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(Circulation Research. 2000;87:106.)
© 2000 American Heart Association, Inc.

Cellular Biology

Transmission of Information From Cardiac Dihydropyridine Receptor to Ryanodine Receptor

Evidence From BayK 8644 Effects on Resting Ca²⁺ Sparks

Hideki Katoh, Klaus Schlotthauer, Donald M. Bers

From the Department of Physiology, Loyola University Chicago, Maywood, Ill.

Correspondence to Donald M. Bers, PhD, Department of Physiology, Loyola University Medical School, 2160 S First Ave, Maywood, IL 60153. E-mail dbers{at}lumc.edu

	Abstract

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Abstract—Coupling between L-type Ca²⁺ channels (dihydropyridine receptors, DHPRs) and ryanodine receptors (RyRs) plays a pivotal role in excitation-contraction (E-C) coupling in cardiac myocytes, and Ca²⁺ influx is generally accepted as the trigger of sarcoplasmic reticulum (SR) Ca²⁺ release. The L-type Ca²⁺ channel agonist BayK 8644 (BayK) has also been reported to alter RyR gating via a functional linkage between DHPR and RyR, independent of Ca²⁺ influx. Here, the effect of rapid BayK application on resting RyR gating in intact ferret ventricular myocytes was measured as Ca²⁺ spark frequency (CaSpF) by confocal microscopy and fluo 3. BayK increased resting CaSpF by 401±15% within 10 seconds in Ca²⁺-free solution, and depolarization had no additional effect. The effect of BayK on CaSpF was dose-dependent, but even 50 nmol/L BayK induced a rapid 245±12% increase in CaSpF. Nifedipine (5 µmol/L) had no effect by itself on CaSpF, but it abolished the BayK effect (presumably by competitive inhibition at the DHPR). The nondihydropyridine Ca²⁺ channel agonist FPL-64176 (1 µmol/L) did not alter CaSpF (despite rapid and potent enhancement of Ca²⁺ current, I_Ca). In striking contrast to the very rapid and depolarization-independent effect of BayK on CaSpF, BayK increased I_Ca only slowly (=18 seconds), and the effect was greatly accelerated by depolarization. We conclude that in ferret ventricular myocytes, BayK effects on I_Ca and CaSpF both require drug binding to the DHPR, but postreceptor pathways may diverge in transmission to the gating of the L-type Ca²⁺ channel and RyR. (Circ Res. 2000;87:106-111.)

Key Words: Ca²⁺ channel • sarcoplasmic reticulum • excitation-contraction coupling • confocal microscopy • FPL-64176

	Introduction

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In cardiac muscle, Ca²⁺-induced Ca²⁺ release (CICR) from the sarcoplasmic reticulum (SR) is pivotal in excitation-contraction (E-C) coupling.^{1 2} In skeletal muscle, depolarization causes SR Ca²⁺ release, and the dihydropyridine receptor (DHPR) is thought to be the membrane potential sensor that transmits a direct intermolecular signal to the ryanodine receptor (RyR), causing SR Ca²⁺ release.^{3 4} In both muscle types, the DHPR and RyR are in relatively close physical proximity, but exactly how they communicate is unclear.

Spatially localized [Ca²⁺]_i elevations (Ca²⁺ sparks) at the sarcomere level can be detected by laser scanning confocal microscopy, allowing direct visualization of SR Ca²⁺ release events in cardiac myocytes.^{5 6 7 8 9} In cardiac muscle E-C coupling, it is generally thought that Ca²⁺ current (I_Ca) via single L-type Ca²⁺ channels (DHPRs) goes into a restricted space, triggering local SR Ca²⁺ release via RyRs.^{1 2} Ca²⁺ sparks evoked by I_Ca are believed to summate spatially and temporally, giving rise to the normal whole-cell twitch Ca²⁺ transient.^{6 7 8} Possible alterations in E-C coupling in hypertrophic and failing rat heart^{10 11 12} emphasize the importance of understanding the basis of cardiac DHPR-RyR interactions.

Evidence suggests that the intracellular loop between domains II and III of the skeletal muscle DHPR is important in transmitting a gating signal to the skeletal RyR.³ Peptides from this II-III loop can also alter ryanodine binding and RyR gating in skeletal RyR.^{13 14} The analogous cardiac II-III loop peptides also alter cardiac RyR gating in lipid bilayers and intact myocytes.¹⁵ This raises the possibility of a physical and/or functional link between cardiac DHPR and RyR.

