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Calcium and Arrhythmogenesis

Department of Medicine, Physiology and Biophysics, University of Calgary, Calgary, Alberta, Canada; and Center of Molecular Therapeutics, Department of Pharmacology, Columbia University, New York, New York

ABSTRACT

I. INTRODUCTION

A. Overview of Ca2+ Transport in the Cardiac Cell

1. Structural aspects

2. Model of ECC

3. ECC coupling in atrial and Purkinje cells from normal hearts

4. Reversal of ECC

II. MOLECULAR BUILDING BLOCKS OF EXCITATION-CONTRACTION COUPLING

A. Ca2+ Flux Through the Sarcolemma

1. Ca2+ entry through voltage-gated channels

2. Na+/Ca2+ exchange, Ca2+ entry, and Ca2+ efflux

3. Stretch-sensitive Ca2+ flux

B. Intracellular Ca2+ Cycling

1. SR Ca2+-ATPase pump

2. Ryanodine-sensitive SR-Ca2+ release channels (RyR)

A) POTASSIUM AND CHLORIDE CHANNELS IN THE SR MEMBRANE.

B) RYANODINE RECEPTORS AND CALCIUM OVERLOAD OF THE SR.

3. IP3-dependent Ca2+ release

4. Mitochondria Ca2+ transport

C. Intracellular Ligands and Buffers

1. Sarcolemmal Ca2+ binding

2. Intracellular ligands

III. FUNCTIONAL CONSEQUENCES OF CALCIUM CYCLING

A. Macroscopic Events

1. The cardiac cycle: cytosolic Ca2+ transients and force development

B. Microscopic Events

1. Ca2+ sparks in normal cardiac ventricular muscle

A) CALCIUM SPARKS IN ATRIAL AND PURKINJE CELLS.

2. Microscopic Ca2+ waves in normal muscle

3. Ca2+ waves in intact whole hearts

C. Regulation of Ca2+ Transport

IV. CALCIUM AND ARRHYTHMOGENESIS

A. Inherited Mutations That Cause Ca2+-Dependent Arrhythmias

1. RyR2 mutations and arrhythmias

A) FUNCTIONAL IMPLICATIONS.

2. CASQ mutations and arrhythmias

A) FUNCTIONAL IMPLICATIONS.

3. Ca2+ channel mutations and arrhythmias

4. Ankyrin B mutations and arrhythmias

5. Long QT syndromes and Ca2+-dependent arrhythmias

B. Automaticity

1. Normal automaticity

A) SAN CELLS.

B) ATRIAL PACEMAKER CELLS.

C) AV NODAL CELLS.

D) NORMAL PURKINJE FIBERS.

2. Abnormal automaticity

3. Triggered activity

A) ROLE OF CALCIUM IN EADS.

B) ARRHYTHMIAS, DADS, AND CALCIUM WAVES IN MYOCARDIUM.

C. Re-entrant Excitation

1. Ca2+ and impulse propagation

2. Ca2+ and gap junction permeability

D. Nonuniform ECC and Electromechanical Alternans

V. SUMMARY

GRANTS

ACKNOWLEDGMENTS

REFERENCES

	ABSTRACT

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	I. INTRODUCTION

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Membrane voltage and [Ca²⁺]_i changes have been linked for many decades. However recently, some human ventricular arrhythmias have been associated selectively with mutation of the ryanodine receptor (RyR), the primary release channel of intracellular Ca²⁺ stores in the cardiac cell (see sect. IIB2). It is the goal of this review to discuss the role of cellular Ca²⁺ transport in cardiac arrhythmias. We review the basis for Ca²⁺-dependent arrhythmias by reviewing the building blocks of excitation-contraction coupling. Then, we review what is known to date about the possible role of Ca²⁺ in different arrhythmias.

A. Overview of Ca²⁺ Transport in the Cardiac Cell

1. Structural aspects

The cell border is delineated by a glycoprotein layer overlying the sarcolemma, which invaginates the cell near the Z lines of the myofibrils. The resultant transverse tubules (t tubules) are rich in dihydropyridine-sensitive Ca²⁺ channels (DHPR) and Na⁺/Ca²⁺ exchange proteins. The t tubules make contact with a longitudinal network of tubules with lipid membranes called the sarcoplasmic reticulum (SR), which is a prominent Ca²⁺ storage organelle. Terminal cisternae of the SR abutting the t tubules contain Ca²⁺ channels with a high affinity for ryanodine (RyRs) that are involved in Ca²⁺ release from the SR. The RyR are so large that they form ultrastructurally recognizable junctional "foot proteins" in close proximity to the DHPR (Fig. 1). The longitudinal SR envelops the myofibrils and is densely covered by SERCA, SR Ca²⁺ pump molecules, which drive Ca²⁺ into the SR where it is buffered in the longitudinal SR by calreticulin and in the junctional SR by calsequestrin, a protein with intermediate affinity for Ca²⁺. The pump rate of SERCA depends on the [Ca²⁺]_i. The Ca²⁺ sensitivity of the pump is controlled by the degree of phosphorylation of the regulatory protein, phospholamban, in the SR membrane. The contractile proteins arranged in sarcomeres in the myofibrils occupy 60% of the intracellular space. Mitochondria adjacent to the sarcomeres occupy the remainder of the cell.

View larger version (80K): [in this window] [in a new window]   FIG. 1. A: Ca2+ release units in cardiac muscle (chick myocardium). Dyads are formed by junctional sarcoplasmic reticulum (SR) with feet on their cytosolic surface and containing calsequestrin (CSQ), associating with the surface membrane or the membrane of t tubules (T). Corbular SR contains the same components but does not associate with the cell membrane. B and C: peripheral couplings; docked, but not yet fully differentiated (embryo 2.5 days). D: freeze fracture of cell membrane arrows surrounding junctional domains containing dihydropyridine receptor (DHPR) particles. [From Franzini-Armstrong et al. (162).]

The action potential initiates the release of Ca²⁺ by mechanisms that are specialized in different cardiac tissues. The most well known specialization of the release units is found in ventricular myocytes, which is penetrated in a highly organized manner by transverse tubules of the cell membrane. The t tubules in these cells make contact with the terminal cisternae of the SR (70, 162, 518). The latter specialized junctional domains of the SR, containing calsequestrin (CASQ) and carrying protein "feet" on their cytoplasmic surface, contact t tubules in the form of dyads. Similar domains of the SR not associated with the cell membrane form so-called corbular SR. The molecular composition of the dyad is now beginning to be revealed. A linking protein junctophyllin is thought to be involved in the docking of t tubules on the dyads (518). While a majority of DHPR and RyR are colocalized in dyads (478), the observation that a significant fraction of RyR is not colocalized with DHPR (40% in adult rat myocytes) suggests that these RyR could be present in the corbular SR. On the luminal side of the junctional domains, the intra SR proteins, CASQ and junctin and triadin, are colocalized with RyR. Studies of the macromolecular protein assemblies have suggested that complete assembly of RyR/DHPR on the one side of the dyad and RyR/TrD (triadin)/JnC (junctin)/CASQ on the luminal side develops during maturation of the animal allowing efficient coupling between surface membrane depolarization and Ca²⁺ delivery to the myofibrils throughout an adult cardiac myocyte.

