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(Circulation Research. 1995;77:765-772.)
© 1995 American Heart Association, Inc.

Articles

New Insights Into the Gating Mechanisms of Cardiac Ryanodine Receptors Revealed by Rapid Changes in Ligand Concentration

Rebecca Sitsapesan, Richard A.P. Montgomery, Alan J. Williams

From the Department of Cardiac Medicine, The National Heart & Lung Institute, London, UK.

Correspondence to Dr Rebecca Sitsapesan, Department of Cardiac Medicine, The National Heart & Lung Institute, Dovehouse St, London SW3 6LY, UK.

	Abstract

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Abstract We have developed a novel technique for incorporation of sheep cardiac sarcoplasmic reticulum (SR) Ca²⁺-release channels into planar phospholipid bilayers in order to investigate changes in [Ca²⁺] on a physiological time scale and have investigated whether the rate of change of cytosolic [Ca²⁺] has a direct effect on the gating of the cardiac SR Ca²⁺-release channel. Vesicles of heavy SR were incorporated into planar phospholipid bilayers painted on glass pipettes, and an established technique for rapid solution exchanges at excised membrane patches was modified to allow solution changes to be made at the bilayer within 10 ms. For a given change in [Ca²⁺], we demonstrate that the open probability (Po) is similar whether the cytosolic [Ca²⁺] is increased rapidly (10 ms) or slowly (1 s) and appears to be no different from the Po measured under steady state conditions that were recorded by using conventional bilayer techniques. We also demonstrate that no desensitization or inactivation occurs at -40 mV when the channel is activated by Ca²⁺ alone or in the presence of other channel activators, ATP or EMD 41000. However, at +40 mV, rapid channel activation followed by inactivation was observed. The probability of such voltage-dependent inactivation appears to depend on the mechanism of channel activation.

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

	Introduction

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Evidence suggests that Ca²⁺-induced Ca²⁺ release is the mechanism for excitation-contraction (EC) coupling in cardiac muscle.^{1 2 3 4} The Ca²⁺ entering the cell via the voltage-activated sarcolemmal Ca²⁺ channels triggers the opening of Ca²⁺-release channels in the sarcoplasmic reticulum (SR).^{3 4} However, the precise mechanisms controlling the gating of the SR Ca²⁺-release channels under conditions of EC coupling are not fully understood. The intracellular location of the Ca²⁺-release channel prevents a detailed knowledge of the physiological environment at the cytosolic and luminal channel face and of the changes that may occur during the action potential. As the Ca²⁺-release channel is not accessible for patch clamping while maintaining the physiological environment of the channel, our understanding of the conduction and gating of the Ca²⁺-release channel has been gathered from reconstitution of the channels into planar phospholipid bilayers.

Elegant experiments by Fabiato¹ have demonstrated that in canine skinned cardiac cells, the amount of Ca²⁺ released from the SR depends on the rate of change of "trigger Ca²⁺." He has postulated that this phenomenon could result from Ca²⁺ binding to activation and inactivation sites on the Ca²⁺-release channel that have different affinities for Ca²⁺ and different rate constants for Ca²⁺ binding. Fabiato initially suggested that the rate constant for Ca²⁺ binding to the inactivating site was lower than that for Ca²⁺ binding to the activating site and that the inactivation site had a higher affinity for Ca²⁺ than did the activation site. Therefore, a slow rate of change of trigger Ca²⁺ at the SR membrane would be expected to inactivate the SR Ca²⁺-release channels before the [Ca²⁺] had reached a level sufficient for optimal activation of the channels. If Fabiato's predictions are correct, then steady state gating measurements of Ca²⁺-release channels incorporated into planar phospholipid bilayers may bear no resemblance to the gating of the channels during EC coupling, when the cytosolic [Ca²⁺] changes in milliseconds. Therefore, we wanted to investigate the mechanisms of cardiac SR Ca²⁺-release channel gating when the channels were activated by cytosolic Ca²⁺ on a physiologically relevant time course. In order to carry out such an investigation, we incorporated vesicles of heavy SR into phosphatidylethanolamine lipid bilayers painted on glass pipette tips. By modifying a method for making rapid solution changes at the tip of a patch pipette,⁵ solution changes could be made at the bilayer within 10 ms. This time course is similar to the rate of rise of trigger Ca²⁺, which activates the channel in the cell.⁶ Therefore, any inactivation or desensitization mechanisms resulting simply from the rapid binding of trigger Ca²⁺ to sites on the Ca²⁺-release channel during EC coupling should be apparent. We also compared the effects of such rapid changes in [Ca²⁺] with changes on a much slower time course (1 s). Our experiments suggest that the rate of change of cytosolic Ca²⁺ does not directly determine the final open probability (Po) of the sheep cardiac SR Ca²⁺-release channel. We also find that the cardiac SR Ca²⁺-release channel inactivates in a voltage-dependent manner. The inactivation occurs at positive membrane potentials and occurs only infrequently with Ca²⁺ as the sole activating ligand.