BayK 8644 (BayK) is a dihydropyridine L-type Ca²⁺ channel agonist¹⁶ that can indirectly modulate resting RyR gating (ie, via DHPR-RyR interaction).^{17 18} BayK converts postrest potentiation to postrest decay in canine and ferret ventricular myocytes secondary to a rapid loss of SR Ca²⁺ during rest.^{17 18 19} The loss of SR Ca²⁺ at rest was found to be due to a dramatic increase in Ca²⁺ spark frequency (CaSpF) that occurred even in the complete absence of extracellular Ca²⁺ and could be competitively blocked by nifedipine. BayK had no effect on single isolated RyR channel gating in lipid bilayers.¹⁸ BayK also increases ryanodine binding in intact ventricular myocytes, but this effect was abolished after homogenization.¹⁷ Thus, an intact physical DHPR-RyR linkage may be needed for the effect of BayK on SR Ca²⁺ release.

Our working hypothesis is that BayK binds to DHPR and that this signal is transmitted to the RyR (independent of Ca²⁺ entry), increasing resting RyR opening and CaSpF. Here, we provide new information about (1) the kinetics of this effect of BayK, (2) whether I_Ca activation modulates the BayK effect, (3) whether FPL-64176 (FPL, a benzoylpyrrole Ca²⁺ channel agonist that does not compete at the DHPR) exerts the same effect, and (4) comparative kinetics and depolarization dependence of BayK on resting CaSpF versus I_Ca. We find that BayK rapidly increases resting CaSpF and is not mimicked by FPL and that the effect is independent of depolarization or Ca²⁺ entry. All 4 of these effects are in striking contrast to BayK effects on I_Ca, suggesting a divergent transduction pathway.

	Materials and Methods

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Ferret ventricular myocytes were prepared as described previously,²⁰ and experiments were performed at 22°C. Standard Tyrode’s solution contained (mmol/L) NaCl 140, KCl 6, MgCl₂ 1, HEPES 5, glucose 10, and CaCl₂ 2. In the 0Ca-0Na solution, LiCl replaced NaCl, CaCl₂ was omitted, and 10 mmol/L EGTA was added. The pH was adjusted to 7.4 with NaOH or LiOH. Cells were loaded with the Ca²⁺ indicator fluo 3 by exposure to 20 µmol/L fluo 3 acetoxymethyl ester (Molecular Probes) for 20 minutes at 22°C, with 30 minutes allowed for deesterification. Unless they were voltage-clamped, cells were field-stimulated (0.5 Hz) to steady state before cessation and quick solution switch (time constant, 300 ms) to 0Ca-0Na solution for Ca²⁺ spark measurement.

CaSpF was then measured during a 30-second rest period (15 images). After the first 10 seconds of rest in 0Ca-0Na/EGTA, the cell was field-stimulated for 10 pulses (at 1 Hz) to depolarize cells during exposure to the test solution. With Li⁺ replacing Na⁺ in this solution, action potentials are still readily activated.²¹ SR Ca²⁺ content was evaluated by rapid application of 10 mmol/L caffeine dissolved in 0Ca-0Na solution with 1 mmol/L EGTA to the cell via a quick-switcher.²¹

Confocal fluorescence imaging was performed as described^{9 18} with a laser scanning confocal microscope (LSM410, Zeiss) coupled to an inverted microscope (Axiovert 100, Zeiss) with an x40 oil-immersion objective (NA=1.3), excitation at a wavelength of 488 nm, and emission at >515 nm. Line scans (512 pixels/line, 0.25 µm/pixel) were acquired at 250 lines/s and were processed with IDL software (Research Systems) with [Ca²⁺]_i calculated^{5 9} with a fluo 3 K_d=1.1 µmol/L and resting [Ca²⁺]_i=150 nmol/L.^{22 23} Visually identified Ca²⁺ sparks were accepted if local [Ca]_i change (5 adjacent pixels) exceeded 60 nmol/L with duration at half-amplitude 8 ms.^{9 18} Ca²⁺ sparks counted per line scan image were normalized spatially (per µm³) and temporally (per second) as CaSpF (pL^-1xs^-1). Global [Ca²⁺]_i transients (depolarization- or caffeine-induced) were derived from average fluorescence intensities along the scanned line.

