1Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, 833 34 Bratislava, Slovak Republic; and 2Department of Physiology, Texas Tech University Health Sciences Center, Lubbock, Texas 79430
Submitted 31 March 2003 ; accepted in final form 24 June 2003
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ABSTRACT |
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excitation-contraction coupling; cardiac myocytes; magnesium; calcium signaling
In the present study, we explored the effects of Mg2+ on dynamic properties of RyRs reconstituted in lipid bilayers (19, 51, 57). The activity of the channel in response to rapid photolytically induced changes in free Ca2+ concentration was assessed in the presence of ATP and various concentrations of free Mg2+ (0.6-1.3 mM). Our study showed that binding of Mg2+ to the Ca2+ activation sites slowed the kinetics of RyR activation and dramatically reduced the probability of activation by brief physiologically relevant Ca2+ stimuli. We conclude that Mg2+ is a key determinant of the dynamic behavior of the RyR channel that is expected to have a major impact on local Ca2+ signaling between the L-type Ca2+ channels and RyRs in the heart.
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METHODS |
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Flash photolysis experiments. Fast changes of the Ca2+ concentration in the microenvironment of the reconstituted channel were performed by flash photolysis of caged Ca2+, as described previously (19, 20, 57), except, in the present study, we used the Ca2+ cage compound nitrophenyl (NP)-EGTA (Molecular Probes, Eugene, OR) instead of DM-Nitrophen. NP-EGTA is highly selective for Ca2+ over Mg2+ (12) and, thus, permitted us to conduct experiments in the presence of various Mg2+ concentrations. After incorporation of a single RyR, NP-EGTA (added to a final concentration of 2.25-3 mM) containing the appropriate amount of CaCl2 (Orion) was added to the cytoplasmic (cis) side of the channel. The amount of CaCl2 in the NP-EGTA-Ca2+ mixture was adjusted to attain 0.1-1.5 µM free Ca2+ in the cis-solution. Intense (0.5- to 2.5-mJ) and brief (9-ns) UV laser flashes produced by a pulsed, frequency-tripled Nd:YAG laser (Spectra-Physics, Mountain View, CA) were applied through fused silica optical fibers (600 µm diameter) positioned in front of the bilayer (100 µm diameter). Thus the whole volume between the optical fibers and the bilayer was illuminated evenly and instantaneously.
The concentration of steady-state free Ca2+ in the cis- chamber was determined with a Ca2+-selective minielectrode (20). The local Ca2+ changes near the bilayer were calibrated by transformation of the bilayer aperture to a Ca2+ electrode with the use of Ca2+ ionophore resin (ETH 129, Fluka), as described previously (20, 57). The potential of the Ca2+ electrode was measured using a patch-clamp amplifier (Axopatch 200A, Axon Instruments) in current-clamp mode.
Fluorescence measurements of Ca2+ concentration with Rhod-5N. To validate our theoretical reconstructions of the photolytic Ca2+ signals, we performed fluorescence measurements of Ca2+ by using pulsed local field fluorescence microscopy (36) and the rapid-response Ca2+ indicator Rhod-5N (Molecular Probes). To measure Rhod-5N fluorescence, the tip of a small-diameter (400-µm) short-working-distance multimode optical fiber (3M) was micropositioned at the edge of the volume illuminated by the UV laser used for flash photolysis. Excitation light generated by a pulsed laser (Nanolase, Uniphase, San Jose, CA; 532-nm wavelength, 900-ps pulse duration, 12-kHz repetition rate, 400-W peak power) was applied through the fiber to the experimental solution. Emitted fluorescence was collected through the same optical fiber, passed through a barrier filter, and measured by an avalanche photodiode (EGG). The signal was digitized with an analog-to-digital converter (model PCI 6110, National Instruments, Austin, TX) at 500-kHz sampling frequency and 125-kHz bandwidth. The Ca2+ sensitivity and Ca2+ off-rate of Rhod-5N (Table 1) were determined fluorometrically according to Escobar et al. (14), giving an approximate value for the Ca2+ on-rate of the dye of
10,000 M-1 · s-1. The high value for the dissociation constant of Rhod-5N suggests that, at 0.1-250 µM Ca2+, the indicator behaves in a linear fashion. However, the intermediate rate of Ca2+ binding might underestimate the peak value of Ca2+, and the fast, but finite, rate of Ca2+ unbinding might overestimate the duration of the Ca2+ spike.