The ultrastructure of Purkinje and atrial cells differs from the above structure described for ventricular myocytes in that they have no t tubules and their SR presents in two forms, subsarcolemmal junctional SR and corbular SR located in the core of the cell (505) (Fig. 1). The diameter of a Purkinje cell is 30–40 µm. The diameter of a normal ventricular myocyte is 15–25 µm, whereas an atrial cell is 13–15 µm. Furthermore, the density of the myofibrils is lower in the Purkinje cell compared with that in ventricular myocytes. Accordingly, it has been shown that for both the small atrial myocyte and the large Purkinje cell, ECC relies on a process where surface membrane depolarization and Ca²⁺ delivery to myofibrils deep inside the myocyte depend on Ca²⁺ release by the SR, which starts at the cell membrane and which is propagated by chemical transmission to the depth of the cell (513).

2. Model of ECC

A descriptive model of ECC (Fig. 2) has been developed to explain the functional properties of the cardiac cell (476). During the action potential Ca²⁺ enter the cell through a protein that is sensitive to dihydropyridine, a DHPR or "L-type Ca²⁺" channels. In ventricular cells the number of Ca²⁺ channels is estimated to be 15/µm² and yet only 3% are open at peak currents (317). The amount of Ca²⁺ entering the cell per second depends on action potential duration and heart rate. Ca²⁺ influx at t tubules is 2.3x that of the cell surface, but more Ca²⁺ entry occurs per square micron at the cell surface than at t tubules (69). Ca²⁺ entering via L-type Ca²⁺ channels in the t tubules start ECC by triggering release of Ca²⁺ from the RyR in the terminal cisternae. In the rat (48), the ratio of RyR to L-type Ca²⁺ channels is 7:1 and would fit spatially with random coupling of the L-type Ca²⁺ channel to Ca²⁺ release via RyR (79). The ratio of superficial L-type Ca²⁺ channels versus t-tubular L-type channels is 1:3 (586), and it is anticipated then that one t-tubular L-type channel faces 10 RyRs. Ca²⁺-induced Ca²⁺ release (CICR) is proportional to the free intra-SR Ca²⁺ content and dictates the force of the cardiac contraction. Free Ca²⁺ in the SR has recently been spatially resolved (484), and data show that intra-SR Ca²⁺ diffusion is rapid, and local Ca²⁺ in SR in normal ventricular cells is never less than 50% of the diastolic value. The released Ca²⁺ activates contraction of the sarcomere. This contraction is short lived due to the rapid elimination of Ca²⁺ from the cytosol (Fig. 3A). About two-thirds of the Ca²⁺ is resequestered by the SR (45); the remainder leaves the cell mostly via the low-affinity, high-capacity, Na⁺/Ca²⁺ exchanger, while a low-capacity, high-affinity Ca²⁺ pump lowers the cytosolic Ca²⁺ level further during the diastolic interval (84). In the steady-state, the sum of the Ca²⁺ efflux through the membrane balances the influx during the action potential. It follows, then, that the Ca²⁺ content of the SR depends on the heart rate and on the duration of the action potential. Furthermore, a fraction of the Ca²⁺ involved in activation of the heartbeat recirculates into the SR and becomes available for activation of the next beat. Thus force of the heartbeat will depend on the force of the previous one. In addition, it takes time for the Ca²⁺ release process to recover completely from the last release so that sequestered Ca²⁺ can again be released from the SR. Therefore, the force of the heartbeat will also depend strongly on heart rate, on the duration of the diastolic interval, as well as on the duration of the action potential (594).

View larger version (29K): [in this window] [in a new window]   FIG. 2. Diagram of the excitation-contraction coupling system in the cardiac cell. During the action potential Ca2+ enters the cells as a rapid influx followed by a maintained component of the slow inward current. Ca2+ entry does not lead directly to force development as the Ca2+ that enter are rapidly bound to binding sites on the SR that envelops the myofibrils. The rapid influx of Ca2+ via the t tubules is thought to induce release of Ca2+ from a release compartment in the SR, by triggering opening of Ca2+ channels in the terminal cisternae, thus activating the contractile filaments to contract. Relaxation follows because the cytosolic Ca2+ is sequestered again in an uptake compartment of the SR and partly extruded through the cell membrane by the Na+/Ca2+ exchanger and by the low-capacity high-affinity Ca2+ pump. The force of contraction is thus determined by the circulation of Ca2+ from the SR to the myofilaments and back to the SR, and by the amount of Ca2+ that has entered during the preceding action potential. The relaxation rate of the twitch depends on the rate of Ca2+ dissociation from the myofilaments and on the rates of Ca2+ sequestration and extrusion. It is important to note that the process of Na+/Ca2+ exchange is electrogenic so that Ca2+ extrusion through the exchanger leads to a depolarizing current.

View larger version (50K): [in this window] [in a new window]   FIG. 3. A, a: superimposed tracings are force (thick black line) and intracellular calcium (Cai) transient (thin black line) recordings of the electrically stimulated trabecula. Bottom tracing illustrates the slow change in Cai occurring in normal muscle during the diastolic period (between vertical dotted lines). A, b: force at increased gain and sarcomere length during the twitch and subsequent diastolic pause. Note that no sarcomere length fluctuations (>1.3 nm) occur (535). B: enlarged confocal image depicting the characteristics of line scans during propagation of one microscopic Ca2+ wave (top panel a) and during initiation and propagation of another (bottom panel b) in normal muscle. In Ba, the Ca2+ wave has an asymmetric appearance, as if it encounters a border or failed to propagate in one direction. In Bb, the wave begins as a "V," indicating equal propagation in both directions; however, this wave stops propagating. The black arrows in both panels mark the same position in the two scans, indicating that the two waves started at the same place. The white arrows indicate the position of sparks at the leading edge of the wave in A. [From Wier et al. (590).]

