Ryanodine Receptor Permeation and Gating : Glowing Cinders That Underlie the Ca ²⁺ Spark

Abstract

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 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 × 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 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}({\mathrm{resting}}\;[{\mathrm{Ca}}^{2}+]\;=\;100\;{\mathrm{nM}})\end{equation*}\end{document} . 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 (IpTxA). This resembles the prolonged subconductance of IpTxA-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 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 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. At a steady state Ca2+ concentration of pCa 7, the applied RyR gating scheme predicts that spontaneous single channel events occur at low open probability (P o). (The gating scheme does not consider other regulatory factors [e.g., Mg2+] that may impact the stationary P o of the channel.) Most single channel open events are brief and bursts of open events are rare (Fig. 1 A). Every RyR channel opening elevates the local Ca2+ concentration. However, nearly all local Ca2+ elevations would not be detected as Fluo-3 fluorescence signals. The largest local Ca2+ elevations induced by bursts of RyR openings are just barely detectable at the fluorescence level. The same RyR gating scheme was also used to predict the response of five RyR channels to a trigger Ca2+ pulse (10 μM for 500 μs). The trigger Ca2+ pulse was applied to synchronize the opening of the RyR channels. Simultaneous opening of multiple RyR channels elevates the local Ca2+ concentration to levels consistent with that predicted to underlie the Ca2+ spark (Fig. 1 B). These local Ca2+ concentrations generate Fluo-3 fluorescence signals reminiscent of the experimentally observed Ca2+ spark. Conclusions 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.