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(Circulation Research. 2000;87:201.)
© 2000 American Heart Association, Inc.

Molecular Medicine

Evidence for Ca²⁺ Activation and Inactivation Sites on the Luminal Side of the Cardiac Ryanodine Receptor Complex

Li Lien Ching, Alan J. Williams, Rebecca Sitsapesan

From the Department of Cardiac Medicine, National Heart & Lung Institute, Imperial College of Science, Technology & Medicine, London, UK.

Correspondence to R. Sitsapesan, Department of Cardiac Medicine, National Heart & Lung Institute, Imperial College of Science, Technology & Medicine, Dovehouse St, London SW3 6LY, UK. E-mail r.sitsapesan{at}ic.ac.uk

	Abstract

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Abstract—We have used tryptic digestion to determine whether Ca²⁺ can regulate cardiac ryanodine receptor (RyR) channel gating from within the lumen of the sarcoplasmic reticulum (SR) or whether Ca²⁺ must first flow through the channel and act via cytosolically located binding sites. Cardiac RyRs were incorporated into bilayers, and trypsin was applied to the luminal side of the bilayer. We found that before exposure to luminal trypsin, the open probability of RyR was increased by raising the luminal [Ca²⁺] from 10 µmol/L to 1 mmol/L, whereas after luminal trypsin exposure, increasing the luminal [Ca²⁺] reduced the open probability. The modification in the response of RyRs to luminal Ca²⁺ was not observed with heat-inactivated trypsin, indicating that digestion of luminal sites on the RyR channel complex was responsible. Our results provide strong evidence for the presence of luminally located Ca²⁺ activation and inhibition sites and indicate that trypsin digestion leads to selective damage to luminal Ca²⁺ activation sites without affecting luminal Ca²⁺ inactivation sites. We suggest that changes in luminal [Ca²⁺] will be able to regulate RyR channel gating from within the SR lumen, therefore providing a second Ca²⁺-regulatory effect on RyR channel gating in addition to that of cytosolic Ca²⁺. This luminal Ca²⁺-regulatory mechanism is likely to be an important contributing factor in the potentiation of SR Ca²⁺ release that is observed in cardiac cells in response to increases in intra-SR [Ca²⁺].

Key Words: ryanodine receptors • Ca²⁺ release • cardiac excitation-contraction coupling • sarcoplasmic reticulum

	Introduction

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The involvement of the intra–sarcoplasmic reticulum (SR) [Ca²⁺] as a regulator of excitation-contraction (EC) coupling in cardiac cells is currently a topic of considerable interest. Ca²⁺ influx through L-type Ca²⁺ channels acts as a trigger for SR Ca²⁺ release and allows contraction of cardiac cells to occur in a graded manner.^{1 2} Increasing evidence now indicates that the loading of the SR directly alters the "gain" of EC coupling: a higher SR Ca²⁺ content leads to a greater SR Ca²⁺ release for a given Ca²⁺ trigger.^{3 4 5} Intuitively, this is expected, inasmuch as a larger SR/cytosolic Ca²⁺ gradient would lead to a greater Ca²⁺ current per ryanodine receptor (RyR) channel opening. Recently, however, detailed quantitative studies have revealed the less expected results that the dependence of SR Ca²⁺ release on SR Ca²⁺ content is not a linear relationship.^{4 6} Shannon et al⁶ have found that the relationship between SR Ca²⁺ content and both the gain of EC coupling and fractional SR Ca²⁺ release becomes extremely steep at higher SR [Ca²⁺]. These results indicate that the gating of RyR channels is altered by changes in SR Ca²⁺ content.

