Do inactivation mechanisms rather than adaptation hold the key to understanding ryanodine receptor channel gating?

Abstract

The use of flash photolysis of caged Ca2+ to rapidly elevate [Ca2+] at the cytosolic face of a cardiac ryanodine receptor (RyR) channel reconstituted into a bilayer gives rise to a rapid increase in open probability (Po; Gyorke and Fill 1993). This is followed by a decline in Po, which occurs much more slowly (τ = 1.3 s). After the decline in Po, the channels can be reactivated by a second Ca2+ stimulus. These results led to the suggestion that RyR channels can adapt to a maintained Ca2+ stimulus. The report was of great interest for two main reasons. First, the concept that Ca2+-induced Ca2+ release (CICR) was a process that was smoothly graded and dependent on the magnitude of the trigger rather than an all-or-nothing process was difficult to explain. Not long before the publication of the Gyorke and Fill report (1993), Stern 1992 had brought this issue to the forefront of discussions on excitation–contraction (EC) coupling in cardiac muscle. He elegantly described how global models of EC coupling needed to be discarded because they were inherently unstable and that “local control” models, where discrete units of RyR channels were activated individually, could be operating. Stern 1992 argued that diffusion of Ca2+ away from the Ca2+ activation sites on RyR, coupled with the process of “stochastic attrition,” led to the inactivation of the Ca2+ release units. The contraction of the whole- cell, therefore, resulted from the summation of the individual packets of Ca2+ released from the separate release units. Stern's local control model of cardiac EC coupling made sense of seemingly irreconcilable discrepancies and stimulated workers in the field to investigate, extend, or disprove his theory. It was at this time that Gyorke and Fill 1993 presented adaptation as a self-regulating property of RyR channels that could serve as a “molecular control mechanism for smoothly graded Ca2+-induced Ca2+-release in heart.” On the face of it, they had uncovered a mechanism that would close the RyR channels to allow reaccumulation of Ca2+ within sarcoplasmic reticulum (SR) stores, and that could explain why CICR was not an all-or-nothing positive feedback process. Gyorke and Fill's paper (1993) was closely followed by the first report of Ca2+ sparks by Cheng et al. 1993. The improved temporal and spatial resolution of in situ SR Ca2+-release events afforded by the use of the confocal microscope accelerated the integration of theoretical models of EC coupling with experimental data. Here was evidence that local control of individual Ca2+ release units could provide smoothly graded CICR. This was an exciting time for cardiac physiologists and biophysicists, and the idea that adaptation of RyR might refine existing models of EC coupling was appealing. The second reason for the interest in this work was the proposal that the phenomenon of adaptation was not merely the response of RyR to a rapid step change in [Ca2+], but that the Ca2+ spike that preceded the maintained increase in [Ca2+] itself affected channel gating (Lamb et al. 1994; Lamb and Laver 1998). Thus, Lamb and co-workers (Lamb et al. 1994; Lamb and Laver 1998) argued adaptation was the overall response of RyR channels to both the Ca2+ spike and the longer lasting increase in [Ca2+]. The idea that the Ca2+ spike influenced the occurrence of adaptation was fueled by subsequent reports that adaptation did not occur when rapid changes in cytosolic [Ca2+] were produced using methods other than flash photolysis of a caged Ca2+ compound (Schiefer et al. 1995; Sitsapesan et al. 1995; Laver and Curtis 1996). The issue was further confounded by the more recent report that Ca2+ spikes, in the absence of a maintained elevation in [Ca2+], could activate RyR channels (Zahradnikova et al. 1999), although previously Gyorke and Fill 1994 had claimed vigorously that Ca2+ spikes could not activate RyR channels. In fact, this was used as evidence indicating that the fast Ca2+ spike did not influence the effect of the more maintained change in [Ca2+] (Gyorke and Fill 1994). There are certain issues that need to be resolved before we can understand how RyR channels respond to rapid changes in [Ca2+], and before we can hope to understand how SR Ca2+ release is triggered and terminated. First, does the Ca2+ spike that precedes the maintained increase in [Ca2+] in flash photolysis experiments affect the subsequent gating behavior of RyR? Second, what is the response of the RyR channel to a maintained step increase in [Ca2+]? Finally, does the gating behavior of RyR channels and the evidence from triggered Ca2+ release in intact cells support the proposal that RyR channels adapt to a maintained increase in [Ca2+]?