3. ECC coupling in atrial and Purkinje cells from normal hearts

In rabbit atrial cells, immunostaining for either RyR or the L-type Ca²⁺ channel can be seen near the cell's sarcolemma (84a). Nonjunctional SR elements visualized with RyR staining are in transverse arrays along the Z lines (284, 610). In rat atrial cells, a 0.5- to 1-µm gap exists between junctional and nonjunctional RyR (341). This is in contrast to the unique ultrastructure of the latent atrial pacemaker cell, where subsarcolemmal SR cisternae are prominent and directly opposed to one another in adjacent cells. Differing from normal ventricular cell ultrastructure, where the presence of t tubules ensures reasonably synchronous Ca²⁺ release throughout the cell, atrial cells exhibit spatial Ca²⁺ gradients. In response to electrical stimulation, Ca²⁺ increases first at the cell's periphery and then after a 30- to 50-ms delay, increases in the central regions of the cell (38, 232, 284, 329, 341, 596). Preferential activation of Ca²⁺ sparks along the cell's periphery and then propagation to the cell's core (487, 596) is consistent with the biphasic nature of the human atrial Ca²⁺ transient under voltage clamp (198) and the ability of peripheral Ca²⁺ release to more efficiently regulate Na⁺/Ca²⁺ exchanger function (328). Furthermore, in rat atrial cells, a set of "eager" Ca²⁺ release sites that have a fixed edge location and preset activation pattern are thought to reflect clusters of RyRs that are closely coupled to Ca²⁺ channels and display a high sensitivity to trigger Ca²⁺ (341). In voltage-clamped cAMP-stimulated atrial cells, L-type Ca²⁺ channel evoked peripheral Ca²⁺ release occurs within 1–4 ms, and this Ca²⁺ then propagates to the interior of the cell at 230 µm/s (596). The efficacy of Ca²⁺ currents to trigger peripheral Ca²⁺ release is fivefold greater than that needed for triggering center Ca²⁺ release. Thus, for rat cells, it is thought that the mechanism of Ca²⁺ release from junctional SR differs from that in nonjunctional SR. Recent spark data suggest that peripheral atrial Ca²⁺ sparks are brighter and occur more frequently than central sparks (597). The opposite appears to be the case in feline atrial cells (284) where under permeabilized conditions, Ca²⁺ spark frequencies in the two regions are similar (488). Thus, in the intact cell, mechanisms that cause Ca²⁺ release in central SR RyRs are not efficient, most probably because of structural components. An interesting report by MacKenzie et al. (343) states that inherent intracellular calcium buffers in the normal rat atrial cell (for instance, mitochondria and SERCA pumps) prevent global Ca²⁺ transients under normal-paced conditions. Furthermore, membrane depolarization evoked peripheral Ca²⁺ release only propagates to the cell's core when the cell is hormonally (e.g., endothelin) stimulated.

As a result of the different structure of the Purkinje cell, coupling of excitation of the cell membrane with Ca²⁺ release in the core of these (large) cells differs substantially from that of myocytes. Immunostaining experiments in rabbit Purkinje cells show that RyRs are subsarcolemmal as well as within the cell consistent with earlier reports (106, 256, 505, 513). In fact, canine Purkinje cells contain both RyR3 and RyR2 isoforms with the RyR3 protein being located in a subsarcolemmal region. Here the action potential of the Purkinje cell precedes rapid Ca²⁺ entry into the subsarcolemmal space. The latter induces Ca²⁺ release which in some species propagates into the core of the Purkinje cells (64). In rabbit Purkinje cells, experiments with ryanodine suggest that Ca²⁺ changes in the central core of cells are best explained by simple buffered Ca²⁺ diffusion and not Ca²⁺ propagation (106). However, in rabbit Purkinje cells, evoked Ca²⁺ transients and sparks are only seen to originate at peripheral cellular components, suggesting that RyRs in the cell center of this type of cell are "silent" (106). This is consistent with voltage-clamp studies of single rabbit Purkinje cells which show a single-component Ca²⁺ transient (497). However, in canine Purkinje cells, electrically evoked Ca²⁺ transients are multiphasic (61, 64). An action potential evokes a sudden increase in Ca²⁺ particularly along the periphery. In some Purkinje cells from normal hearts, if electrically evoked peripheral release is spatially and temporally inhomogeneous, a local Ca²⁺ wave is produced and can propagate as a traveling Ca²⁺ wave the length of the aggregate as well as towards the cell's core (64, 513). Finally, while fundamentals of ECC have been described above, it is important to remember that the time course of the action potential (AP) of the cell can have significant modulatory effects on ECC efficiency. Not only does AP duration affect the time course of the evoked Ca²⁺ transient (58, 463), but altering the rate of early repolarization can affect both the magnitude and time course of SR Ca²⁺ release (464).

4. Reversal of ECC

In a ventricular myocyte, the distance between Ca²⁺ release sites on the terminal cisternae of the SR to the Ca²⁺ transport molecules at the surface of the t tubules is less than 300 nm (i.e., from 40 nm to DHPR to 300 nm to Na⁺/Ca²⁺ exchangers). It follows that the transport molecules face the largest variation of [Ca²⁺]_i within the cell. This review discusses potential consequences of the SR-related [Ca²⁺]_i changes on function of membrane Ca²⁺ transport and how this feedback may be involved in modulation of action potential waveform and the development of arrhythmias. We anticipate that the cross-talk between the surface membrane and the SR is strongest in the working myocyte, given their high density of t tubules. In addition, it has become clear that rapid mechanical perturbations of the contracting cell may cause rapid Ca²⁺ dissociation from troponin C (TnC). Consequently, Ca²⁺ released from the myofilaments may also trigger Ca²⁺ release from the SR by CICR. While normal CICR occurs during the action potential, Ca²⁺ dissociation from the myofilaments may take place when the cell is repolarized. In that case, CICR could elicit a [Ca²⁺]_i transient that in turn affects a different set of membrane channels.

	II. MOLECULAR BUILDING BLOCKS OF EXCITATION-CONTRACTION COUPLING

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A. Ca²⁺ Flux Through the Sarcolemma

1. Ca²⁺ entry through voltage-gated channels

There are several types of ion channels that are Ca²⁺ permeable. What has been termed as a background Ca²⁺ channel was originally defined in bilayer experiments (453, 454) (B-type Ca²⁺ channels). Under these conditions this channel spontaneously opens, has a relatively low conductance, is not blocked by nisoldipine, and is reasonably selective for Ba²⁺. Further investigations into resting Ca²⁺ influx into adult cardiac cells have shown the existence of spontaneously active Ca²⁺- and Ba²⁺-permeable but Ni²⁺-insensitive single channels in both cell-attached and inside-out patches (108). These latter channels are activated by phenothiazines such as chlorpromazine, trifluoperazine, and H₂O₂ but at very negative holding potentials (13, 314). A short report has stated that the voltage-independent B-type Ca²⁺ channel is regulatory in ceramide-induced rat myocyte apoptosis (201). While whole cell clamp data do not reveal such macroscopic inward currents in myocytes, some have suggested that this Na⁺-independent Ca²⁺ channel contributes importantly to tonic Ca²⁺ entry in the quiescent rat trabecula (307). The molecular nature of these background Ca²⁺ channels is unknown at this time.

The L-type (L for long lasting, I_CaL) and T-type (T for transient, I_CaT) Ca²⁺ currents were initially described in neuronal tissues. Bean et al. (31) first described multiple cardiac Ca²⁺ channels in canine atrial cells. At that time two types of Ca²⁺ currents carried by Ba²⁺ were recognized. Subsequently, I_CaL and I_CaT have been recorded in cardiac tissues of most species under various conditions. However, within the same species, the density of I_CaL and I_CaT varies depending on the location of the myocyte within the heart. Hagiwara et al. (189) first described the large density of both the L- and T-type channels in rabbit sinoatrial node (SAN) cells. Zhou and Lipsius (639) described large T-type currents in latent atrial pacemaker cells. Studies of cells dispersed from canine ventricles revealed a large peak T/L current density ratio in Purkinje cells dispersed both from free-running fiber bundles and the subendocardium of the left ventricle (LV) (212, 213, 548). In contrast, myocytes dispersed from mid and epicardial layers have a smaller T/L current ratio (548). Notably, T currents have not been observed in human atrial (366, 556), human ventricular (57, 366), or human Purkinje cells (P. Boyden, unpublished data).