By reconstituting single RyR into bilayers, several investigators have already demonstrated that increasing the luminal [Ca²⁺] leads to increases in open probability (P_o).^{3 7 8 9 10} The mechanism for this effect, however, is a matter of considerable debate at present because investigators are divided into 2 camps. It was originally suggested that changes in luminal [Ca²⁺] cause changes in RyR gating by interacting with luminal binding sites on the channel.⁷ Subsequent work provided further evidence for this hypothesis.^{3 8 11} Other investigators, however, have suggested that luminal Ca²⁺ modulates P_o because it flows through the open channel and acts via cytosolic Ca²⁺ binding sites and does not interact with luminal sites.^{9 10} Unequivocal proof of the site of action of luminal Ca²⁺ has been confounded by the fact that RyR channels can be activated and inactivated by cytosolic Ca²⁺, making it very difficult to completely exclude the possibility that luminal Ca²⁺ has some access to cytosolic Ca²⁺ binding sites even at high positive trans-membrane potentials. Therefore, in the present study, we used a different approach to examine this question, namely, tryptic digestion of sites exposed on the luminal side of the bilayer. Elegant experiments in the squid giant axon set a precedent for using proteolysis as a means of separating activation and inactivation gating processes in ion channels.¹² Our experiments provide compelling evidence to suggest that there are luminal Ca²⁺ binding sites located either on the cardiac RyR channels or on a closely associated protein that regulates RyR gating. Moreover, our results indicate for the first time the existence of luminal inhibition sites.

	Materials and Methods

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Single-Channel Experiments
SR membrane vesicles were prepared from sheep hearts and fused with planar phosphatidylethanolamine lipid bilayers as previously described.¹³ Vesicles were incorporated in a fixed orientation such that the cis chamber corresponded to the cytosolic side of the channel and the trans chamber corresponded to the SR lumen. The trans chamber was held at ground, and the cis chamber was held at potentials relative to ground. After vesicle fusion, the cis chamber was perfused to give a symmetrical cis/trans solution containing 250 mmol/L CsOH and 140 mmol/L PIPES, pH 7.2. The pH and free [Ca²⁺] of the solutions were measured at 23°C by using a Ca²⁺ electrode as previously described.¹⁴ Experiments were performed at 23±1°C.

Single-channel data were displayed on an oscilloscope and stored on digital audio tape (DAT, Intracel). The holding potential was switched between +40 and -40 mV every 30 seconds. A channel event–detection program (Satori, Intracel) was used to analyze the data. Current recordings were filtered at 1 kHz and digitized at 2 kHz. P_o was determined for both holding potentials from a total of 2 minutes of recording by using a 50% threshold analysis.¹⁵ The average P_o for multiple channels was calculated as previously described.⁸ The total number of channels in the bilayer was obtained by fully opening the channels with the caffeine analog, EMD 41000 (Merck Pharmaceuticals). P_o values are quoted as mean±SEM for n4. For n=3, mean±SD is given.

Materials
All chemicals were AnalaR or the best available grade from Sigma Chemical Co or BDH.

	Results

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Effects of Luminal Ca²⁺ on Channels Activated by Cytosolic Ca²⁺ Plus ATP
We find that the gating of RyR channels activated solely by cytosolic Ca²⁺ is not affected by changes in luminal [Ca²⁺].^{7 8} Therefore, we have examined the effects of luminal Ca²⁺ on native sheep cardiac RyR channels activated by cytosolic Ca²⁺ plus ATP. The effects of luminal Ca²⁺ under these conditions can be observed in Figure 1. In the presence of symmetrical 10 µmol/L Ca²⁺ and in the absence of other channel activators, P_o was 0. The addition of 1 mmol/L ATP to the cytosolic channel side increased P_o to 0.157±0.100 at +40 mV and 0.110±0.93 (mean±SEM, n=8) at -40 mV. When channels were thus activated synergistically by cytosolic Ca²⁺ and ATP, increasing the luminal [Ca²⁺] was then able to cause further increases in P_o at both +40 and -40 mV. After the addition of 1 mmol/L luminal Ca²⁺, P_o was 0.369±0.132 at +40 mV and 0.254±0.100 at -40 mV (mean±SEM, n=8). Figure 1 (lower traces) illustrates the typical response of the channels to increases in luminal [Ca²⁺] from 10 µmol/L to 1 mmol/L. The results demonstrate that the effects of luminal Ca²⁺ on sheep cardiac RyR channels activated by the combination of micromolar cytosolic Ca²⁺ and ATP are similar to the effects observed in sheep skeletal⁸ and canine cardiac³ channels. It is important to note that under these experimental conditions, luminal Ca²⁺ was no less effective at activating channels at +40 mV than at -40 mV. These results correspond with our previous observations with skeletal RyRs and indicate that luminal Ca²⁺ does not have to flow through the channel and bind to cytosolic Ca²⁺ binding sites to regulate P_o.⁸

