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Letters to the Editor

Glowing Cinders That Underlie the Ca²⁺ Spark

Schneider 1999 recently addressed the question of whether Ca²⁺ sparks arise from the opening of a single ryanodine receptor (RyR) channel or the simultaneous opening of several channels. The discussion highlighted the importance of single RyR channel permeation and gating in the interpretation of Ca²⁺ spark data. The Schneider 1999 perspective inspired us to extend this theoretical discussion by using a published kinetic model of modal RyR gating to actually simulate RyR channel gating and permeation that may underlie a Ca²⁺ spark in cardiac muscle.

	The Single Channel Ca2+ Spark Interpretation

Top The Single Channel Ca2+... The Multichannel Ca2+ Spark... Simulating the RyR Channel... Conclusions REFERENCES

 
Cheng et al. 1993 was the first to propose that the spontaneous Ca²⁺ spark is the elementary intracellular Ca²⁺ release unit that underlies excitation–contraction coupling in cardiac muscle. They estimated that the local Ca²⁺ flux underlying the Ca²⁺ spark would need to be 2 x 10^–17 mol/s, assuming a volume of 10 fl (i.e., an 2-µm cube), duration of 10 ms (time to peak), and a final [Ca²⁺] of 300 nM . This type of calculation predicts that the underlying unitary RyR channel Ca²⁺ current would need to be 1–4 pA to generate the observed Ca²⁺ spark (Cheng et al. 1993; Pratusevich and Balke 1996; Blatter et al. 1997; Jiang et al. 1998; Schneider 1999). An early estimate of the unitary Ca²⁺ current through the cardiac RyR channel was 2.5 pA (at 0 mV with 50 mM charge carrier; Rousseau and Meissner 1989). This lead Cheng et al. 1993 to propose that the Ca²⁺ spark may arise from the opening of a single RyR Ca²⁺ release channel. The RyR channel, however, is a poorly selective Ca²⁺ channel, and thus other ions (e.g., K⁺ and Mg²⁺) are likely to compete with Ca²⁺ for occupancy of the pore. Consequently, the unitary Ca²⁺ current must be smaller under more physiological conditions (1 mM lumenal Ca²⁺, 150 mM K⁺, and 1 mM Mg²⁺). Tinker et al. 1993 used a RyR permeation model to estimate that the unitary Ca²⁺ current was 1.4 pA (at 0 mV, 1.2 mM lumenal Ca²⁺ charge carrier in symmetrical 120 mM K⁺ and 0.5 mM Mg²⁺). This updated estimate lead Blatter et al. 1997 to propose that simultaneous opening of two RyR channels may generate the Ca²⁺ spark. If Ca²⁺ sparks arise from the opening of one or two RyR channels, then certain pharmacological manipulations that alter single channel properties should be reflected at the Ca²⁺ spark level. Cheng et al. 1993 reported that lower amplitude, long duration Ca²⁺ sparks occur in the presence of ryanodine. This resembles the ryanodine-induced long-lasting subconductance states observed at the single channel level. Shtifman et al. 1999 reported that prolonged small-amplitude Ca²⁺ sparks occurred after application of Imperatoxin A (IpTx_A). This resembles the prolonged subconductance of IpTx_A-modified RyR channels in bilayers (Tripathy et al. 1998).

In summary, the single channel Ca²⁺ spark interpretation is largely based on two lines of evidence: first, the relatively large estimates of unitary RyR channel Ca²⁺ current and, second, the parallel pharmacological actions at the spark and single channel levels.

	The Multichannel Ca2+ Spark Interpretation

Top The Single Channel Ca2+... The Multichannel Ca2+ Spark... Simulating the RyR Channel... Conclusions REFERENCES

 
The hypothesis that multiple RyR channels open simultaneously to generate the Ca²⁺ spark is consistent with the clustered arrangement of RyR channels in heart (Sun et al. 1995; Franzini-Armstrong and Protasi 1997). It is also consistent with the stereotypic amplitude of the Ca²⁺ spark. If Ca²⁺ sparks were generated by spontaneous openings of a single channel, then the distribution of Ca²⁺ spark amplitudes should be exponential in nature because single channel open times are distributed exponentially. Observed Ca²⁺ spark amplitudes, however, are normally distributed. There is also a curious lack of small Ca²⁺ spark events that is not easy to reconcile with the single channel spark hypothesis. Recently, Mejía-Alvarez et al. 1999 have directly measured the amplitude of unitary Ca²⁺ current through a single cardiac RyR channel under quasi-physiological ionic conditions. The unitary Ca²⁺ current was considerably smaller than previously predicted (0.35 vs. 1.4 pA; Tinker et al. 1993). This suggests that Ca²⁺ sparks may arise from 3 to 10 RyR channels opening simultaneously.