Cardiac L- and T-type Ca²⁺ channels differ in the following biophysical properties. 1) Voltage range of activation: the T-channel activation occurs at more negative voltages than the L channel, e.g., in 5 mM [Ca²⁺]_o the threshold for activation is –50 and –30 mV for T and L, respectively (548). 2) Voltage range of inactivation: in 5 mM [Ca²⁺]_o, the T channel can be inactivated by membrane depolarization positive to –70 mV. The L channel remains fully available for activation at potentials more negative than –40 mV. 3) Mechanism of inactivation: T channel inactivates solely by membrane depolarization. For the L channel, both membrane depolarization and Ca²⁺ participate in the inactivation process.

Voltage-dependent inactivation of the L-type Ca²⁺ current is clearly evident as channels incorporated into lipid bilayers inactivate even when Ca²⁺ is buffered (453) and as noted from the dependence of the time course of Ba²⁺ current decay on voltage (14, 187). In fact, inactivation of L-type Ca²⁺ current can occur at voltage steps where "apparent" activation is absent. The molecular determinants of voltage-dependent inactivation of Ca²⁺ channels are less well understood than those of K⁺ or Na⁺ channels. In studies using Ba²⁺ as a charge carrier, several critical locations throughout the channel protein have been implicated in the fast (tens to hundreds of milliseconds) voltage-dependent inactivation process. They are the I-II linker, the proximal COOH terminus, the EF hand area in IC, and all four S6 regions (37, 40, 41, 59, 202, 203, 504, 624, 628). One model proposed suggests that a domain of the I-II linker docks to one or all of the S6 segments at the cytoplasmic end (85, 512). Importantly, this mechanism is not involved in the channel's "recovery from inactivation," only the channel's response to depolarization. Critical of course to these mechanisms is that Ba²⁺ permeating through these proteins show only voltage-dependent inactivation and no ion-dependent inactivation. However, recent data suggest that inactivation of the L-type Ca²⁺ channel when Ba²⁺ is the charge carrier may not be all due to a voltage-dependent process (157, 357).

The molecular basis of the cardiac L-type Ca²⁺ channel structure is due to the combination of the _1C-subunit [Ca_v1.2; see Ertel et al. (146) for nomenclature] (four 6-transmembrane segments joined by intracellular linkers with cytoplasmic NH₂ and COOH termini) with ₂-, ₂/-, and -subunits. Alternative splicings of the -subunit have been reported (for review, see Ref. 324) and two missense mutations in one exon appear to lead to abnormal Ca²⁺ current function in cells of patients with Timothy's syndrome (see sect. IVA3). Perhaps in some acquired diseases, alternatively spliced proteins constitute the remodeled Ca²⁺ channels in arrhythmogenic substrates. The -subunit (33 kDa) is also expressed in skeletal muscle and in expression systems can have a modest effect on Ca²⁺ channel currents (112). Other studies have shown it can modulate Ca_v3.1, T-type Ca²⁺ channels (196). Its role in modulation of cardiac Ca²⁺ channels is minimal.

The Ca_v1.2 NH₂ terminus can act as an inhibitory particle (490) as well as a site for modulation by Ca²⁺-binding proteins such as CaBP1 (635, 636), Ca²⁺/calmodulin protein kinase II (CaMKII) (227), calmodulin (CaM) (227, 636), and Cav subunits (260). Some have suggested that a reduction in this inhibition can be caused by protein kinase C (PKC), which increases Ca_v1.2 Ca²⁺ currents (491); alternatively, phosphorylation of the NH₂ terminus by PKC has been proposed to decrease the L-type Ca²⁺ current (613).

Other major sites of modulation of the _1C-subunit function are within the COOH terminus since it is the target of several kinases that regulate Ca_V1.2 L-type Ca²⁺ currents. Both PKA and CaMKII increase L-type Ca²⁺ current and change channel modal gating (139, 439, 621), and both effects are thought to be due to phosphorylation of the _1C COOH terminus (227, 613). Additionally, Src kinase phosphorylation of the neuronal _1C-isoform at a COOH-terminal residue leads to potentiation of the L-type Ca²⁺ current (36). However, the mechanisms by which specific kinases modulate L-type Ca²⁺ currents differ. For example, the major target for PKA in _1C has been identified as Ser¹⁹²⁸ (122, 370); however, recent data suggest that phosphorylation of this site may not be required for adrenergic stimulation of L-type Ca²⁺ currents (166). Although CaMKII activation leads to the same shift in modal gating as that caused by PKA stimulation (139), and CaMKII also phosphorylates the COOH terminus, the specific targets are unknown. One report suggests that Ser¹⁵¹⁷ may be the target (147), but definitive biochemical evidence is lacking. PKC also phosphorylates Ser¹⁹²⁸ (613), although the effects of this phosphorylation on L-type Ca²⁺ currents are unknown.

The _1C COOH terminus also contains a binding pocket for CaM (278), which mediates Ca²⁺-dependent inactivation and Ca²⁺-dependent facilitation of L-type Ca²⁺ channels (417, 643). The interaction between CaM and _1C is constitutive (145, 278). T-type Ca²⁺ currents do not show Ca²⁺-dependent inactivation (136, 213, 508); the _1G- and _1H-subunits lack the determinants for CaM binding in their respective COOH termini.

The voltage sensor for activation is the highly charged S4 segment. Ca²⁺ binding sites formed by glutamates in the pore loop of each repeat are critical for selectivity of the channel (144). Sections of the ₁-subunit pore-forming segments, intracellular loops, and COOH terminus all contribute to Ca²⁺ channel inactivation. Mutating single amino acids in IIIS6 and IVS6 domains have a significant effect on current decay, suggesting that an area in the inner channel mouth is a key player in channel inactivation. Further point mutations in the intracellular I-II loop and the IVS5-IVS6 linker both affect Ca²⁺ channel inactivation. Notably, these sections of the protein are also critical sites of -subunit and/or G protein interactions.

Currently, four potential -subunits (Ca_{v 1}-₄) have been recognized, but in cardiac cells the ₂-subunit predominates, providing a rate-limiting step in the expression of Ca²⁺ channel proteins (102). Ca_V subunits are entirely cytoplasmic, and each subunit includes a variable NH₂ terminus, a conserved core that includes an interacting Src homology domain, a guanylate kinase (GK)-like domain (90, 361, 402, 526), and a divergent COOH terminus. Both the NH₂ terminus and SH3 domain contribute to isoform-specific regulation of channel inactivation (361). Interestingly, the GK domain contains the binding pocket for the ₁ interaction domain (AID) on the ₁-subunit. The role of the Ca_V COOH terminus is still largely unknown, but ₂ is a target for several kinases known to modulate L-type Ca²⁺ currents (e.g., the _2a COOH terminus is phosphorylated by PKA on Ser⁴⁷⁸ and Ser⁴⁷⁹). This phosphorylation appears to contribute to cAMP-dependent regulation of the channel (72).