I_Ca was recorded by whole-cell ruptured-patch voltage clamp as described²⁴ with pCLAMP (Axon Instruments), filtered at 10 kHz, and sampled at 1 kHz. Patch electrodes had resistances of 1.0 to 1.5 M, with an internal solution composed of (mmol/L) CsCl 125, MgCl₂ 1, HEPES 20, EGTA 10, MgATP 10, and GTP 0.3 (pH 7.2). Cells were superfused with Tyrode’s solution in which KCl and NaCl were replaced by CsCl and TEA-Cl, respectively, to better isolate I_Ca. Action potentials were recorded in current-clamp mode with physiological pipette and bath solutions and higher-resistance electrodes (10 to 20 M).

Nifedipine (Sigma) and (±)BayK (Calbiochem) were dissolved in ethanol, and FPL (Alexis) was dissolved in DMSO (final ethanol and DMSO concentrations <0.1%). Caffeine was dissolved directly in 0Ca-0Na solution. Results were expressed as mean±SEM for the indicated number (n) of myocytes, and a value of P<0.05 was considered significant (Student’s t test).

	Results

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Rapid BayK Application: CaSpF and SR Ca²⁺ Content
To prevent Ca²⁺ influx from affecting CaSpF, we removed extracellular Ca²⁺ and added 10 mmol/L EGTA immediately after the interruption of electrical stimulation. Furthermore, to avoid any change in SR Ca²⁺ load or [Ca²⁺]_i at the beginning of the rest period (for control versus experimental), identical solutions and stimulation protocols preceded the rest. During rest, 0Ca-0Na inhibited Ca²⁺ extrusion via Na⁺/Ca²⁺ exchange and minimized SR Ca²⁺ loss.²⁰

Under control 0Ca-0Na/EGTA conditions, CaSpF during 30 seconds of rest remained nearly constant (Figures 1A and 2). Thus, CaSpF at resting membrane potential is not altered by complete removal of [Ca²⁺]_o. Furthermore, stimulation in 0Ca-0Na/EGTA did not produce any detectable changes in [Ca²⁺]_i. After this control measurement, Tyrode’s solution was restored and the cell stimulated to return to the initial steady state. When the protocol was repeated with 500 nmol/L BayK (Figures 1B and 2), CaSpF increased rapidly by 401±15% at maximum in <10 seconds (P<0.01, n=11). Field stimulation after 10 seconds of rest in BayK had no effect, indicating that the BayK effect on CaSpF was voltage-independent, in marked contrast to the effect of BayK on I_Ca.^{16 25}

View larger version (101K): [in this window] [in a new window]   Figure 1. Rapid BayK application increases resting Ca2+ sparks. Confocal line-scan images (along long cell axis) were obtained after 4, 12, and 28 seconds of rest after 1-Hz stimulation in control (A) and with 500 nmol/L BayK (B). Single line scans were stacked from left to right. Selected [Ca2+]i line plots are from marked sites.

View larger version (26K): [in this window] [in a new window]   Figure 2. BayK effects on CaSpF. A, Time course of CaSpF during 30 seconds of rest in control and in the presence of 500 nmol/L BayK. Data are mean±SEM, n=11, paired. Tick marks indicate 10 pulses at 1 Hz.

The lack of increased CaSpF on stimulation could have been due to the BayK effect being maximal already at 10 seconds with 500 nmol/L BayK. To test this possibility, BayK concentration was lowered to 50 nmol/L, which increased CaSpF by only 245±12% (P<0.01, n=6; Figure 3) at the maximum point. Depolarization still had no effect on CaSpF with 50 nmol/L BayK. There was no apparent change in time course of BayK effect on CaSpF between 50 and 500 nmol/L BayK. These results suggest that the peak BayK effect on CaSpF was dose-dependent and rapid, but independent of both depolarization and Ca²⁺ influx.

View larger version (26K): [in this window] [in a new window]   Figure 3. Low-concentration BayK also alters CaSpF. Same as Figure 2 except 50 nmol/L BayK was used (n=6, paired). Tick marks indicate 1-Hz stimulation.

To measure SR Ca²⁺ content, we applied caffeine (10 mmol/L) either after the last steady-state pulse or after the 30-second rest period (±500 nmol/L BayK). Figure 4 shows that after 30 seconds in control 0Ca-0Na/EGTA, there was a small (5%) loss of SR Ca²⁺ content (versus steady state). In the presence of BayK, the SR Ca²⁺ content in 0Ca-0Na/EGTA was 16% lower than steady state and significantly lower than control. This is consistent with previous findings on control SR Ca²⁺ loss in ferret myocytes²⁰ and also with equilibrium exposure to BayK,¹⁸ in which the faster loss of resting SR Ca²⁺ content with BayK is due to the higher CaSpF.