	Materials and Methods

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Heavy SR vesicles were isolated from sheep hearts as previously described.⁷ The method for rapid solution changes at a bilayer was basically as described previously.⁸ A glass pipette (trans chamber) of 50- to 80-µm tip diameter was placed with the tip in a 250-µL cis chamber. The trans chamber was backfilled with solution from the cis chamber by suction. Phosphatidylethanolamine lipid bilayers were painted across the pipette tip in 250 mmol/L CsOH and 140 mmol/L PIPES, pH 7.2, at 5 µmol/L free [Ca²⁺] with no applied pressure to the pipette. Bilayers were visualized as flat membranes at the outside edge of the pipette rather than inside the pipette tip. After bilayer formation, an osmotic gradient was established by the addition of 100 µL of 3 mol/L KCl to the cis chamber. Membrane vesicles were added to the cis chamber and stirred. Excess fluid in the cis chamber was allowed to overflow. After SR Ca²⁺-release channel incorporation into the bilayer, the trans chamber was moved into the flow of one of two parallel streams of solution flowing from a length of glass theta tubing (the perfusion pipette [outer diameter, 1.5 mm]) at a distance of 100 to 300 µm from the theta tubing and 50 to 100 µm from the interface between the two solutions. Each barrel of the theta tubing was connected to a number of reservoirs (containing the appropriate perfusion solutions) via a length of polythene tubing. The theta capillary was firmly clamped to a solenoid switching device. Rapid changes in solution were effected by a lateral movement (200 to 300 µm) of the theta tubing, allowing the adjacent solution to flow over the bilayer. The time between activation of the solenoid switching device and the movement of the solution interface across the bilayer depends primarily on the distance of the bilayer pipette from the interface between the two solutions flowing from the theta tubing. Junction potential changes were measured after the experiments to determine the time of the solution change in relation to the activation of the solenoid switching device. The speed of the change in liquid junction potential at the trans chamber pipette was used to determine the speed of the solution change across the entire bilayer.⁸ As described previously,⁸ the speed of the liquid junction potential change was very dependent on the tip diameter of the bilayer pipette (trans chamber). Therefore, we carried out experiments on pipettes with as small a tip diameter as possible. In practice, it is very difficult to paint bilayers on pipettes with tip diameters of <50 µm. For pipettes of 80 µm, the time taken from the start of the junction potential change to the peak of the change was <10 ms. Therefore, the pipette tip diameters used were in the range of 50 to 80 µm so that solution changes could be made within 10 ms.

The large diameter of the trans pipette tip favors the rapid diffusion of ions between the pipette and perfusing solutions; thus, diffusional changes can mask junction potential changes. When this is the case, the trans chamber is filled with 5% to 10% agar in 210 mmol/L KCl, and positive pressure is applied to the pipette, thus allowing a more accurate measurement of the time course of the junction potential change.

The perfusion solutions were essentially the same as the solution in the trans chamber but also included 1 mmol/L BAPTA and various additions of CaCl₂ to obtain the required free [Ca²⁺]. The free [Ca²⁺] of all solutions was measured with a Ca²⁺ electrode (Orion 93-20). The switching protocol was as follows: Unless otherwise stated, bilayers were perfused alternately every 10 s with a solution containing 0.1 µmol/L Ca²⁺ and the channel-activating perfusate.

Slow changes in [Ca²⁺] (1 s) were not produced by lateral movement of the theta tubing but by switching to a different reservoir of solution. The tip of the bilayer pipette was placed in the flow of the low [Ca²⁺] solution (0.1 µmol/L) flowing from one side of the theta tubing (perfusion pipette). The rate of flow of solution was then increased from 0.9 mL/min to 6 to 8 mL/min. The inflow to the same side of the theta tubing was then switched from the reservoir containing 0.1 µmol/L free Ca²⁺ to the reservoir containing 100 µmol/L free Ca²⁺. The time course of the solution change at the bilayer was estimated by monitoring the time course of the current evoked by the change in liquid junction potential at the tip of a high-resistance (15 to 30 M) pipette.