View larger version (45K): [in this window] [in a new window]   Figure 1. Effect of luminal Ca2+ on representative RyR activated by ATP in the presence of 10 µmol/L cytosolic Ca2+. The bilayer was held at +40 mV (left) and -40 mV (right). Arrows indicate the closed channel level. Experimental conditions were as follows: 10 µmol/L cytosolic and luminal Ca2+ (top traces) plus 1 mmol/L ATP (middle traces) plus 1 mmol/L luminal [Ca2+] (bottom traces).

Effects of Luminal Trypsin
Figure 2 illustrates a representative experiment in which the effects of 1 mmol/L luminal Ca²⁺ on RyR gating were compared before and after luminal incubation with 400 µg/mL trypsin. To serve as an internal control, the RyRs were first shown to be responsive to ATP and luminal Ca²⁺ before trypsin incubation. Figure 2 (left) illustrates a typical experiment in which channels were first activated by 1 mmol/L ATP and 10 µmol/L cytosolic Ca²⁺ (top trace) and subsequently activated further by increasing luminal [Ca²⁺] from 10 µmol/L to 1 mmol/L (bottom trace). Trypsin (400 µg/mL) was added to the luminal side of the bilayer, in the continued presence of 1 mmol/L luminal Ca²⁺. After 6 minutes of incubation, the reaction was stopped by the removal of trypsin and 1 mmol/L luminal Ca²⁺ by perfusion with solution containing 250 mmol/L Cs⁺ and 10 µmol/L Ca²⁺. The gating of the channels after perfusion is shown in the right panel (top trace) of Figure 2. The right panel (lower trace) also demonstrates that readdition of 1 mmol/L luminal Ca²⁺ now results in a marked lowering of P_o. For example, at -40 mV, P_o was 0.092±0.102 (mean±SEM, n=4) after perfusion and was reduced to 0.016±0.011 (mean±SEM, n=4) after readdition of 1 mmol/L luminal Ca²⁺. The reduction in P_o caused by 1 mmol/L luminal Ca²⁺ in trypsin-treated channels was observed at both +40 mV and -40 mV. Reversal of the Ca²⁺-induced inhibition was also observed at both holding potentials after lowering the free luminal [Ca²⁺] to 90 nmol/L and is demonstrated in a representative experiment in Figure 3. After incubation with trypsin, P_o was 0.053±0.038 and 0.016±0.011 (mean±SEM, n=4) at +40 mV and -40 mV, respectively, in the presence of 1 mmol/L luminal Ca²⁺ and was 0.110±0.096 and 0.103±0.104 (mean±SEM, n=4) at +40 mV and -40 mV, respectively, after lowering the luminal free [Ca²⁺] to 90 nmol/L.

View larger version (35K): [in this window] [in a new window]   Figure 2. Comparison of effects of luminal Ca2+ on RyR gating before and after luminal trypsin incubation. Channels were voltage-clamped at -40 mV. Arrows indicate the fully closed channel level. Po was increased by addition of 1 mmol/L luminal Ca2+ (left). The luminal side of the channel was then treated with 400 µg/mL trypsin in the presence of 1 mmol/L Ca2+ for 6 minutes. The reaction was stopped by perfusing away the trypsin and the luminal Ca2+. After trypsin incubation, the channels were still activated by 1 mmol/L ATP in the presence of 10 µmol/L cytosolic and luminal Ca2+, albeit to a lower Po (right panel, top trace). However, readdition of 1 mmol/L luminal Ca2+ reduced Po (right panel, bottom trace).

