cells and is responsible for the entry of Ca into the

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cell that triggers contraction. Inhibitors of L-type Ca channels will depress conduction through the AV node, thus making them useful drugs for the treatment of many supraventricular arrhythmias. T-type Ca channels are prevalent in the conduction system and are probably involved in automaticity. Enhanced function of these channels may lead to an increase in automaticity, as is observed in repetitive monomorphic ventricular tachycardia and some forms of atrial tachycardias. The newly described ICa(TTX) activates over a voltage range more negative than the sodium channel and thus may be important in “boosting” or enhancing conduction and contraction. The main intracellular Ca channel resides in the sarcoplasmic reticulum and is responsible for the ultimate release of the Ca that activates the contractile apparatus. Oscillatory behavior of this channel influences the sarcolemmal membrane, causing delayed aftercontractions and arrhythmias, such as those seen in digoxin toxicity. The molecular characterization of each of these channels has begun, and important structural data already has been obtained. The continued molecular determination of these channels and their regulation will enhance our knowledge of their normal function and dysfunction in disease states, leading to the development of new therapeutic agents to treat arrhythmias and contractile dysfunction.

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ACKNOWLEDGMENT The authors apologize in advance to our many colleagues whose outstanding scientific contributions to our understanding of the general field of cardiac calcium channels could not be included in this concise review because of the inevitable space limitations. The authors also thank all past and present members of their laboratories for the numerous and valuable contributions to this review.

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