Single-channel current recordings were filtered at 2 kHz and digitized at 4 kHz. Po was monitored by 50% threshold analysis. For each rapid switching experiment, Po values from six data sweeps were averaged in the first 100 ms after switching to the activating solution and in the 5 to 10 s following activation. In experiments in which the [Ca²⁺] was changed over 1 s, Po was measured in the 5 to 10 s following activation. Due to the difficulty of maintaining a bilayer during these slow-change experiments, only one to five changes were carried out and averaged for each experiment. Ensemble averaging of data was performed by the SCAN computer analysis program (John Dempster, University of Strathclyde) after filtering the data at 200 Hz and digitizing at 400 Hz.

	Results

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Fig 1A illustrates the rapid activation of a typical single sheep cardiac SR Ca²⁺-release channel that results from changing the cytosolic free [Ca²⁺] from 0.1 to 100 µmol/L. At 0.1 µmol/L [Ca²⁺], the Po of the sheep cardiac SR Ca²⁺-release channel is essentially zero. The bilayer was held at +40 mV in the pipette (corresponding to the conventional -40 mV cis relative to trans) in symmetrical 250 mmol/L cesium PIPES. No change in Po was observed even 5 to 10 s after channel activation. The Po measured in the first 100 ms after channel activation (Po_early, .215±.056 [mean±SEM]; n=9) was no different from the Po measured in the 5 to 10 s after activation (Po_late, .232±.064 [mean±SEM]; n=9). This was the case for channel activation at all [Ca²⁺]s investigated. The respective values for Po_early and Po_late were as follows: .022±.006 and .029±.013 (mean±SEM, n=6) at 10 µmol/L Ca²⁺; .088±.059 and .110±.018 (mean±SEM, n=5) at 50 µmol/L Ca²⁺; and .145±.083 and .154±.069 (mean±SD, n=3) at 500 µmol/L Ca²⁺. Indeed, the relation between Po and [Ca²⁺] obtained by rapid activation of the channels appears to be the same as that for steady state Po measurements obtained by use of the conventional bilayer technique under otherwise identical conditions.⁹

View larger version (128K): [in this window] [in a new window]   Figure 1. Activation of sheep cardiac SR Ca2+-release channels. A, Rapid activation of a representative single channel incorporated into a bilayer painted on a pipette. Four successive switches from 0.1 to 100 µmol/L are shown. The bilayer was held at -40 mV cis relative to trans. Channel openings are upward. Fluctuations in baseline noise result from the gating of SR K+ channels, which incorporate into the bilayer together with the Ca2+-release channels. The large artifact occurs when the rapid switching system is activated. The arrow indicates the time of the start of the change in [Ca2+] at the bilayer. This time is obtained from the time course of the current evoked by the junction potential change as described by Sitsapesan et al,8 which reflects the time for the interface between two solutions to move across the face of the bilayer. The delay between the switching artifact and the start of the change in liquid junction potential is primarily governed by the distance of the bilayer pipette (trans chamber) from the interface of the two solutions flowing from the perfusion pipette. The time base of the junction potential change is expanded and shown in the inset. The inset demonstrates that the junction potential change (which occurs at the arrow in the main figure) was complete within 10 ms. The arrow indicates the start of the junction potential change on switching from a 210 to a 105 mmol/L KCl solution. B, Rapid switching (within 10 ms) from 0.1 to 100 µmol/L Ca2+ at a bilayer containing five channels. The arrow indicates the time of the change in [Ca2+] at the bilayer. The bilayer was held at -40 mV cis relative to trans. Channel openings are upward. C, Activation of a single Ca2+-release channel when the [Ca2+] is increased from 0.1 to 100 µmol/L Ca2+ over 1 s. The top tracing shows the time course of the current evoked by the junction potential change. The lower section of the figure shows four successive changes from 0.1 to 100 µmol/L free Ca2+. The holding potential was -40 mV cis relative to trans. Channel openings are upward.