View larger version (39K): [in this window] [in a new window]   Figure 3. Effects of lowering the luminal [Ca2+] after trypsin incubation at +40 mV (left) and -40 mV (right) in a typical experiment in which 3 RyR channels have been incorporated into the bilayer. Arrows indicate zero current level. After trypsin incubation, increasing luminal [Ca2+] from 10 µmol/L to 1 mmol/L reduces Po, and the top traces illustrate the low Po at both +40 and -40 mV under these conditions. The inhibitory effect of luminal Ca2+ was removed by lowering luminal [Ca2+] to 90 nmol/L with 10 mmol/L EGTA (bottom traces).

The addition of trypsin to the luminal side of the bilayer caused a time-dependent effect on channel gating at both +40 mV and -40 mV and is shown in Figure 4. P_o appeared to increase immediately after luminal trypsin addition but was followed within 1 minute by a gradual decline in P_o. For example, at -40 mV, average P_o was 0.303±0.216 (mean±SEM, n=4) in the first 30 seconds immediately after the addition of trypsin (400 µg/mL) to the luminal chamber. After 3 and 6 minutes of incubation, P_o declined to 0.108±0.059 and 0.031±0.019 (mean±SEM, n=4), respectively. A similar decrease in P_o with time was observed at +40 mV.

View larger version (19K): [in this window] [in a new window]   Figure 4. Time-dependent effects of trypsin incubation on the Po of a typical cardiac RyR channel. The histogram shows average Po calculated in 30-second segments at a holding potential of -40 mV. The channels were activated by 10 µmol/L cytosolic Ca2+ and 1 mmol/L ATP in the presence of 1 mmol/L luminal Ca2+. Arrow indicates the time of luminal trypsin addition. The initial apparent increase in Po was followed by a decline in Po over the 6-minute incubation period.

Effects of Luminal Trypsin on Conductance
Figure 5 illustrates the changes in RyR current amplitude at +40 and -40 mV that occur with changes in luminal [Ca²⁺] and luminal trypsin addition. Addition of 1 mmol/L luminal Ca²⁺ caused a reduction in current amplitude at both +40 mV and -40 mV (Figure 5b), as previously observed^{7 8} and expected from a knowledge of the conductance and relative permeabilities of divalent and monovalent cations in the cardiac RyR.¹⁶ This was followed by a further reduction in current amplitude by trypsin (Figure 5c). For example, at -40 mV, current amplitude was reduced from 18.68±0.37 to 17.61±0.51 pA (mean±SEM, n=4) after increasing luminal [Ca²⁺] from 10 µmol/L to 1 mmol/L and was further reduced to 14.01±0.67 pA at -40 mV (mean±SEM, n=4) after trypsin incubation. Removal of trypsin and returning the free luminal [Ca²⁺] to 10 µmol/L by perfusion of the luminal chamber restored current amplitude to control levels (Figure 5d). Readdition of 1 mmol/L luminal Ca²⁺ again lowered current amplitude to the same level that was observed before trypsin incubation (results not shown). Thus, although the effects of trypsin incubation on RyR gating were irreversible, the effects on single-channel conductance were reversible, indicating that the conductance-induced changes were not caused by tryptic digestion of the channel.

View larger version (29K): [in this window] [in a new window]   Figure 5. Effects of luminal Ca2+ and trypsin on the current amplitude of a representative channel at -40 mV (top) and +40 mV (bottom). C indicates the closed channel level; O, open channel level; and T, open channel level in the presence of 400 µg/mL luminal trypsin. a, Channel is activated by 1 mmol/L ATP in the presence of 10 µmol/L cytosolic and luminal Ca2+. b, Addition of 1 mmol/L luminal Ca2+ decreases current amplitude at both holding potentials. c, Current amplitude is further decreased at both +40 and -40 mV after trypsin (400 µg/mL) addition. d, Removal of trypsin and luminal Ca2+ by perfusion restores single-channel current amplitude to control levels. The traces shown were chosen to illustrate current amplitude changes and are not representative of average Po values.