In summary, the multichannel Ca²⁺ spark interpretation is based on three lines of evidence: first, the stereotypic nature of the Ca²⁺ spark; second, new smaller estimates of unitary RyR channel Ca²⁺ current; and, third, tantalizing correlations with the clear physical clustering of RyR in heart.

	Simulating the RyR Channel Gating that Underlies the Ca2+ Spark

Top The Single Channel Ca2+... The Multichannel Ca2+ Spark... Simulating the RyR Channel... Conclusions REFERENCES

 
A published kinetic Markovian scheme of RyR channel gating was used to generate simulated single RyR channel records. The simulated gating reflects single RyR channel measurements made in planar lipid bilayer studies (e.g., Sitsapesan and Williams 1995). The unitary Ca²⁺ current was fixed at 0.35 pA (Mejía-Alvarez et al. 1999). To predict free [Ca²⁺] fluctuations, a multicompartment unidimensional diffusion model was evaluated (Cannell and Allen, 1984; Pizarro et al. 1991). The diffusion model includes Ca²+ binding/unbinding to known buffers and SR Ca²⁺ reuptake. The only entity allowed to diffuse is the Ca²⁺ ion. The predicted fluorescence (Fluo-3) signals due to the local Ca²⁺ fluxes produced by the simulated single RyR channel activity were calculated and are presented in Fig. 1.

View larger version (22K): [in this window] [in a new window] [Download PPT slide]   Figure 1 Single RyR channel gating and Ca2+ spark simulations. (A) Steady state single channel simulation of RyR gating, predicted local Ca2+ flux, and resulting fluorescence signal. Individual single channel openings fail to elevate local Ca2+ sufficiently to be detected as a fluorescence signal. Local Ca2+ elevations induced by a burst of single channel openings generates a small but detectable fluorescence signal. (B) Triggered nonstationary gating simulation of five RyR channels. The trigger stimulus was applied at the arrow. The predicted local Ca2+ flux and resulting fluorescence signals are shown. Brief simultaneous opening of multiple RyR channels generate fluorescence signals reminiscent of experimentally observed Ca2+ sparks. The kinetic RyR channel gating scheme is from Villalba-Galea et al. 1998 and was based on the model proposed by Zahradníková and Zahradník 1996. The RyR channel mean open time was 0.5 ms and the unitary current amplitude was 0.3 pA, which corresponds to 9.4 x 105 ions/s. Simulations were run under steady state conditions (pCa 7) assuming the presence of a single RyR channel. Simulations of triggered RyR channel activity were run assuming the presence of five RyR channels. The applied trigger Ca2+ pulse (10 µM for 500 µs) was from pCa 7. This trigger Ca2+ stimulus (arrows) mimics that which may be generated by an opening of a single DHPR Ca2+ channel. For simplicity, the simulation assumed that the trigger Ca2+ pulse and RyR-mediated Ca2+ release occur in different pools. A multicompartment diffusion model was used to evaluate how the simulated single channel behavior (i.e., the underlying driving Ca2+ waveform) impacts local Fluo-3 (100 µM) fluorescence. Model parameters include: . The Ca2+ flux through a single RyR channel was 1.55 x 10–15 µM/ms. If five RyR channels open simultaneously for 500 µs in a volume of 10–15 liters, then the [Ca2+] was 775 nM. This value is considerably higher than that needed to generate a Ca2+ spark. However, the inclusion of parameters like binding and diffusion result in a [Ca2+] of 232 nM, which corresponds to a F/F0 of 1.53.

	Conclusions

Top The Single Channel Ca2+... The Multichannel Ca2+ Spark... Simulating the RyR Channel... Conclusions REFERENCES

 
Our simulations suggest that individual openings of a single RyR channel under steady state conditions at a resting Ca²⁺ level are unlikely to generate detectable local Ca²⁺ release events. Barely detectable Ca²⁺ release events occasionally occur when bursts of open events (lasting many milliseconds) occur. This implies that an abnormally long opening of a single RyR channel would generate a prolonged detectable local Ca²⁺ release. Simultaneous opening of multiple RyR channels generated fluorescence signals that were consistent with the observed Ca²⁺ spark waveform. We propose that the stereotypical Ca²⁺ sparks are generated by the simultaneous opening of multiple RyR channels. This proposition is consistent with our recent estimates of unitary Ca²⁺ current, the stereotypical nature of the spark, and the clustering of RyR channels in the diadic space. We also propose that pharmacological manipulations that generate small-prolonged local Ca²⁺ fluxes could arise from the opening of single RyR channels.

	REFERENCES

Top The Single Channel Ca2+... The Multichannel Ca2+ Spark... Simulating the RyR Channel... Conclusions REFERENCES

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This Article Full Text (PDF, 57K) PPT slides of all figures Alert me when this article is cited Citation Map Services Email this article Similar articles in this journal Similar articles in PubMed Alert me to new content in the JGP Download to citation manager Citing Articles Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Search for Related Content PubMed PubMed Citation Social Bookmarking                            What's this?