As the Po of sheep cardiac channels activated solely by cytosolic Ca²⁺ is very low, ensemble currents constructed from many data sweeps cannot be properly distinguished from the zero current level. Therefore, we have shown the effect of rapidly changing the [Ca²⁺] from 0.1 to 100 µmol/L at a bilayer containing five Ca²⁺-release channels (Fig 1B). Two important observations can be made from this figure. First, it is very rare for all five channels to open simultaneously and second, there is no obvious change in channel activity with time.

Fig 1C illustrates another representative channel in which the [Ca²⁺] was changed more slowly (over 1 s) from 0.1 to 100 µmol/L. The resultant Po was no different from that obtained when the [Ca²⁺] was changed within 10 ms. The Po 5 to 10 s after the slow change in [Ca²⁺] was .268±.081 (mean±SEM, n=9). Therefore, we find no difference in the final Po when the [Ca²⁺] is changed rapidly (10 ms) or relatively slowly (1 s). The rate at which the channels activate appears to be similar to the rate of change of [Ca²⁺] at the cytosolic channel face. To determine exactly whether the rate of channel activation mirrors the rate of change of cytosolic [Ca²⁺] would require ensemble currents from many data sweeps. However, as mentioned earlier, the low Po values that result from Ca²⁺-activation of the sheep cardiac SR Ca²⁺-release channel coupled with the bursting behavior of the channel yield ensemble currents that are much noisier when the channels are activated by Ca²⁺ but are not clearly differentiated from the closed channel level. In addition, the slow changes (1 s) in [Ca²⁺] are more difficult to carry out than the fast changes (10 ms), and we are not able to carry out enough changes to a higher [Ca²⁺] before the bilayer breaks in order to obtain an ensemble average.

It could be argued that time-dependent changes in Po will be observed only if higher Po values are attained. However, the sheep cardiac Ca²⁺-release channel cannot be fully activated with Ca²⁺ as the sole ligand. Maximal activation occurs with 100 µmol/L cytosolic Ca²⁺ to a Po of .2.^{9 10} Therefore, we have activated the sheep cardiac SR Ca²⁺-release channel with Ca²⁺ plus a second ligand to induce almost full activation. Fig 2 shows the effect of rapidly switching from 0.1 µmol/L Ca²⁺ to 100 µmol/L Ca²⁺ plus 1 mmol/L ATP (Fig 2A) or 100 µmol/L Ca²⁺ plus 100 µmol/L EMD 41000 (Fig 2B) with a holding potential of -40 mV cis relative to trans. Under the above experimental conditions, neither ATP nor EMD 41000 can fully activate the channel in the absence of activating levels of cytosolic Ca²⁺. ATP and EMD 41000 bind to distinct sites on the Ca²⁺-release channel,^{11 12} but both agents can almost fully open the channel in the presence of activating cytosolic [Ca²⁺]. Fig 2 demonstrates that at -40 mV, when the channels are fully activated by two distinct mechanisms (ATP or EMD 41000 activation), there is no evidence for any time-dependent inactivation or desensitization even over several seconds. This is quite clear from the ensemble current in each case. For channels activated by 100 µmol/L Ca²⁺ plus 1 mmol/L ATP, Po_early was .841±.087 (mean±SEM, n=5), and Po_late was .874±.072 (mean±SEM, n=5). For channels activated by 100 µmol/L Ca²⁺ plus 100 µmol/L EMD 41000, Po_early was .936±.110 (mean±SD, n=3) and Po_late was .930±.115 (mean±SD, n=3). Fig 2C illustrates three consecutive sweeps of data, where three channels in a bilayer were activated by 100 µmol/L Ca²⁺ plus 1 mmol/L ATP for 5 s. Unlike the situation in Fig 1B, all three channels are frequently open simultaneously. These results clearly indicate that even under conditions where Po is maximized and when the channel is activated by different mechanisms, no desensitization to the agonists occurs. This figure also clearly demonstrates that the channels shut very rapidly when the agonists are removed.