The _1C-subunit (Ca_v1.2) when expressed alone is sufficient for L-type channel activity, but when expressed with a cytosolic -subunit (52), peak currents increase fourfold, apparently by accelerating the opening of the pore and reducing the rate of channel closure (295, 371, 389, 391, 415). Furthermore, there is a shift in the activation curve, a slowing of activation, and an enhancement of the inactivation process (415). _2a-Subunits slow inactivation (243) due to palmitoylation of the NH₂-terminal residues, and subsequent tethering to the membrane (509). A region of I-II intracellular linker the AID of the -subunit forms the primary binding site for the accessory -subunits (429). It is thought that this AID region forms an -helix that becomes buried within a conserved domain common to all -subunits (90, 402, 555). Interestingly, functional studies have revealed that while the AID region is not necessary for -subunit modulation of Ca²⁺ currents, the tethering of subunits to the AID region optimizes subunit orientation which in turn increases local -subunit concentration (346, 526). Finally, cardiac L-type Ca²⁺ current facilitation also occurs when the _2a-subunit is coexpressed with the _1C-subunit (113).

The two-component ₂/-subunit (170 kDa) remains linked in vitro, with ₂-subunit being an extracellular highly glycosylated protein; is the short membrane-spanning protein with a fully glycosylated extracellular portion. Several members of this family exist with ₂/ and ₂/₂ being expressed in heart (170, 348). This subunit complex affects both ionic and gating currents of the expressed Ca²⁺ channels by increasing the number of functional channels at the cell surface (15, 25, 86, 281, 495). In one series of experiments, coexpression of ₂/ with _{1C 3}-subunits prevented voltage-dependent facilitation (424). In these latter studies, this voltage-dependent facilitation was due to an increase in the number of channels that were able to produce gating current, as well as the number of channels that opened in response to voltage (424). Small molecules like the Ras-related G protein Gem have been shown to bind to -subunits (33). Such binding apparently interferes with the subunit's ability to traffic the -subunit to the membrane. Interestingly, gabapentin, a compound that has been shown to bind specifically to the 2/-subunit (282), appears to have no consistent effect on expressed L-type Ca²⁺ currents (348).

Evidence suggests that the cardiac L-type Ca²⁺ channel protein in rabbit myocytes exists in two forms. One form is full length and comprises 20% of all _1C-subunits in rabbit membranes (122, 171, 173), while the other form, truncated at its COOH terminus, comprises 80% of all ₁-subunits. The truncated form of the channel cannot be directly phosphorylated by PKA (122, 200), but remains in situ near the remaining -subunit protein (169). Functionally, it has been shown that removal of the COOH terminus from the full-length protein results in an increase in channel activity (280, 582). Several peptides designed to mimic residues of the distal COOH terminus of the Ca_v1.2 protein also inhibit expressed Ca²⁺ currents, illustrating that a specific domain of residues 2024–2171 of the subunit functions to inhibit channel conductance (170). It is unclear as to whether the truncated Ca²⁺ channel and its COOH terminus remain fully functional in the in situ myocyte. However, this mechanism of modulation of L-type Ca²⁺ current amplitude has the potential of being an important contributor to ECC. For example, activation of N-methyl-D-aspartate (NMDA) receptors and subsequent L-type Ca²⁺ channel-mediated Ca²⁺ influx induces such COOH-terminal truncation resulting in sustained changes in Ca²⁺ channel activity (200).

In addition to _1C (Ca_v1.2), mRNA and protein from _1D (Ca_v1.3) subunits have been measured in heart tissues (448, 530, 604). Ca_v1.3 Ca²⁺ channel proteins are sparse, but this type of Ca²⁺ protein may serve a specific functional role. Expression studies have shown that currents mediated by Ca_v1.3 proteins (plus _2a-subunits) activate more quickly and decay more slowly than those of Ca_v1.2 proteins (288). Furthermore, steady-state inactivation and activation voltage relations of Ba²⁺ currents through Ca_v1.3 channels are shifted in the hyperpolarizing direction. These specific kinetic differences between Ca_v1.2 and Ca_v1.3 currents account in part for the decreased sensitivity of expressed Ca_v1.3 currents to dihydropyridine block (288). Finally, mice lacking _1D-subunit proteins are deaf and exhibit sinoatrial dysfunction and bradycardia, suggesting a role for _1D-proteins in SAN pacemaker activity (347, 425, 633). Interestingly, these mutant mice also show altered atrial Ca²⁺ currents and have inducible atrial fibrillation but no change in effective refractory period (ERP) (634). While Purkinje cells have not been studied in these mice, canine Purkinje cells express this protein, and two types of L-type Ca²⁺ currents have been described (548) suggesting that pacemaking in Purkinje cells may also involve Ca²⁺ currents through Ca_v1.3 channels.

The molecular basis of neuronal and cardiac T-type Ca²⁺ channels has been defined (111, 416). In both cases, the low-voltage T-type Ca²⁺ channel protein (_1H, _1G) (Ca_v3.3 and Ca_v3.2) has high sequence identity with the _1C-subunit particularly in the membrane-spanning regions (416). Charged residues of the S4 regions are conserved between _1C and _1H, _1G while a ring of glutamates important in _1C channel selectivity has been partially replaced by aspartates. T-type Ca²⁺ currents inactivate with voltage but not by Ca²⁺ (136, 508). Some have suggested that a "ball-and-chain" type mechanism involving the amino side of the COOH terminus contributes to T-type channel inactivation (74, 508). Perhaps more importantly in terms of possible targets for pharmacological modulation, intracellular loop motifs involved in -subunit binding (306, 429) or Ca²⁺ binding (124) of the L-type _1C protein are missing in both the _1G and _1H proteins. In canine Purkinje cells, the large T currents most likely are due to Ca_v3.2 based on their kurtoxin sensitivity (448).