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To examine the extent to which the measurement of peak Ca2+ concentration on NP-EGTA photolysis is affected by the kinetics of Rhod-5N, we have measured the fluorescence in response to a series of 10 photolytic stimuli that photolyzed 4.5% of NP-EGTA per stimulus. Steady-state fluorescence before the flash and peak fluorescence in response to the flash are plotted in Fig. 1A. The amplitude and time course of free Ca2+ concentration signals and of dye fluorescence in response to the photolyzing laser pulses were computed in Mathematica (version 4.2, Wolfram Research, Champaign, IL) from steady-state Ca2+ concentrations before and after the flash and from the concentration of total NP-EGTA (57) with the use of the kinetic parameters from Table 1. The steady-state fluorescence calculated from the measured Ca2+ sensitivity of Rhod-5N and from the photolysis fraction of 4.5% was in a very good agreement with the measurements. The values of peak fluorescence after photolysis were compared with the theoretical values on the basis of three assumptions: 1) instantaneous photolysis and Ca2+ binding by the dye, 2) realistic kinetics of photolysis and of NP-EGTA complexation (13) but instantaneous Ca2+ binding by the dye, and 3) realistic kinetics of all processes (all values from Table 1). The finite rate of photolysis decreased the peak Ca2+ concentration quite evenly in the whole range. The finite rate of Ca2+ binding to Rhod-5N led additionally to a significant (up to 50%) underestimation of peak Ca2+, especially at lower steady-state Ca2+ values. However, the peak fluorescence was in good agreement with predictions for which the values of on- and off-rates of Ca2+ binding to Rhod-5N were used (see above).
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Figure 1B shows Rhod-5N fluorescence transients induced by flash photolysis of caged Ca2+ in the absence and presence of 3 mM MgATP and in the presence of 0.6 mM Na2ATP at a high time resolution. The measured fluorescence signals are overlaid with the theoretically reconstructed fluorescence of the Ca2+-Rhod-5N complex together with the estimated Ca2+ concentration transients that would occur under the same conditions but in the absence of Rhod-5N. The calculations were performed using the kinetic parameters presented in Table 1. The theoretically predicted fluorescence traces corresponded well with the experimentally measured data. Because of the finite rates of binding and unbinding of Ca2+ from the indicator, the time course of the fluorescence transient was considerably slowed with respect to the Ca2+ signal. The depression of the Rhod-5N fluorescence and Ca2+ signals in the presence of ATP could be attributed to the buffering action of ATP. Taken together, these results suggest that the brief photolytic Ca2+ spikes cannot be reliably measured, even with the fastest available Ca2+ indicators. However, the amplitude and the time course of these signals can be reconstructed with reasonable accuracy from the preand postflash steady-state free Ca2+ concentration near the bilayer surface with the use of the rate constants for Ca2+ binding and photolysis of NP-EGTA.
Data analysis. Experimental data were analyzed with the program Origin (version 7.0, OriginLab, Northampton, MA) on a personal computer. Values are means ± SE.
Free concentrations of ions and ligands in mixtures were calculated with the WinMaxC program (version 2.40; http://www.stanford.edu/~cpatton) (3).
The apparent Ca2+ sensitivity of peak Po (Popeak) at different Mg2+ concentrations in response to a Ca2+ stimulus was described according to Laver et al. (27) as follows
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The upper limit of probability of RyR activation (ps) for experiments that yielded no active responses was calculated from the cumulative binomial distribution as follows
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RESULTS |
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To determine how the presence of luminal Ca2+ affects activation of RyRs in response to a Ca2+ increase, we compared the peak Po in response to sustained Ca2+ elevations to 1.4, 3.5, and 10 µM in the presence of 3 mM ATP and 0.6 mM free Mg2+ in the absence and presence of 1 mM luminal Ca2+. Table 2 illustrates that luminal Ca2+ did not have a significant effect on peak Po at any of the sustained Ca2+ levels. To determine how the effect of luminal Ca2+ on the steady-state activity of RyRs is achieved, we compared the time courses of Po in response to Ca2+ elevations to 3.5 µM in the absence and presence of luminal Ca2+ at a longer time scale. Figure 3 and Table 2 show that luminal Ca2+ increased Po at the end of the episodes (1.1 s after the onset of the Ca2+ stimulus) to 300% of control. This difference was due to a 50% decrease in the adaptation rate as well as an increase in the steady-state Po, which was quite prominent (5 times) at a sustained Ca2+ concentration of 3.5 µM. Although in the absence of luminal Ca2+ the decrease of RyR Po was relatively fast and complete, as previously observed (51), it was much slower and more incomplete in the presence of luminal Ca2+.