Effects of Heat-Inactivated Trypsin on RyR Function
To examine whether the change in RyR response to luminal Ca²⁺ observed after treatment with trypsin was the result of its proteolytic activity, control experiments were performed with the use of heat-inactivated trypsin. Trypsin was boiled for 10 minutes, and the loss of enzymatic activity was qualitatively tested with SDS-PAGE by comparing its ability to cleave BSA with that of the active enzyme. We found that heat-inactivated trypsin produced a similar reversible reduction in Cs⁺ current amplitude at both +40 and -40 mV, as was observed with active trypsin. For example, at -40 mV, current amplitude was 18.87±0.61 pA before and 15.58±0.70 pA (mean±SD, n=3) after luminal addition of 1 mmol/L Ca²⁺ and 400 µg/mL inactivated trypsin. The effects of heat-inactivated trypsin on luminal Ca²⁺ modulation of RyR gating is shown in a representative experiment in Figure 6. There was no significant difference in the effects of heat-inactivated trypsin at +40 or -40 mV, but for clarity, only the effects at -40 mV are shown. The figure demonstrates that the channels respond normally to changes in luminal [Ca²⁺] before incubation with heat-inactivated trypsin; increasing the luminal [Ca²⁺] from 10 µmol/L to 1 mmol/L produced an increase in P_o (top and middle traces). P_o was 0.113±0.019 at +40 mV and 0.069±0.063 at -40 mV (mean±SD, n=3) in the presence of 10 µmol/L luminal Ca²⁺. After increasing luminal Ca²⁺ to 1 mmol/L, the P_o just before the addition of heat-inactivated trypsin was 0.448±0.217 at +40 mV and 0.261±0.150 at -40 mV (mean±SD, n=3). Subsequent incubation of the channels with 400 µg/mL heat-inactivated trypsin for 6 minutes did not cause any time-dependent reduction in P_o, as was observed with active trypsin (see Figure 4). For example, the P_o at +40 mV after perfusing away the heat-inactivated trypsin from the luminal channel side and reestablishing the luminal [Ca²⁺] at 1 mmol/L was 0.447±0.030 (mean±SD, n=3). The results with the heat-inactivated trypsin indicate that the decrease in P_o caused by incubation with active trypsin is the result of tryptic digestion of luminal sites.

View larger version (35K): [in this window] [in a new window]   Figure 6. Effect of heat-inactivated trypsin on RyR. The holding potential is -40 mV. Arrows indicate zero current level. Channels were activated by 1 mmol/L ATP in the presence of 10 µmol/L cytosolic and luminal Ca2+ (top trace). Luminal Ca2+ (1 mmol/L) further increased Po (middle trace). After 6-minute luminal incubation with heat-inactivated trypsin (400 µg/mL) in the presence of 1 mmol/L luminal Ca2+, heat-inactivated trypsin was removed by perfusion. Readdition of 1 mmol/L luminal Ca2+ did not cause the channel to become inhibited as was observed with active trypsin. Po was similar to that observed before incubation (bottom trace).

	Discussion

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Cardiac RyRs localized in junctional SR membranes are thought to complex with a number of other proteins, including junctin, triadin, and calsequestrin.¹⁷ Some of these proteins have a luminal location or have luminally exposed domains and may still be associated with RyRs after reconstitution into bilayers. This may occur with native RyRs (as in the present study) and even with "purified" RyRs, inasmuch as it has been shown that certain proteins are still bound to RyRs even after purification with CHAPS solubilization.¹⁷ Therefore, it is possible that the effects of luminal Ca²⁺ observed in our experiments do not result from the direct effect of luminal Ca²⁺ on the channel itself but are mediated by an associated protein to which Ca²⁺ binds.