2. Na⁺/Ca²⁺ exchange, Ca²⁺ entry, and Ca²⁺ efflux

The cardiac Na⁺/Ca²⁺ exchanger protein transports Ca²⁺ across the sarcolemma in exchange for Na⁺ and is important in maintaining Ca²⁺ homeostasis in the myocyte. Na⁺/Ca²⁺ exchanger activity has been shown to affect various components of normal ECC [i.e., Ca²⁺spark frequency, SR Ca²⁺ release, and SR load (47, 176, 331)]. Normally, Na⁺/Ca²⁺ exchange works in the so-called forward mode, i.e., extruding Ca²⁺ in exchange for extracellular Na⁺. Reverse-mode operation of Na⁺/Ca²⁺ exchanger could provide additional Ca²⁺ influx into the cell. In the forward mode, Ca²⁺ are being transported out against their electrochemical gradient, and therefore, this mode of activity requires an expenditure of energy. It is generally accepted that the Na⁺ transcellular distribution indirectly provides the energy. Stoichiometric determinations have shown that three Na⁺ are transported for one Ca²⁺. Thus the exchanger is electrogenic. A recent study suggested that the stoichiometry may be closer to 4:1 Na⁺/Ca²⁺ (163, 261), but these data have been challenged (211). Under normal conditions, the reversal potential of the Na⁺/Ca²⁺ exchanger is –30 mV (279). Accordingly, negative to this potential Na⁺ flux is inward and Ca²⁺ flux is outward generating an inward current. Positive to –30 mV, the Na⁺/Ca²⁺ exchanger works in reverse mode, and outward current is generated. For the NCX1 transporter protein, it has been estimated that the turnover rate can be up to 5,000/s (209, 393) with a K_D for [Ca²⁺]_i of 6 µM (354). Recent data derived from the steady-state voltage and Ca²⁺ dependence of the Na⁺/Ca²⁺ exchanger protein have suggested that within <32 ms of an action potential upstroke, peak Ca²⁺ in a submembrane space is >3.2 µM (578). Thus Na⁺/Ca²⁺ exchanger current influences both the atrial and ventricular action potential (248). Furthermore, a component of observed transmural electrical heterogeneity of the left ventricle has been ascribed to basal differences in I_Na/Ca currents across the wall (647).

The Na⁺/Ca²⁺ exchanger protein is now considered to consist of nine transmembrane segments with a large (550 amino acids) intracellular loop (loop f) between segments 5 and 6 (392). The Na⁺/Ca²⁺ exchanger gene NCX1 undergoes alternative splicing (NCX1.1, NCX1.3) in the COOH terminus of its large intracellular loop. Splice variants function differently with respect to regulating properties, and expression of NCX1.3 was found to protect against severe Ca²⁺ overload conditions (230). Distinct regions of the protein have been shown to be involved in the Na⁺/Ca²⁺ translocation process (135), while other regions, particularly loop f, are involved in the intrinsic regulation of the Na⁺/Ca²⁺ exchanger by Na⁺ and Ca²⁺ (355). Ca²⁺-dependent regulation of exchanger activity is via a high-affinity binding site (0.022–0.4 µM) that is distinct from the Ca²⁺ transport site (354), is 130 amino acids in length, and is located in the center of loop f (316). Ca²⁺-dependent regulation of Na⁺/Ca²⁺ exchanger activity is apparently allosteric in ferret cells such that when [Ca²⁺]_i levels are reduced (approximately <150 nM) Na⁺/Ca²⁺ current deactivates (577). A corollary is that when [Ca²⁺]_i is elevated, steady-state activation of Na⁺/Ca²⁺ exchanger current increases, by as much as 67% for a doubling of [Ca²⁺]_i. Such activation in normal cells promotes Ca²⁺ efflux with concomitant production of inward currents. If Ca²⁺ transients occur as traveling Ca²⁺ waves between cells, activated Na⁺/Ca²⁺ exchanger current would contribute to the occurrence of Ca²⁺-activated membrane currents.

Binding of the regulator Ca²⁺ decreases Na⁺-dependent inactivation of the Na⁺/Ca²⁺ exchanger (208). Intrinsic regulation of the Na⁺/Ca²⁺ exchanger by Na⁺ originally observed by Hilgemann (206) was termed Na⁺-dependent inactivation. This process is enhanced at low intracellular pH and diminished by micromolar Ca²⁺ (207). Mutagenesis studies suggest that the exchanger inhibitory peptide (XIP) binding site is located on loop f of the protein and is involved in the Na⁺-dependent inactivation of the exchanger (355). However, recent work with split exchanger proteins suggests that endogenous XIP region is not located between amino acids 265 and 672, since the activity of split exchanger with these loop residues deleted is still blocked (405). Other regulators of exchanger function include free radicals, pH, lipid products, as well as several kinases.

3. Stretch-sensitive Ca²⁺ flux

Stretch-activated ion channels have been described in both atrial and ventricular cells of several species (34, 226, 626). The channel is permeable to monovalent cations and Ca²⁺ (275) and thus can provide a source of Ca²⁺ influx. In single cells and isolated tissues from normal hearts, stretch has been observed to lead to a gradual (10 s) increase in [Ca²⁺]_i (167, 533) as well as increases of inositol trisphosphate (IP₃) and inositol tetrakisphosphate (IP₄), both of which may modulate [Ca²⁺]_i levels (119) and subsequent force development. In adult guinea pig cells, large stretch-induced [Ca²⁺]_i changes are blocked by streptomycin (34, 168), a blocker of mechanosensitive transduction currents in hair cells (399), are not sensitive to ryanodine or tetrodotoxin (TTX), but sensitive to extracellular Ca²⁺. Interestingly, streptomycin also blocks stretch-induced atrial tachyarrhythmias in the isolated heart (19a), presumably by inhibiting mechanosensitive cation channels in atrial myocytes (168, 275, 276). In rat cells, stretch produces a slow increase (minutes) in the electrically evoked Ca²⁺ transient (222). Stretch of either rat myocytes or trabeculae increases both the frequency of SR Ca²⁺ release (seen as Ca²⁺ sparks) as well as the level of Akt and endothelial nitric oxide synthase (eNOS) phosphorylation. Thus it has been proposed that in response to stretch, myocytes generate nitric oxide (NO), which acts locally to modify ECC efficiency (419). Interestingly, sensitivity of a myocyte to stretch increases with age and degree of cellular hypertrophy (258).

B. Intracellular Ca²⁺ Cycling

1. SR Ca²⁺-ATPase pump

Two Ca²⁺ can be transported by the cardiac SR Ca²⁺ pump for each ATP molecule consumed (524), although other stoichiometries have been reported. ATP binds to high-affinity binding sites on the cytoplasmic side of the pump. The terminal phosphate of ATP is transferred to aspartate-351 on the pump protein, and the bound Ca²⁺ are "occluded." ATP hydrolysis of the protein alters the structure such that Ca²⁺ cannot return to the cytoplasmic side. Phosphorylation also reduces the Ca²⁺ affinity of the pump such that Ca²⁺ can be released into the lumen of the SR.

The cardiac Ca²⁺ pump protein is the same as that from slow-twitch muscle (66, 67, 344) and has 10 membrane-spanning regions where each region, M1-M5, has additional -helical "stalk" regions on the cytoplasmic side. Most of the 96-kDa protein is on the cytoplasmic side of the SR membrane including a -strand, phosphorylation (aspartate-351) and nucleotide binding sites, stalk domains, and a hinge region. The crucial high-affinity Ca²⁺ binding sites were initially proposed to reside in the highly anionic stalk region (67); however, more recent data suggest that they are not in the stalk, but within the transmembrane regions M4-M6 and M8 (99, 100). It is likely that in the membrane these transmembrane domains may be arranged in a cylinder to form an ion channel through the SR bilayer (344).

