Luminal calcium regulation of ryanodine receptor channels

The mechanism that links surface depolarization and RyR-mediated SR Ca release is called EC coupling. The features of EC coupling differs between cardiac and skeletal muscle. The specific mechanisms that control EC coupling are debated. Defining these mechanisms is significant and may also represent points of therapeutic intervention.;The overall goal of my research is to define how single RyR channels are regulated by changes in luminal Ca2+ concentration. To this end, function of single RyR channels was measured in planar lipid bilayers. Experimental manipulations applied included removing/replacing casequestrin (CSQ), changing cytosolic and luminal Ca2+ concentrations, swapping CSQ and RyR isoforms, comparing wildtype and mutant CSQs as well as defining the actions of certain cardiotoxic agents that target CSQ.;The following hypothesis was tested. The RyR1 channel function is not controlled by CSQ-dependent luminal Ca2+ regulation. The RyR2 channel is regulated by luminal Ca2+ acting at both RyR2-resident and RyR2-remote sites. The RyR2-remote Ca2+ sensor is CSQ and its operation depends on the structure-function of the CSQ isoform present. The RyR2-resident site does not distinguish between Ca2+ and Mg2+ limiting its physiological significance. The RyR2-remote site is Ca2+ specific and operates at physiologically relevant luminal Ca2+ levels (0.1 to 1 mM). Lastly, tricyclic antidepressants are not CSQ-specific experimental probes and thus their cardiac toxicity is not solely due to their actions on CSQ Ca2+ binding.;We found no significant luminal Ca2+ regulation of single RyR1 channels in the presence of either CSQ isoform tested. Thus, RyR1 regulation via the RyR1-CSQ1 complex is not significant implying CSQ1's chief role in skeletal muscle may be that of an intra-SR Ca2+ buffer.;Specifically, we found 2 luminal Ca2+ regulatory mechanism(s). One was CSQ-independent and presumed to be RyR2-resident. The other mechanism was CSQ2-dependent and presumed to be RyR2-remote processes. This RyR2-remote mechanism was independent of Ca2+ dependent CSQ2 oligomerization or CSQ2 monomer Ca2+ binding affinity. The key Ca2+ sensitive step was a Ca2+ dependent triadin-CSQ interaction. This indicates that CSQ2 is not the Ca2+ sensor per se. Instead, CSQ2 seems to act more like a required cofactor that needs to be present for luminal RyR2-remote Ca2+ regulation to operate.;The CSQ independent RyR2-resisend mechanism did not distinguish between luminal Ca2+ and Mg2+ and this limit the likelihood that it plays a major physiological role during normal EC coupling. Since the intra-SR free Mg2+ is like that in the cytosol (∼1 mM), there is ample intra-SR Mg2+ present to operate this mechanism as the intra-SR Ca2+ concentration changes over its normal physiological range (0.1 to 1 mM).;We found that AMT and NRT increased the mean open time and Po of CSQ-free RyR2 channels in a dose dependent fashion with EC50's of 4.2 and 8.9 muM, respectively. We also showed that high concentrations of these drugs evoked frequent long openings and concluded that these actions on RyR2 function likely contributes to their adverse cardiac side effects.;Several additional findings include: (1) The disparate actions of 2 catecholaminergic polymorphic ventricular tachycardia linked CSQ mutants on single RyR2 function indicate that either alteration or elimination of CSQ2-dependent luminal RyR2 regulation can generate the CPVT phenotype. (2) Luminal Ca2+ regulation of single RyR2 channels involves luminal Ca2+ dependent changes not only in the frequency of individual open events but also in the frequency of high Po bursts. (3) Like the tricyclic antidepressants, the antipsychotic trifluoperazine (TFP) activation of single RyR2 channels in CSQ independent and this likely contributes to the cardiotoxic side effects associated with TFP overdose. (4) Robust RyR2 activation by AMT and TFP indicate that these drugs may represent new interesting pharmacological probes for use in future Ca2+ release studies.