We find that the P_o of sheep cardiac RyR channels activated by 1 mmol/L ATP in the presence of 10 µmol/L cytosolic Ca²⁺ can be further increased by increasing luminal [Ca²⁺] from 10 µmol/L to 1 mmol/L. The results are similar to the effects of luminal Ca²⁺ in native canine cardiac channels.^{3 11} A direct comparison with the effects of luminal Ca²⁺ on sheep skeletal RyRs under identical experimental conditions indicates that sheep cardiac RyRs are less sensitive to changes in luminal [Ca²⁺] than are sheep skeletal channels.⁸ Sheep skeletal channels could be almost fully activated by 1 mmol/L luminal Ca²⁺, whereas in the present study, we found that P_o was increased to only 0.4. Indeed, experiments with RyR channels, purified as previously described,¹³ provide no further clues as to the precise location of the luminal Ca²⁺ binding sites on the RyR channel complex (see online supplementary information, available at http://www.circresaha.org).

As shown previously for skeletal⁸ and cardiac¹¹ RyRs activated synergistically by cytosolic ATP and Ca²⁺, we find that the stimulatory effects of luminal Ca²⁺ are not significantly different at +40 mV or at -40 mV. The different holding potentials produce a marked difference in the amount of Ca²⁺ flux through the channel^{9 18} ; therefore, as we suggested in our earlier study,⁸ our control results with luminal Ca²⁺ argue against the possibility of luminal Ca²⁺ acting by binding to cytosolic sites.

Trypsin incubation altered RyR function in at least 2 respects. First, trypsin reduced conductance; second, the response of the channel to luminal Ca²⁺ was altered. The 2 effects appear to be the result of different interactions of trypsin with the channel because the conductance change was reversible after perfusing away the trypsin, whereas the gating change was irreversible. Further distinction between the 2 effects was obtained by the use of heat-inactivated trypsin. Heat-inactivated trypsin also caused a similar reversible reduction in conductance but did not cause the trypsin-induced change in RyR gating. These data lead us to conclude that the conductance effect is the result of a protein-protein interaction between RyR and trypsin, whereas the change in the response of RyR to luminal Ca²⁺ involves cleavage of exposed luminal sites. Therefore, the results strongly suggest that the increase in P_o observed in response to an increase in luminal [Ca²⁺] is due to the binding of Ca²⁺ to luminal sites on the RyR protein complex and not the result of Ca²⁺ flowing through the channel to act on cytosolic sites. Furthermore, the fact that trypsin completely abolishes any stimulatory effect of luminal Ca²⁺ and does not leave a residual stimulatory effect argues against any contribution to a luminal Ca²⁺-induced increase in P_o by Ca²⁺ flowing through the channel and accessing cytosolic binding sites.

The unexpected reduction in P_o caused by increasing luminal [Ca²⁺] after incubation with trypsin indicates that there are also luminal Ca²⁺ binding sites on the RyR protein complex that mediate the inhibition of RyR activity. The simplest explanation for our results is that there are luminal Ca²⁺ activation and Ca²⁺ inhibition sites but that trypsin cleavage of the RyR protein complex damages the activation site(s) while allowing the inhibitory site(s) to remain functional. Changing the holding potential and, therefore, the Ca²⁺ flux through the channel does not affect the inhibitory action of luminal Ca²⁺, indicating that the sites mediating the inhibition do not have a cytosolic location. This is the first time that an inhibitory effect of luminal Ca²⁺ has been observed in cardiac RyRs, although high luminal [Ca²⁺] has been reported to inhibit purified rabbit skeletal RyRs.^{9 10} It is possible that skeletal inhibitory luminal Ca²⁺ binding sites exhibit a relatively higher affinity for Ca²⁺ than do the cardiac inhibitory sites or that the purification procedure has led to alterations to luminal activation sites. The fact that 1 mmol/L luminal Ca²⁺ causes activation of the cardiac RyR before trypsinization but channel inactivation after trypsinization indicates either that the luminal activation sites have a higher affinity for Ca²⁺ or that they transduce a greater change in P_o than do the inhibition sites.