View larger version (27K): [in this window] [in a new window]   Figure 4. BayK accelerates rest-dependent loss of SR Ca2+. Peak [Ca2+]i of caffeine-induced [Ca2+]i transient was used as a measure of SR Ca2+ content. Caffeine (10 mmol/L) was applied rapidly in 0Ca-0Na/EGTA solution 1 second after steady-state stimulation (SS) and after 30 seconds of rest in the absence (control, Ctl) and presence of BayK (n=6, paired *P<0.01).

BayK Effect on RyR Gating Is Mediated via Dihydropyridine Receptor
Next, we tested whether the BayK effect could be inhibited by nifedipine competition at the DHPR. Figure 5A illustrates that when 500 nmol/L BayK and 5 µmol/L nifedipine were included together in the test solution, the effect of BayK on CaSpF was completely abolished (see also Reference ¹⁸ ). Rapid application of 5 µmol/L nifedipine alone did not alter CaSpF (Figure 5B). These results indicate that the BayK effect on RyR is mediated via the DHPR. Figure 5A shows that action potentials were still activated in 0Ca-0Na solution and also when BayK and nifedipine are included (although plateaus are lower than control, because inward I_Ca and Na/Ca exchange current are prevented).

View larger version (30K): [in this window] [in a new window]   Figure 5. Nifedipine inhibits BayK effect on CaSpF but has no effect alone. A, CaSpF during 30 seconds in control and with 500 nmol/L BayK plus 5 µmol/L nifedipine (n=5, paired). Inset shows action potentials recorded in control and in 0Ca-0Na with or without 500 nmol/L BayK plus 5 µmol/L nifedipine. B, Same as in A, but in the absence and presence of 5 µmol/L nifedipine (n=7, paired). In these experiments, [Ca2+]o before 0Ca-0Na was 3 mmol/L (rather than 2 mmol/L) to improve detection of possible inhibitory effect of nifedipine on CaSpF (control CaSpF was slightly higher than in Figures 2, 3, and 5A).

To further test the hypothesis that the DHPR, rather than altered Ca²⁺ channel gating, mediates the BayK effect on CaSpF, we used FPL, another potent L-type Ca²⁺ channel agonist with effects on I_Ca similar to those of BayK. In contrast to BayK, FPL does not compete at the dihydropyridine binding sites, indicating that FPL activates I_Ca at a site distinct from dihydropyridines.^{26 27} Steady-state exposure to 1 µmol/L FPL doubled I_Ca amplitude at 0 mV (from 8 to 16 A/F) and shifted peak I_Ca from +10 to -10 mV (Figure 6A). FPL also altered I_Ca activation and inactivation and caused large tail currents even at the first pulse after 10 seconds of exposure to FPL (Figure 6B). These FPL effects on cardiac I_Ca confirm previous reports.^{26 27}

View larger version (25K): [in this window] [in a new window]   Figure 6. The nondihydropyridine FPL-64176 increases ICa but not resting CaSpF. A, Peak ICa current-voltage relationship at steady-state FPL exposure (holding Em=-80 mV). B and C, FPL effects on ICa after 10 seconds of perfusion at rest (pulses from -80 to 0 mV for 150 ms at 1 Hz) were fit to a single exponential curve (n=4). D, CaSpF as a function of rest duration in the absence and presence of 1 µmol/L FPL (n=6, paired).

After resting cells had been exposed to FPL for 10 seconds, 10 pulses to 0 mV (1 Hz) further enhanced the FPL effect on I_Ca amplitude (Figures 6B and 6C). I_Ca amplitude was already 80% of maximum at the first pulse and gradually increased to 99% of maximum (achieved after 1 minute at 1 Hz).

Figure 6D shows that despite the dramatic changes of I_Ca, rapid application of FPL in 0Ca-0Na/EGTA caused no detectable change in CaSpF during the same protocol as used for BayK in Figures 2 and 3. These results indicate that FPL binds to Ca²⁺ channels during rest but does not alter RyR gating during rest or depolarization. This supports the idea that BayK binding to the DHPR may be the first, essential step for the BayK effect on RyR gating during rest (and FPL cannot mimic BayK).