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Mg2+ effects on nonstationary RyR Ca2+ dependency. Previous studies performed at steady state demonstrated that Mg2+ can inhibit RyR activity by at least two mechanisms (25, 27): 1) displacing Ca2+ from the high-affinity Ca2+ activation site(s) and 2) acting at the low-affinity Ca2+ inhibition site(s).
The action of Mg2+ on the activation of RyRs can be visualized in experiments employing two consecutive sustained Ca2+ stimuli. An example of such an experiment in the presence of two different free Mg2+ concentrations (0.6 and 0.94 mM) is presented in Fig. 4. To optimally activate RyR in the presence of 0.6 mM Mg2+, Ca2+ concentration was increased photolytically from 1.5 µM to a sustained level of
10 µM in the presence of 1 mM luminal Ca2+. The second flash produced an incremental increase in Ca2+ concentration from
10 to
60 µM. The first stimulus resulted in a rapid and large increase in Po (
5ms, Po
0.64). Elevation of Ca2+ concentration by the second flash produced only a slight additional increase in Po, indicating that the channel had already been almost fully activated by the first Ca2+ stimulus. Increasing Mg2+ concentration to 0.94 mM dramatically reduced the rate and extent of RyR activation (to
15 ms and Po
0.17) by the primary Ca2+ stimulus (Fig. 4B). However, the second stimulus produced a much greater increase in Po than in the case with lower Mg2+ concentration. This result suggests that the apparent sensitivity of the RyR was reduced by elevated Mg2+ concentration because of competition between Ca2+ and Mg2+ for the activation sites. The slowed time course of channel activation at higher Mg2+ concentration would then reflect the dynamics of reequilibration of Ca2+ and Mg2+ on activation sites during the Ca2+ stimulus. The reduction of the maximum Po during the second Ca2+ stimulus is likely to reflect stationary inhibition at the low-affinity Ca2+ inhibition sites by elevated Mg2+ concentration.
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We have attempted to describe the dependence between the peak Po, Ca2+ concentration, and Mg2+ concentration by the model of Laver et al. (27). The best fit of the whole data set by Eq. 1, the parameters of which are given in Table 3, is shown in Fig. 4C. Although the curves were in qualitative accordance with the observations, the relatively large 2 value of the fit by this model had only a chance of P = 0.045 to arise as a result of random deviations from the model. In addition, the errors of the model parameters were larger than their values. Therefore, we attempted to fit the data by a simpler model, assuming the same mechanisms of Mg2+ action but describing the interaction of Ca2+ and Mg2+ at the Ca2+ activation sites by the Hill equation for the simultaneous action of an agonist and a competitive antagonist (Eq. 2). The best fit of the data by Eq. 2 is shown in Fig. 4C. The probability that the deviations between the model and the data were due to random processes was P = 0.61. The model also provided acceptable parameter errors. The probability that Eq. 2 is a better description of the data than is Eq. 1 was P = 0.90. Thus, consistent with the results of previous studies at steady state (25, 27), the responses of RyRs to rapid and sustained Ca2+ elevations are inhibited by Mg2+ at Ca2+ activation and inhibition sites. However, a phenomenological model of competition between Ca2+ and Mg2+ that does not specify distinct binding sites but, instead, describes the cooperativity of binding by an apparent Hill slope gives a better description of the effect of Mg2+ on RyR activation by Ca2+.