View larger version (27K): [in this window] [in a new window]   Figure 2. Activation of a single SR Ca2+-release channel by switching from 0.1 µmol/L Ca2+ to 100 µmol/L Ca2+ plus 1 mmol/L ATP (A) or from 0.1 µmol/L Ca2+ to 100 µmol/L Ca2+ plus 100 µmol/L EMD 41000 (B). In each case, the solution change at the bilayer is indicated by the arrow. In panel a, four sweeps of data are shown, and in panel b, the ensemble current generated by averaging 54 sweeps (ATP) or 46 sweeps (EMD 41000) is illustrated. The bilayers were held at -40 mV cis relative to trans. The dotted lines indicate the zero current level. In panel C, the activation of multiple channels by switching from 0.1 µmol/L Ca2+ to 100 µmol/L Ca2+ plus 1 mmol/L ATP for 5 s is illustrated. Three consecutive sweeps are shown. The first and second arrows indicate the change to and from the activating solution, respectively. The holding potential was -40 mV cis relative to trans. The dotted lines indicate the zero current level.

Although we observed no desensitization or inactivation to any of the agonists used (Ca²⁺, ATP, or EMD 41000) at -40 mV, we did observe a voltage-dependent inactivation at +40 mV cis. This only occurred in 17% (4 of 24) of the channels activated by cytosolic Ca²⁺ as the sole ligand. However, 56% (5 of 9) of those channels that were activated by Ca²⁺ plus ATP or EMD 41000 exhibited voltage-dependent inactivation at +40 mV cis but not at -40 mV cis. Therefore, the inactivation may be more likely to occur at high Po values or after the channel has entered a particular long-lived open state. Fig 3 illustrates the voltage-dependent inactivation of two channels in a bilayer activated by 100 µmol/L Ca²⁺ plus 1 mmol/L ATP. In tracing a, the bilayer was held at -40 mV, and no decline in Po occurred. After switching back to 0.1 µmol/L Ca²⁺, the holding potential was then changed to +40 mV, and the bilayer was rapidly exposed to 100 µmol/L Ca²⁺ plus 1 mmol/L ATP (tracing b). The channels activated rapidly and then closed. Switching to 0.1 µmol/L Ca²⁺ and then back to 100 µmol/L Ca²⁺ plus 1 mmol/L ATP, as shown in tracing c, did not result in any further openings. Channel openings at +40 mV could be restored only by changing the holding potential to -40 mV briefly. This removed the inactivation, and on changing the holding potential back to +40 mV, channels could again be activated briefly by switching to 100 µmol/L Ca²⁺ plus 1 mmol/L ATP as shown in tracing d. As before, no further openings were observed unless a negative holding potential was applied (as shown in tracing e).

View larger version (30K): [in this window] [in a new window]   Figure 3. A representative example of voltage-dependent inactivation of cardiac SR Ca2+-release channels. Rapid switching from 0.1 µmol/L Ca2+ to 100 µmol/L Ca2+ plus 1 mmol/L ATP at the bilayer occurs at the arrow. The dotted lines indicate the open channel levels. For tracing a, the holding potential was -40 mV cis relative to trans, and no inactivation was observed. In all the following tracings, the holding potential was +40 mV cis relative to trans. For tracing b, the channels were activated when perfused with 100 µmol/L Ca2+ plus 1 mmol/L ATP but then rapidly shut. Repeatedly removing the agonists and switching back to 100 µmol/L Ca2+ plus 1 mmol/L ATP did not open the channels (tracing c). In tracing d, the holding potential was briefly changed to -40 mV before activating the channels at +40 mV. As before, once the channels were inactivated, the channels could not be reactivated by perfusion with 100 µmol/L Ca2+ plus 1 mmol/L ATP (tracing e).

	Discussion

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We have investigated the effects of rapid changes in [Ca²⁺] and other activating ligands at the cytosolic side of sheep cardiac SR Ca²⁺-release channels incorporated into planar lipid bilayers. We demonstrate that the channels can be rapidly activated and inactivated by fast application and removal of agonists. We estimate the changes in ligand concentration to occur in <10 ms, as judged by the speed of the current change evoked by the junction potential change when switching between two different solutions. This is similar to the time course of the rise in intracellular [Ca²⁺], which triggers the opening of the Ca²⁺-release channels during EC coupling.⁶ When the [Ca²⁺] is changed rapidly (<10 ms), we find that the relation between Po and cytosolic [Ca²⁺] is essentially identical to that obtained by conventional bilayer techniques when the [Ca²⁺] is changed more slowly and steady state Po measurements are made (3-minute consecutive recordings).^{9 10} Under both conditions, peak Po values for cardiac SR Ca²⁺-release channels from the sheep are obtained at 100 µmol/L cytosolic Ca²⁺, and the EC₅₀ value is close to 40 µmol/L. In addition, with rapid activation of the channels, we find no time-dependent change in Po at -40 mV; the Po in the first 100 ms after activation is no different from the Po 5 to 10 s after activation for all [Ca²⁺] values investigated. When the [Ca²⁺] is changed more slowly over 1 s, the final Po that is attained is no different from that when the Po is changed within 10 ms. Therefore, the rate of change of [Ca²⁺] at the cytosolic face of the sheep cardiac SR Ca²⁺-release channel does not appear to affect the degree of channel activation. The rate at which the channel is activated appears to be related simply to the rate of change of cytosolic [Ca²⁺]. However, we cannot state this unequivocally because of the difficulties in assessing the time course of activation with low open probabilities, particularly with slow changes in [Ca²⁺] because too few changes in [Ca²⁺] could be performed for ensemble averaging.