Rapid BayK Effects on I_Ca
Figure 7A shows I_Ca traces before and after exposure to 500 nmol/L BayK for 5, 10, and 20 seconds before depolarizations. The first pulse after 5 seconds of BayK perfusion showed larger I_Ca, but I_Ca increased progressively during the following 9 pulses. Longer resting exposure to BayK enhanced I_Ca amplitude more markedly at the first pulse. Steady-state current-voltage relationships (Figure 7C) show that BayK increased I_Ca amplitude at 0 mV from 8.7±1.2 to 20.3±1.7 A/F and shifted peak I_Ca from +10 to 0 mV. BayK altered I_Ca activation and inactivation, and these BayK effects on I_Ca agree with previous work.^{16 27 28}

View larger version (27K): [in this window] [in a new window]   Figure 7. Effect of BayK on ICa. A, ICa measured before (Ctl) and after exposure to 500 nmol/L BayK for indicated rest durations (voltage steps from -80 to 0 mV for 150 ms at 1 Hz). B, Rest and depolarization dependence of ICa increase after BayK exposure (n=4 to 6, paired) are fit to single-exponential functions (=18 seconds for the dotted and 2 to 4 seconds for the solid lines). C, Peak ICa current-voltage relationship at steady-state BayK exposure (holding Em=-80 mV).

BayK application induced both rest-dependent and depolarization- (or pulse-) dependent effects on I_Ca amplitude (Figure 7B). Rest-dependent I_Ca activation was slow (=18 seconds) and incomplete until 1 minute. The depolarization-dependent increase of I_Ca was much faster (=2.5 to 4 seconds). The maximal BayK effect on resting CaSpF was achieved within 10 seconds (Figure 2), whereas at this time the BayK effect on I_Ca was only 43%. Furthermore, the BayK effect on CaSpF (RyR gating) was independent of depolarization, but the BayK effect on I_Ca was strongly depolarization-dependent. These results revealed marked differences between BayK effects on CaSpF and I_Ca.

	Discussion

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In 4 major ways, we extend our original steady-state findings that BayK increases resting SR Ca²⁺ release.^{17 18} We measured (1) onset kinetics of BayK effects on CaSpF and I_Ca, (2) depolarization dependence of BayK effects on CaSpF and I_Ca, (3) whether the nondihydropyridine Ca²⁺ channel agonist FPL could mimic BayK, and (4) disparities in kinetics and depolarization dependency of BayK effects on CaSpF and I_Ca. BayK effects on CaSpF were maximal within 10 seconds and were unaffected by depolarization, whereas effects on I_Ca were slower in onset and highly depolarization-dependent. Both effects of BayK could be blocked by the dihydropyridine nifedipine. However, the nondihydropyridine FPL did not alter resting CaSpF, despite increasing I_Ca.

Kinetics of BayK Effect on Resting CaSpF
BayK was applied in 0Ca-0Na/EGTA solution to rule out possible CICR, which could have been enhanced by BayK at negative membrane potential. To reduce transverse tubule [Ca²⁺] to negligible levels in our conditions requires 1 second.^{18 29 30} This does not affect our conclusions here, because resting Ca²⁺ sparks are not due to Ca²⁺ influx, CaSpF is unaltered in 0Ca-0Na/EGTA, and all comparisons are at times >2 seconds.¹⁸ Nevertheless, this geometric constraint limits temporal resolution such that we can only claim BayK effects on CaSpF as maximal in <10 seconds (for both 50 and 500 nmol/L BayK).

Rampe et al³¹ found biphasic BayK association and dissociation rates in canine sarcolemma, with a rapid association rate constant [k_on 2.2x10⁶ (mol/L)^-1 · s^-1]. On the basis of this k_on (and k_off=0.012 s^-1),³¹ our 10-second exposure to BayK would result in 99% and 65% saturation with 500 and 50 nmol/L BayK, respectively (in line with our time course of BayK effect on the CaSpF). Ca²⁺ agonist effects are also less dependent on Ca²⁺ channel state than is the case for Ca²⁺ antagonist.³²

BayK Binding to DHPR Is Necessary for BayK Effect on RyR Gating
Nifedipine (5 µmol/L) inhibited the BayK effect on CaSpF (Figure 5A) but did not alter CaSpF by itself (Figure 5B). This indicates that BayK binding to the DHPR is necessary for altering RyR gating and agrees with steady-state findings.¹⁸ To further test this hypothesis here, we used the Ca²⁺ channel agonist FPL, which does not compete for binding at the DHPR.²⁶ Although BayK and FPL produce comparable effects on I_Ca (Figures 6 and 7), FPL had no effect at all on resting CaSpF. This has 2 relevant implications: (1) BayK binding to the DHPR is an essential step in altering RyR gating and (2) similar alterations in Ca²⁺ channel gating properties are not sufficient to mimic the effect of BayK on CaSpF.