The rate of the cardiac SR Ca²⁺ pump is highly regulated by phosphorylation of the protein phospholamban (522). In the dephosphorylated state, phospholamban interacts with the SR Ca²⁺ pump near the phosphorylation site of the pump (246), acting as an inhibitor of the Ca²⁺ pump activity. Phosphorylation removes the inhibitory effect and increases the pumping rate (205). Phospholamban is a homopentamer; the monomer has 52 amino acids and exhibits one hydrophobic and one hydrophilic domain. A proposed structural model states that the pentamer could have a hydrophilic pore through the SR membrane with phosphorylation sites on the cytoplasmic surface (494). Kovacs et al. (290) have obtained evidence that dephosphorylation of phospholamban can form Ca²⁺-sensitive channels in lipid bilayers. However, it is not clear whether or how the ionophoretic property might be related to the function of phospholamban in cardiac SR.

Phospholamban is phosphorylated by cAMP-dependent protein kinases (523). Studies from the intact perfused hearts show that -adrenergic stimulation via PKA reduces the K_m for Ca²⁺ and thus accelerates relaxation of the muscle (362). Ca²⁺-calmodulin dependent protein kinases and protein kinase C (PKC: Ca²⁺/phospholipid dependent) (244) also phosphorylate phospholamban at threonine-17 (579). The PKC site on phospholamban is Ser-10, but there is no evidence that this site is ever phosphorylated physiologically. Whether Thr-17 phosphorylation increases V_max or Ca²⁺ affinity is controversial. This stimulation can result in an increase in SR content. The cardiac Ca²⁺ pump has two ATP binding sites: a high-affinity site (K_d 1 µM) that is the substrate site and a second lower affinity site (K_d 200 µM) that serves as a regulatory role (127, 138). The substrate for the Ca²⁺ pump is probably MgATP, but other nucleotides can be used (525). Therefore, the ATP level would have to be low to prevent ATP binding to the substrate site. However, decrease of the ATP level during ischemia slows SR Ca²⁺ pumping and relaxation.

2. Ryanodine-sensitive SR-Ca²⁺ release channels (RyR)

Two kinds of Ca²⁺ release channels found in the SR membrane, a Ca²⁺-activated channel and an IP₃-activated channel, are proteins that form a distinct, highly conserved gene family. It is thought that the major mechanism regulating Ca²⁺ release in cardiac cells is CICR. CICR requires that Ca²⁺ provided by the activated L-type Ca²⁺ channel bind to the SR-Ca²⁺ release channel and cause opening of a high-conductance channel allowing rapid Ca²⁺ efflux from the SR. Studies of the SR-Ca²⁺ release channel have been greatly accelerated by the recognition that ryanodine, a plant alkaloid, is a selective and specific ligand for this channel. The RyR functionally constitutes the Ca²⁺ release channel of the SR and structurally represents the "foot" structure linking the t tubules to the SR. The recognition of selective ryanodine binding has allowed purification of several isoforms of RyR (RyR1, RyR2, RyR3) from skeletal (236, 239, 298) and cardiac (238, 297) muscle. Most of what is known about RyR comes from electrophysiological experiments on the channels after they have been incorporated into lipid bilayers (298, 456, 457, 502). Such experiments have suggested a biphasic response of the open probability of the channel (P_o) to activating [Ca²⁺]. RyR2 P_o increases up to micromolar concentrations of [Ca²⁺]_i and then decreases at higher [Ca²⁺] (606, 609) (Fig. 4, A and B). Luminal [Ca²⁺] versus P_o of RyR activity slightly differs between control wild-type RyR and mutated RyR (251) (Fig. 4C). Furthermore, the probability that a single RyR will be activated is determined by the amplitude and duration of Ca²⁺ trigger signals (623). The channel has a high Ca²⁺ conductance but can also conduct other divalent cations such as Ba²⁺ and Mg²⁺ (42, 97, 455) as well as monovalent ions in the absence of Ca²⁺ (502). Compared with the sarcolemmal Ca²⁺ channel (Ca_v1.2) under similar conditions, the SR Ca²⁺ release channel has lower selectivity for Ca²⁺ and 10-fold higher conductance (42). The ability of Ca²⁺ to cause release depends on [Ca²⁺]_i, the rate of rise of [Ca²⁺]_i at its receptor (151), as well as the presence of various nucleotides and Mg²⁺. RyR channels close rapidly either by deactivation (192) or decrease in trigger Ca²⁺. An increase of SR luminal [Ca²⁺] causes a marked increase in the P_o of the Ca²⁺ release channel as well as the cell's resting Ca²⁺ spark frequency (27, 339, 500). Human atrial RyR share similar biochemical properties compared with ovine or canine ventricular counterparts (107). Refractoriness of SR release may be due in part to SR Ca²⁺ refilling mediated by the SR Ca²⁺ pump (521). Ryanodine, at low concentrations (<30 µM), opens the cardiac SR Ca²⁺ release channel in either vesicles or bilayers to a stable subconducting state and the channel no longer responds to Ca²⁺, ATP, Mg²⁺, or ruthenium red (363, 459). This probably is due to the occupation of the high-affinity ryanodine binding sites (K_d 10 nM). Very high concentrations of ryanodine (>100 µM) appear to lock the Ca²⁺ release channel in a closed state.

View larger version (24K): [in this window] [in a new window]   FIG. 4. Dependence of ryanodine receptor (RyR) single-channel activities on cytosolic Ca2+ and SR-luminal Ca2+. A: original current traces from cardiac Ca2+ release channels at three differing Ca2+ levels. Upward deflections indicate openings from closed state (small bar at left). B: average single-channel open probability (Po) values determined as in A at +35 mV (closed symbol) and –35 mV (open symbol). See Ref. 606 for more information. C: Po-luminal [Ca2+] relationship of wild-type RyR2 expressed in HEK293 cells compared with the Po-luminal [Ca2+] relationship of RYR2 channels with mutations linked to VT (L433P and R176Q/T2504M). These mutations displayed a leftward shift of the Po-luminal [Ca2+] relationship without a change in the sensitivity to cytosol [Ca2+]. See Ref. 251 for details.

Novel high-resolution imaging electron microscopic techniques have allowed exciting progress in the structural understanding of SR Ca channels IP₃R as well as RyR1, -2, and -3 (468, 481). Future progress will be facilitated by the development of crystallization procedures for these protein complexes (619). These studies have revealed that SR Ca²⁺ channels are strikingly similar tetrameric structures. We will review here the structure of SR Ca²⁺ release channels based on data from both cardiac RyR2 (629) and skeletal RyR1 (468).

Three-dimensional reconstruction of RyR1 has revealed a transmembrane domain and a large cytoplasmic assembly (Fig. 5A). The transmembrane domain is shaped as a square prism that is linked by columns in a narrower region to the cytoplasmic assembly (468). The cytoplasmic assembly itself forms a square that is rotated 45 degrees with respect to the prism. The center of the assembly gives access to a trough that connects to the Ca²⁺ channel in the depth of the transmembrane domain. The corners of the cytoplasmic assembly form the so-called "clamp." The sides form the "handle." A series of interconnected tubular structures form a rhomboid structure on the t-tubular surface of the clamp linking four domains (337).