We have also investigated the effects of rapid changes in cytosolic Ca²⁺ plus a second activating ligand. The sheep cardiac SR Ca²⁺-release channel is usually activated only to a Po value close to .2 in the presence of a single activating ligand.^{9 10} In order to achieve high Po values, the channel must be synergistically activated by Ca²⁺ plus a second ligand, invoking an apparently different gating scheme.^{11 13 14} In the presence of Ca²⁺ plus EMD 41000 or Ca²⁺ plus ATP, the cardiac SR Ca²⁺-release channel can be activated to very high Po values. EMD 41000 and ATP are thought to activate the channel by binding to distinct sites^{11 12} ; therefore, Po is probably elevated by different mechanisms. What is clear from these experiments is that under the present ionic conditions, at -40 mV, when the channel is activated to high Po values by Ca²⁺ plus ATP or Ca²⁺ plus EMD 41000, no discernible time-dependent change in Po is observed.

Why do Györke and Fill¹⁵ observe a time dependent decline in Po or "adaptation" after rapid increases in cytosolic [Ca²⁺], whereas we do not observe any change in gating up to 10 s after increasing the [Ca²⁺] at -40 mV? At cis-positive potentials, we find that some channels inactivate in a voltage-dependent manner; however, this phenomenon is quite different from the adaptation reported by Györke and Fill, who reactivated channels to the original peak Po simply by lowering the free [Ca²⁺] and then rapidly elevating the [Ca²⁺] by flash photolysis of caged Ca²⁺. Using the flash photolysis technique, Valdivia et al¹⁶ have reported that if the [Ca²⁺] is increased slowly over several seconds, the resultant Po is similar to that measured during steady state conditions. One explanation for these dissimilarities is simply that a species difference exists. Dog cardiac channels adapt to a maintained cytosolic [Ca²⁺], whereas sheep cardiac channels do not. This is not an unlikely situation given the major differences in Ca²⁺ activation reported in the literature for the dog and sheep channels. The sheep cardiac SR Ca²⁺-release channel is characterized by very brief openings (mean open lifetime, 1 ms) with cytosolic Ca²⁺ as the sole ligand. Increasing the free [Ca²⁺] increases Po by increasing the frequency of channel openings.^{9 10} Only rarely does cytosolic Ca²⁺ appear to increase the duration of open states, and this effect has a relatively minor effect on Po and can only be detected as Po approaches .5.⁹ Ca²⁺ alone cannot fully open the channel, and Po values of >.5 are rarely achieved with cytosolic Ca²⁺ alone.^{9 10} In comparison, the mechanisms involved in Ca²⁺ activation of the canine cardiac SR Ca²⁺-release channels appear to be different. Open lifetime durations appear to be an order of magnitude longer in canine cardiac channels when activated by Ca²⁺.^{17 18 19} Cytosolic Ca²⁺ increases Po not just by increasing the frequency of channel opening but also by causing large increases in the duration of the open lifetimes.^{18 19} Therefore, with sheep channels, cytosolic Ca²⁺ increases Po by binding to the closed channel state(s), whereas with canine channels, a Ca²⁺-induced increase in Po results from Ca²⁺ binding to both open and closed channel states. The overall Po-[Ca²⁺] relation for canine cardiac SR Ca²⁺-release channels is shifted to the left (EC₅₀, 1 µmol/L) compared with that for the sheep channels. In addition, in the canine channel, cytosolic Ca²⁺ alone can cause very high open probabilities (>.75),^{17 18} presumably because Ca²⁺ can increase the duration of the open lifetimes. Such apparently distinct mechanisms for Ca²⁺ activation between species may result in different channel gating behavior after rapid changes in [Ca²⁺].