Although BayK could have direct effects on the RyR, our previous data showed no effect on single-channel RyR current amplitude or open probability in lipid bilayer experiments.¹⁸ Although BayK increased ryanodine binding to intact ferret ventricular myocytes, mechanical disruption of SR-sarcolemmal junctions eliminated the effect.¹⁷ BayK also had no influence on SR Ca²⁺ release in skinned guinea pig atrial fibers.³³ Thus, it seems that the effect of BayK on RyR is mediated by the DHPR and a Ca²⁺-independent connection between these receptors.

BayK Effect on RyR Gating Was Depolarization-Independent
The effect of BayK on CaSpF was not influenced by depolarization, in sharp contrast to the effect on I_Ca. Indeed, voltage- and use-dependent effects of dihydropyridines (including BayK) on I_Ca are classically observed.^{25 34} BayK has also been reported to alter gating charge movement attributed to cardiac Ca²⁺ channels.^{35 36} Because the BayK effect on CaSpF was maximal during rest (when no charge movement occurs), it seems unlikely that the BayK effect on CaSpF was mediated by gating charge movement. Because depolarization did not alter CaSpF (or [Ca²⁺]_i), we infer that depolarization per se did not trigger the release of Ca²⁺ from SR under our experimental conditions. Interestingly, BayK may also cause depolarization-independent SR Ca²⁺ release in skeletal muscle.³⁷

Our working hypothesis (Figure 8) is that BayK binding to the DHPR could facilitate protein conformational changes to alter L-type Ca²⁺ channel gating in a manner that depends on 1 gating cycles (eg, providing access to additional interaction sites). In contrast, the DHPR may transmit a physical signal to the RyR that is independent of I_Ca gating or depolarization (and rapid at rest). Our results cannot distinguish whether or not an intermediate protein () is involved (eg, sorcin can bind to both DHPR and RyR and alter RyR gating^{38 39} ). Conversely, direct DHPR-RyR effects cannot be ruled out, because cardiac DHPR peptides can alter RyR gating in both bilayers and intact voltage-clamped myocytes.¹⁵ Thus, the initial step of BayK binding to the DHPR may be the same for both I_Ca and CaSpF, but the functional pathways may diverge between the DHPR and the effector site.

View larger version (36K): [in this window] [in a new window]   Figure 8. Model for BayK-mediated effects on ICa and RyR gating. See text for details.

In ventricular myocytes, there are 4 to 10 times as many RyRs as there are DHPRs.⁴⁰ Thus, even if all DHPRs were coupled in this relatively direct way to RyRs, that would include only 10% to 25% of RyRs. However, activation of 1 RyR may cause sufficiently high local [Ca²⁺] to activate a whole cluster of RyRs via CICR, resulting in a Ca²⁺ spark. Thus, RyRs coupled to DHPRs may have gating properties different from those of uncoupled RyRs. Niggli⁴¹ suggested such a scenario to explain differential triggering of Ca²⁺ sparks.

It is unclear how the functional DHPR-RyR linkage discussed here might alter E-C coupling. BayK actually depresses E-C coupling, ie, less SR Ca²⁺ release for a given I_Ca and SR Ca²⁺ load.^{28 42} This could be due to altered Ca²⁺ responsiveness of the RyR. However, this could also be explained by long single-channel openings induced by BayK and the relatively rapid activation of SR Ca²⁺ release, such that there is wasted I_Ca (that does not trigger Ca²⁺ release). Our data do not indicate any purely voltage-induced Ca²⁺ release,⁴³ because no Ca²⁺ increase accompanied depolarization in 0Ca-0Na solution (in any condition). The BayK-induced increase of resting Ca²⁺ sparks (and RyR gating) is from an extremely low resting probability (0.0001),⁵ whereas the huge CICR during E-C coupling might override this BayK effect. We speculate that the DHPR-RyR interaction responsible for the BayK-induced Ca²⁺ sparks is weak compared with that in skeletal muscle and that its main physiological importance may be to help colocalize these 2 important Ca²⁺ channels in heart.

	Acknowledgments

 
This study was supported by grants from the NIH (HL-30077), the American Heart Association Metropolitan Chicago affiliate, and the Japanese Heart Association. We thank Steve Scaglione and Sarah Wimbiscus for expert technical assistance.

Received March 27, 2000; accepted May 24, 2000.

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