Taken together, the results of these experiments indicate that Mg2+ at submillimolar concentrations has a dramatic impact on the kinetics of the RyR response to elevations in Ca2+ concentration. Apparently, the binding of Mg2+ to the Ca2+ activation sites reduces the availability of the sites to Ca2+, thereby reducing the magnitude and slowing the dynamics of the response. In addition, a steady-state inhibition at the low-affinity inhibition sites is likely to contribute to the overall decrease in RyR Po by elevated Mg2+ concentration. This latter Mg2+ inhibition, however, would not be expected to have an impact on the kinetics of the RyR response to rapid Ca2+ concentration changes.
Mg2+ effects on RyR responses to brief Ca2+ spikes. The profound effects of Mg2+ on RyR kinetics raise the question of whether the Mg2+-bound RyR channel is sufficiently fast to track the rapid Ca2+ changes associated with single L-type Ca2+ channel openings under conditions of normal intracellular Mg2+ and ATP concentrations. To directly address this question, we investigated the impact of 0.6-1.3 mM free cytosolic Mg2+ on the ability of brief photolytically induced Ca2+ spikes to activate the RyR channel in the presence of 3 mM total ATP. In the absence of Mg2+ and ATP, RyRs responded to brief Ca2+ spikes in a manner similar to the response we observed previously using the Ca2+ caging compound DM-Nitrophen (57). As shown in Fig. 5A, rapid and brief (full duration at half-amplitude 70 µs) elevations of Ca2+ concentration from a basal level of
0.15 µM to a peak of
100 µM activated RyR to a peak Po of
0.3, consistent with the results of our previous studies (57). Owing to the buffering action of ATP, addition of 3 mM MgATP (
0.6 mM free Mg2+)to the cis-chamber caused a nearly twofold decrease in the amplitude of the brief Ca2+ stimulus and a similar increase in its duration without a measurable change in the area under the Ca2+ transient (10 µM · ms in both cases). Under these conditions, the shape of the Ca2+ stimulus and its duration and time integral were still comparable to those that have been shown to activate RyRs in the absence of Mg2+ (57). However, RyR activation in response to these Ca2+ stimuli was not observed in this and two other experiments (a total of 147 "null" episodes; Table 4), putting an upper limit for probability of RyR activation under these conditions to <2.1% at the significance level of P = 0.05 (see Eq. 3). A much higher decrease of the stimulus amplitude to 20% of control induced by addition of 3 mM ATP in the absence of Mg2+ decreased the probability of RyR activation to 36% of control (0.09 ± 0.03, data not shown), i.e., less than three times. Therefore, only a minor part of the inhibition of channel activity could have been due to changes in the shape of the Ca2+ stimulus. An increase in the basal Ca2+ concentration to
0.3 µM was required to record RyR responses at a reasonable rate (>5%) at this Mg2+ concentration (i.e., 0.6 mM). We found that photolytic elevations of Ca2+ from a basal level of
0.3 µM to a peak amplitude of
65 µM activated RyRs with
10% probability in the presence of 3 mM MgATP (
0.6 mM free Mg2+; Fig. 5). Luminal Ca2+ did not have a significant effect on the probability of activation (Table 4), consistent with the absence of its effect on the peak probability to sustained Ca2+ elevations. Raising free Mg2+ in the cis- chamber to
0.94 mM reduced the probability of active RyR response to the stimulus to <1% without significantly affecting the amplitude and time course of the Ca2+ stimulus. Adding more Mg2+ to the cis-chamber (
1.3 mM free Mg2+) led to a complete loss of RyR responsiveness to the Ca2+ spike (not shown). We conclude that the ability of RyR to respond to rapid Ca2+ spikes is highly sensitive to the presence of Mg2+. These results also suggest that unbinding of Mg2+ from the Ca2+ activation sites of the RyR may be too slow to allow Mg2+-bound RyR channels to respond to brief elevations in Ca2+ concentration produced by openings of single L-type Ca2+ channels in the presence of physiological levels of MgATP (see DISCUSSION).