The dissimilarities in Ca²⁺-activation mechanisms make direct comparisons difficult between our results and those of Györke and Fill.¹⁵ The decline in Po or adaptation of the canine SR Ca²⁺-release channel to flash photolysis of caged Ca²⁺ occurs predominantly at low [Ca²⁺] (at levels close to 1 µmol/L), but as the [Ca²⁺] approaches 10 µmol/L, the degree of adaptation appears to be slight. However, the Po–cytosolic [Ca²⁺] relation of the sheep SR Ca²⁺-release channel is different from that of the dog, and significant activation of the sheep channel with cytosolic Ca²⁺ cannot be observed below 10 µmol/L. In both steady state and rapid switching experiments, we observe no significant increase in Po when the [Ca²⁺] is increased from 0.1 to 1 µmol/L (data not shown). Therefore, it is not possible to directly compare rapid changes to identical concentrations of Ca²⁺ in dog and sheep channels by using the two different methods. It is only possible to compare activation of the channels with similar Po values. In order to obtain the high Po values that occur with maximal Ca²⁺ activation of the canine cardiac SR Ca²⁺-release channel, we have activated the sheep Ca²⁺-release channel with Ca²⁺ plus a second ligand (ATP or EMD 41000). However, even though the Po levels may be comparable in the two sets of experiments, a direct comparison of the data is not ideal in view of the different mechanisms involved in activating the dog and sheep channels.

It has been suggested that the observed change in Po with time reported by Györke and Fill¹⁵ may reflect an underlying change in [Ca²⁺] due to an initial Ca²⁺ spike before a steady state [Ca²⁺] is reached.^{20 21} Since the activation of the canine Ca²⁺-release channel occurs over a range where very slight changes in [Ca²⁺] (0.1 to 0.5 µmol/L) would be expected to result in large changes in Po, it is very important that this issue is resolved. It is apparent from our experiments that when the activating ligands are rapidly removed (Fig 2C), the Po rapidly declines to zero. Lamb and Stephenson²¹ argue that rapidly lowering the free [Ca²⁺] should indicate whether Ca²⁺-release channels close with a similar or a much faster time constant than seen in the adaptation reported by Györke and Fill. Certainly, our experiments with sheep cardiac SR Ca²⁺-release channels suggest that if a Ca²⁺ spike occurred, lasting approximately a millisecond, then Po would decline rapidly (within tens of milliseconds) rather than in the hundreds of milliseconds over which adaptation occurs. However, the dissimilar mechanisms for Ca²⁺ activation of the cardiac SR Ca²⁺-release channels from the sheep and the dog indicate that inactivation mechanisms may also be different. Canine Ca²⁺-release channels may not close rapidly after a brief Ca²⁺ spike, and this should be tested experimentally rather than extrapolating from results obtained with sheep channels. Another potential cause of non–steady state cytosolic [Ca²⁺] in the flash photolysis experiments is diffusion of Ca²⁺ away from Ca²⁺-activation sites on the Ca²⁺-release channels, as pointed out by Lamb and Stephenson.

Under our experimental conditions, a rapid increase in agonist concentration (either Ca²⁺ alone or Ca²⁺ plus a second ligand) leads to a maintained level of channel activation at -40 mV until removal of the agonists. However, at +40 mV, after switching to the solution containing the agonist(s), the channels may open briefly and then rapidly inactivate. The inactivation can be removed only by changing the holding potential briefly to a negative potential (-40 mV); merely removing the agonist(s) cannot reverse the inactivation. Such voltage-dependent inactivation bears some similarities to inactivation reported previously in SR Ca²⁺-release channels from skeletal muscle at certain holding potentials.^{22 23} Percival et al²² observed inactivation of the and ß isoforms of the chicken skeletal SR Ca²⁺-release channels at cis-positive potentials. Ma²³ also reported "desensitization" of the rabbit skeletal SR Ca²⁺-release channel. However, this phenomenon occurred at negative holding potentials, although the ionic conditions were very similar to those used in our experiments. The voltage-dependent inactivation observed in our experiments appears to be very dependent on the mechanism of channel activation. The inactivation occurred rarely when the channels were activated solely by cytosolic Ca²⁺ (17%) but occurred more readily when channels were opened by Ca²⁺ plus a second ligand (ATP or EMD 41000). As discussed previously, when the sheep cardiac SR Ca²⁺-release channel is activated by Ca²⁺ alone, the mechanism for the increased Po is predominantly an increase in the frequency of channel opening,^{9 10} whereas in the presence of a second ligand binding to the ATP or caffeine site, the Po can be elevated to very high levels, because both the duration and the frequency of channel opening are increased.^{11 13} Therefore, when the channel is synergistically activated by Ca²⁺ plus a second ligand, the different gating scheme that is operating may allow the channel to enter the inactivated state more readily. Possibly the channel has to enter a particularly long-lived open state before entry into the inactivated state. We have demonstrated that the voltage dependence of Po of both the cardiac and skeletal Ca²⁺-release channels from the sheep is very dependent on the luminal [Ca²⁺]^{24 25} and the mechanism of channel activation. Therefore, the holding potential at which voltage-dependent inactivation occurs may be controlled by the luminal [Ca²⁺]. Further experiments are required to establish if this is the case.