The mechanism underlying the reduced current amplitude observed in the presence of trypsin or heat-inactivated trypsin is not known. It is possible that both molecules interact with RyRs near the luminal mouth of the pore and block the flow of permeant ions, but equally, trypsin (and heat-inactivated trypsin) may bind elsewhere on the RyR protein complex, resulting in a conformational change of RyR that leads to the reduction in conductance. Further experiments are required to understand the conductance changes observed with these molecules. The importance of these results lies in the fact that the irreversible change in RyR gating is not associated with any measurable changes in conductance either when Cs⁺ is the permeant ion or when appreciable Ca²⁺ current is evident (see Figure 5), indicating that the trypsin cleavage of luminally located residues does not lead to any major changes in the pore structure of the channel. Therefore, these results provide strong evidence that the Ca²⁺ binding sites involved in the effects of luminal Ca²⁺ on channel gating are distinct from any cation binding sites within the channel pore.

Where are the luminal Ca²⁺ binding sites? The precise location is not yet known, but they must be situated either on RyR or on a protein that remains associated with RyRs after incorporation into the bilayer. Trypsin preferentially cleaves amino acid residues with positively charged side chains.¹⁹ RyRs have a number of lysine and arginine residues in the C-terminal domain that are predicted to have a luminal location and that could be the putative sites for cleavage in the present experiments,^{20 21 22 23} but so also do the luminal domains of junctin and triadin.^{17 24} In a number of cases, these residues are located closely to negatively charged residues, which potentially could form Ca²⁺ binding regions. Trypsin does not cleave calsequestrin under our conditions of trypsin incubation (in the presence of 1 mmol/L Ca²⁺),²⁵ suggesting that the observed changes to luminal Ca²⁺ modulation of P_o are not caused by damage to calsequestrin.

Our observation that luminal Ca²⁺ can regulate cardiac RyR gating from the luminal side of the bilayer and does not require that Ca²⁺ should first flow through the channel before binding to cytosolic sites is crucial for a full understanding of the mechanisms determining the dependence of SR Ca²⁺ release on SR Ca²⁺ content. Unlike the situation in the bilayer, it would be expected that in the restricted space of the cleft region in cardiac cells, the Ca²⁺ flowing through RyRs would feed back to regulate RyR gating by binding to Ca²⁺ activation and inhibition sites. The luminal regulatory sites proposed in the present study would provide an additional distinct mechanism by which the SR [Ca²⁺] could modulate the release mechanism, thus allowing a far greater degree of potentiation in the gain of EC coupling. Therefore, our results may help to explain the nonlinear relationship between SR Ca²⁺ content and SR Ca²⁺ release and the hugely steep increases in Ca²⁺ release and in the gain of EC coupling at high SR Ca²⁺ levels, described by Shannon et al.⁶ Importantly, the SR [Ca²⁺] at which the authors observed steep changes in Ca²⁺ release corresponds closely to the levels of luminal Ca²⁺ that have been shown to activate isolated cardiac RyR channels reconstituted into bilayers.^{3 7 11 26} Because changes in SR load can occur under pathological conditions such as heart failure and ischemia, the above discussion would suggest that small changes in SR Ca²⁺ content may lead to large changes in contractility. Therefore, it is crucial to understand the mechanisms responsible for regulating the gating of the cardiac RyR channel by luminal [Ca²⁺].

In summary, we demonstrate that the cardiac RyR response to luminal Ca²⁺ can be altered by physical protein modifications at the luminal side of the bilayer, indicating that luminal Ca²⁺ can regulate channel gating by binding to luminal sites on the RyR channel complex. Our results reveal for the first time the presence of luminal Ca²⁺ activation and inactivation sites, although the exact location of the sites is yet to be elucidated. Luminal Ca²⁺ binding sites would play an important role in regulating SR Ca²⁺ release and contractility in cardiac muscle by providing an increased potential for increasing the gain of EC coupling.

	Acknowledgments

 
This study was supported by The British Heart Foundation

Received March 9, 2000; accepted June 9, 2000.

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