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When RyRs did respond to the Ca2+ stimuli, the parameters of the responses and the time course of the ensemble averages were not significantly affected by the presence of Mg2+ and ATP. As shown in Fig. 5, C and D, the latency and mean duration of unitary events were not significantly different under control conditions and in the presence of 3 mM MgATP. The activation and deactivation time constants of the ensemble averages were not affected significantly by the presence of MgATP (Table 4). The lack of effects of Mg2+ on the properties of unitary events triggered by Ca2+ spikes suggests that the brief Ca2+ stimulus can activate RyR channels only when Mg2+ is not bound to the Ca2+ activation sites of the channels.
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DISCUSSION |
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In the presence of 3 mM ATP and 1 mM luminal Ca2+, the peak Po of RyR activation in response to sustained stimuli could be quantitatively described by independent action of Mg2+ at the Ca2+ activation site (KMg = 0.25 mM, nH = 2) and at the low-affinity inhibition site (KI = 0.76 mM). Under these conditions, Mg2+ dramatically slowed the rate of activation of RyRs in response to sustained Ca2+ stimuli (Fig. 2). This is expected as a result of competitive interactions between Ca2+ and Mg2+ at the activation sites of the channel. The slowed kinetics of RyR activation in the presence of Mg2+ reflects the dynamics of reequilibration of Ca2+ and Mg2+ at the activation sites during sustained elevations in Ca2+ concentration. At the same time, Mg2+ had no measurable impact on the latency and time course of the RyR response induced by brief Ca2+ stimuli but, instead, markedly reduced the probability of activation of the channels by brief Ca2+ stimuli (Fig. 5). The absence of changes in the mean open time of channels activated in the presence and absence of MgATP might be due to the presence of solely H-mode openings after rapid activation (57) as well as the absence of modulation of RyR gating by ATP in the presence of Mg2+ (9). The lack of changes in the latency of the RyR response to Ca2+ spikes indicates that the brief Ca2+ signals can activate RyR only when the channel is Mg2+ free at the time of the stimulus. It appears that, once bound, Mg2+ remains associated with the RyR activation sites significantly longer than the duration of the Ca2+ spike (0.2 ms), thus effectively blocking the access of Ca2+ to the sites during the Ca2+ stimulus. The strong dependency of RyR responses on Mg2+ concentration is consistent with the previous reports that RyR activation requires simultaneous binding of multiple (i.e., up to 4) Ca2+ ions to their binding sites (28, 57), so that the occupancy of even one Ca2+ binding site by Mg2+ may prevent channel activation. Therefore, our results suggest that the ability of RyRs to respond to physiologically relevant brief Ca2+ stimuli in the presence of 0.5-1 mM Mg2+ is far from optimal. Because competition between Ca2+ and Mg2+ is the mechanism responsible for changes in the kinetics of RyR activation (Fig. 4, Table 3), RyR responsiveness will be modulated by the ratio of free Ca2+ concentration to Mg2+ concentration in the vicinity of the channel. Raising Mg2+ concentration or lowering Ca2+ concentration will result in increased occupancy of the Ca2+-binding sites by Mg2+ and reduced fraction of channels available for activation by the brief Ca2+ pulse and, consequently, in reduced probability of activation. On the other hand, lowering Mg2+ concentration or elevating Ca2+ concentration will result in reduced occupancy of the sites by Mg2+ and increased probability of channel activation.
Luminal Ca2+ did not affect the probability of RyR activation in response to sustained or brief Ca2+ stimuli, suggesting that it has no significant effect on the rate of dissociation of Mg2+ from the cytosolic Ca2+ activation sites or on the Ca2+ sensitivity of RyR-binding sites. On the other hand, luminal Ca2+ had a profound effect on RyR kinetics at a slower time scale, affecting the spontaneous decrease of Po during the maintained Ca2+ elevation, a process known as adaptation (19, 20, 51). The rate of adaptation, which was fast in the absence of luminal Ca2+, as previously observed (51), was decreased by luminal Ca2+. In addition, luminal Ca2+ dramatically increased the steady-state Po; i.e., it decreased the degree of adaptation. The effect of luminal Ca2+ on the rate and extent of adaptation support the role of luminal Ca2+ in Ca2+ release termination at the single-channel level (17, 31, 32, 46, 49). Previously, it has been shown that the steady-state effects of luminal Ca2+ transpire as an increase of Ca2+ sensitivity as well as of maximum Po of RyRs (18). Because adaptation was shown to proceed as a shift from the high-Po H mode to the low-Po L and I modes (42, 55), it can be speculated that luminal Ca2+ shifts the steady-state equilibrium between modes in favor of the H mode; alternatively, it could increase Po within the L mode.