What are the implications of our results to the understanding of the behavior of the sheep cardiac Ca²⁺-release channel during EC coupling? In the bilayer, the rate of change of trigger [Ca²⁺] at the cytosolic face of the channel does not appear to be an important determinant of the final Po value. In the cell, however, other more complex factors may be involved, such as activation of neighboring channels by Ca²⁺ flowing through activated Ca²⁺-release channels, diffusion times away from the activation site, or Ca²⁺ binding to other buffers and proteins in the cytosolic space. Each may contribute to the rate-dependent effect on SR Ca²⁺ release observed by Fabiato.¹ Fabiato's experiments were performed on canine cardiac tissue; therefore, species differences may also contribute to the apparently disparate observations. What is clear from our experiments is that after a slow or a rapid increase in [Ca²⁺] at the cytosolic channel face, the Po of the channels will remain constant, given that the [Ca²⁺] and the environment of the channel remains constant. Therefore, we conclude that adaptation to a maintained Ca²⁺ stimulus is probably not the mechanism by which smoothly graded Ca²⁺-induced Ca²⁺ release occurs in sheep cardiac cells. However, other mechanisms are more likely to control the termination of Ca²⁺ release from the SR during EC coupling. In the absence of information on the structural organization of the Ca²⁺-release channels within the junctional regions of the SR and with conflicting reports of the gating behavior of the Ca²⁺-release channel, Stern²⁶ has convincingly modeled the inactivation of Ca²⁺ release by using arguments based primarily on the rapid diffusion of Ca²⁺ away from the Ca²⁺-activation sites on the channel. We suggest that other mechanisms may also contribute to the regulation of SR Ca²⁺ release. If releasable Ca²⁺ in the SR is depleted more rapidly than it is reloaded, then the reduction in luminal Ca²⁺ will decrease both the conductance of Ca²⁺ through the Ca²⁺-release channels²⁷ and the Po of the channels.²⁴ In addition, our experiments demonstrate that the cardiac Ca²⁺-release channel may be inactivated at cis-positive holding potentials (ie, with the SR lumen negative relative to the cytosol). Present evidence indicates that the skeletal SR K⁺ channel can maintain the SR membrane potential close to zero during Ca²⁺ release.²⁸ However, this parameter cannot be measured in the cell, and it is possible that even a small potential change resulting from the initial rapid release of Ca²⁺ from the SR would be sufficient to inactivate the Ca²⁺-release channel, which would remain inactivated until the normal resting potential was restored.

This is the first report of changes in [Ca²⁺] at the cytosolic face of single cardiac SR Ca²⁺-release channels on a physiological time scale. Our results suggest that in channels derived from sheep, the rate of change in cytosolic [Ca²⁺] does not directly affect the final Po of the channel. Our results also indicate that at cis-negative potentials, there is no desensitization, inactivation, or adaptation to Ca²⁺, ATP, or EMD 41000. At cis-positive potentials, some channels rapidly inactivate in a voltage-dependent manner. The voltage-dependent inactivation is highly dependent on the mechanism of channel activation, and further experiments are required to establish whether this may be a physiologically relevant mechanism of inactivation of Ca²⁺ current from the SR during EC coupling.

	Acknowledgments

 
This study was supported by The British Heart Foundation.

Received February 28, 1995; accepted June 26, 1995.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
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