View larger version (54K): [in this window] [in a new window]   FIG. 5. A: ultrastructure of RyR1 at 9.5 Å resolution. The receptor is composed of a cytosolic assembly linked to a transmembrane assembly (TMA) through a neck region which conveys columns that form the vestibule of the TMA and the Ca2+ channel in the center of the TMA to the regulatory elements in the clamps and handle domain of the cytosolic assembly (see text for further details). [From Samso et al. (468).] B: schematic diagram of the reported mutation sites of RyR1 and RyR2. NH2 terminus, central domain, and transmembrane (channel) regions are denoted. For more information, see text and http://pc4.fsm.it:81/cardmoc/. [From Yano et al. (617).]

Four columns arise from the external peripheral branches of the transmembrane prism. Each column consists of two connections to the handle; in addition, two adjacent monomers are structurally linked. This creates a connection between each rhomboid structure with a column of the prism via a direct pathway as well as via an external arm of the handle. If the structures in this link exhibit elastic properties, one would expect a torsional force on the prism that should depend on the integrity of the rhomboid structure on the t-tubular surface of the clamp. The twist of the transmembrane prism (as in Fig. 5), observed in the closed state of the channel, is consistent with this notion. Releasing the torque on the molecule, by dissociation of the internal connections in the rhomboid structure, would be accompanied by untwisting the transmembrane prism as has been observed during opening of the channel (480).

The peptide sequences involved in the transmembrane domains are known to some extent, although the exact number of transmembrane sequences (6) is still under study. Similarly, the location of the peptide sequences in the cytoplasmic domains is only partially known. The location of the peptide chains in the structure of the Ca²⁺ channel is still far from complete and even farther from conclusions regarding control mechanisms of the P_o of the channel, and therefore, a detailed review of their location (cf. Refs. 332, 334, 350, 466; see also Refs. 35, 191, 333, 334, 465, 467, 629) is beyond the scope of this review. However, the proximity of mutations that affect the channel in skeletal muscle in malignant hyperthermia and central core disease and arrhythmogenic mutations in cardiac muscle suggests that the bridge in the rhomboid structure in the clamp is important to regulation of opening of the channel. The central domain of mutations that is involved in arrhythmias is again found in the bridge within the rhomboid structure of the clamp, suggesting that this structure in the clamp is important in the regulation of the opening probability of the channel.

Similar to what has been hypothesized for RyR1 channel proteins (609), it appears that RyR2 structure involves a critical interdomain interaction that plays a role in modulation of the channel's ability to release Ca²⁺. In this hypothesis, specific domains of the NH₂ terminus interact to "zip" shut regions of the central core region. This zipped conformation has been linked to RyR2 channels with no Ca²⁺ "leak" (235). In disease and with RyR2 mutations, these regions can become unzipped to "leak" Ca²⁺ (see sect. IVA1). However, recent data also suggest that highly reactive free radicals destabilize these interdomain interactions and by themselves can cause partial dissociation of the FKBP12.6 binding protein (616).

Several studies have elucidated the sites for modulation of CICR (see reviews in Refs. 44, 88). Smaller modulatory proteins that have been found to copurify with RyR proteins are triadin (68, 183), sorcin, FKBP12.6 (249), PKA catalytic and regulatory subunits, MAKAP anchoring proteins, protein phosphatase (PP) 1 and PP2A (349), and calmodulin/CaMKII (44, 182). Recently, it has become known that RyR2 can be phosphorylated by at least two kinases, PKA and CaMKII, but each has a distinctive effect on RyR2 function. Ca²⁺ spark frequency of a normal myocyte increases with CaMKII stimulation due to a direct effect of phosphorylation of RyR2 (182). On the other hand, PKA-mediated effects to increase spark frequency appear to be related to an effect on SR load. The role of each of these kinases in abnormal Ca²⁺ spark frequencies accompanying disease awaits further study. It has been suggested that the FKBP12 protein is required for normal function of RyR2 playing a key role in the efficient so-called coupled gating between neighboring RyR2 channels (401). An immunosuppressant agent, FK506, binds to FKBP12 presumably inhibiting its modulation of RyR1, thereby increasing spontaneous [Ca²⁺]_i transients by increasing the rate of release from the SR (358). FKBP12 null mice have RyR2 channels that exhibit abnormal gating in that there is a high occurrence of subconductance states (492). However, others have reported that removal of FKBP12.6 from RyR2 has no effect on RyR single-channel function (26). In rapamycin- or FK506-treated ventricular cells, presumably the loss of association of FKBP12 from RyR2 underlies the substantial increase in resting Ca²⁺ spark frequency (358).

A) POTASSIUM AND CHLORIDE CHANNELS IN THE SR MEMBRANE.  The presence of large Ca²⁺ fluxes through the membrane of the SR requires the existence of other channels which allow large countercurrents to protect against electrical instability of the SR membrane. A large-conductance (150–200 pS) K⁺ channel exists in both ventricular and atrial SR membranes and provides counter ion transport for Ca²⁺ release (1, 107, 159). Activation kinetics are slow with open times of 100 ms (455). There is no inactivation process. Typical K⁺ channel blockers (4-aminopyridine, iberiotoxin, amiodarone) are without effect (420). Ca²⁺ and Mg²⁺ do not alter the channel's activity (455), but its P_o is reduced in low pH. The molecular identity of this protein is unknown at this time.

Additionally, a large-conductance (120 pS) Cl^– channel exists in SR membrane and can be also permeable to Ca²⁺ (516). This Cl^– channel's activity is altered with phosphorylation (125, 270, 458), and some have suggested that phospholamban modulates its conductance (125). The molecular identity of this protein remains unknown.

B) RYANODINE RECEPTORS AND CALCIUM OVERLOAD OF THE SR.  Spontaneous SR Ca²⁺ release was first observed by Fabiato and Fabiato (153) in the form of spontaneous oscillatory contractions in skinned fibers. The spontaneous contractions were initiated by loading the SR using low [Ca²⁺]; the [Ca²⁺] used for the loading was by itself insufficient to induce Ca²⁺ release. The observation that skinned myocyte fragments started to contract in an oscillatory fashion led to the concept that a heavily Ca²⁺-loaded SR is characterized by spontaneous Ca²⁺ release. The importance of this phenomenon is that spontaneous contractions, caused by cytosolic [Ca²⁺]_i oscillations (403, 588), are accompanied by spontaneous oscillations in current and membrane potential in both single myocytes as well as nondriven multicellular cardiac preparations (80, 264, 287). Agents that reduce Ca²⁺ load of the SR (e.g., ryanodine, caffeine, EGTA buffer) abolish spontaneous [Ca²⁺]_i oscillations as well as the oscillatory potentials, current, and contractions (4, 353, 5