Our findings about the effect of Mg2+ on RyR activation by physiologically shaped Ca2+ stimuli have important ramifications for understanding EC coupling in vivo. It has been suggested that RyRs in the SR are locally controlled by the activity of single L-type Ca2+ channels in the juxtaposed t-tubule membrane (see introduction). Our studies indicate that the unbinding of Mg2+ from the Ca2+ activation sites of the RyR channel might be too slow to allow the channels that happen to be Mg2+ bound to respond to openings of single L-type Ca2+ channels and that the fraction of these channels is highly significant in the presence of physiological concentrations of Mg2+ (1 mM). Thus, if our data are considered, it is likely that the majority of L-type Ca2+ channel openings will not activate Ca2+ release sites in cardiac myocytes, consistent with the finding of Zhou et al. (58) that, under physiological conditions, only 1 of
50 L-type Ca2+ channel openings triggers Ca2+ release. An alternative possibility to explain our findings is that the Mg2+ sensitivity of RyRs is lower inside the cell than in the bilayer experiments, e.g., because of phosphorylation of RyRs by protein kinase A, which has been shown to remove the inhibitory effect of Mg2+ on steady-state RyR activity (50). This would result in a lower occupancy of the RyR Ca2+ activation sites by Mg2+ under conditions of normal basal intracellular Ca2+ and Mg2+ concentrations and in a higher ability of the rapid Ca2+ stimulus to activate the RyRs. This possibility, however, is not very likely, if it is considered that the steady-state Ca2+ sensitivity of RyRs in heart cells (apparent KD
15 µM) (4, 31) has been estimated to be close to that determined in lipid bilayers in the presence of millimolar MgATP (apparent KD
20 µM) (18).
Our results suggest that Ca2+ stimuli of any amplitude lasting less than the lifetime of the Mg-RyR complex have a reduced chance to activate RyR. Therefore, if it is not long enough, simultaneous opening of multiple dihydropyridine receptor (DHPR) channels would not increase the probability of RyR activation, despite increasing the local Ca2+ concentration. On the other hand, consecutive DHPR channel openings increase the chance of activating Ca2+ release simply because of increased number of trials (see Eq. 3). Thus our data are consistent with the findings of Collier et al. (8), who showed that synchronized opening of multiple DHPR Ca2+ channels is not required to trigger Ca2+ sparks.
In the presence of 0.6 mM free Mg2+, the probability of RyR activation by a single brief stimulus was 2% at 100 nM and
10% at 300 nM basal Ca2+ in our experiments. The increase in the probability of RyR activation by increased basal Ca2+ in the dyad has a physiological counterpart in the findings of Litwin et al. (29), who observed that triggering of Ca2+ release by Ca2+ current (ICa) is amplified by the presence of reverse Na+/Ca2+ exchange. The interactions of Mg2+ and Ca2+ at the Ca2+-binding sites of the RyR could explain this phenomenon. These changes, together with the presence of multiple DHPRs at the dyads (23), can also be the reason why, during the action potential, probability of release activation at individual release sites was very high under certain conditions (23), even if the coupling fidelity between DHPR and RyR activation was shown to be only several percent (58).
Overall, our results on dynamic properties of RyRs in the presence of Mg2+ seem to support the notion that the coupling between the L-type Ca2+ channels and RyRs in cardiac muscle does not operate in a saturated mode and is a likely site for modulation and failure. For example, our results could explain the more than twofold increase in the fidelity of DHPR-RyR coupling (measured by the number of Ca2+ sparks triggered by ICa) in cardiac myocytes on adrenergic stimulation (48, 52). The enhanced ability of ICa to trigger Ca2+ sparks could be due to activation of more DHPR channels per release unit or to prolonged duration of individual DHPR channel openings. Finally, our results may help rationalize alterations in Ca2+ cycling during such pathological conditions as cardiac acidosis and ischemia, in which changes in basal Ca2+ and Mg2+ concentrations have been documented (22, 37, 38).
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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