Luminal Ca2+ Regulation of Single Cardiac Ryanodine Receptors: Insights Provided by Calsequestrin and its Mutants

Abstract

The luminal Ca2+ regulation of cardiac ryanodine receptor (RyR2) was explored at the single channel level. The luminal Ca2+ and Mg2+ sensitivity of single CSQ2-stripped and CSQ2-associated RyR2 channels was defined. Action of wild-type CSQ2 and of two mutant CSQ2s (R33Q and L167H) was also compared. Two luminal Ca2+ regulatory mechanism(s) were identified. One is a RyR2-resident mechanism that is CSQ2 independent and does not distinguish between luminal Ca2+ and Mg2+. This mechanism modulates the maximal efficacy of cytosolic Ca2+ activation. The second luminal Ca2+ regulatory mechanism is CSQ2 dependent and distinguishes between luminal Ca2+ and Mg2+. It does not depend on CSQ2 oligomerization or CSQ2 monomer Ca2+ binding affinity. The key Ca2+-sensitive step in this mechanism may be the Ca2+-dependent CSQ2 interaction with triadin. The CSQ2-dependent mechanism alters the cytosolic Ca2+ sensitivity of the channel. The R33Q CSQ2 mutant can participate in luminal RyR2 Ca2+ regulation but less effectively than wild-type (WT) CSQ2. CSQ2-L167H does not participate in luminal RyR2 Ca2+ regulation. The disparate actions of these two catecholaminergic polymorphic ventricular tachycardia (CPVT)–linked mutants implies that either alteration or elimination of CSQ2-dependent luminal RyR2 regulation can generate the CPVT phenotype. We propose that the RyR2-resident, CSQ2-independent luminal Ca2+ mechanism may assure that all channels respond robustly to large (>5 μM) local cytosolic Ca2+ stimuli, whereas the CSQ2-dependent mechanism may help close RyR2 channels after luminal Ca2+ falls below ∼0.5 mM.

Unexpected Structural and Functional Consequences of the R33Q Homozygous Mutation in Cardiac Calsequestrin

“…Therefore, based on the present data, we propose that 2 consequences prompted by the mutation concur to the pathogenesis of the homozygous CASQ2 R33Q/R33Q phenotype: (1) reduction of CASQ2 levels (likely attributable to post/translational regulation leading to increased protein degradation, see below) and (2) impairment of RyR2 deactivation, as shown by Terentyev et al 13 and Qin et al 21 Despite a substantial reduction of CASQ2 seems to be central in recessive CPVT, it is still a matter of debate whether a threshold decrease of CASQ2 is required for eliciting the CPVT phenotype or whether even a minor loss of CASQ2 increases propensity to arrhythmias. At variance with data reported in the heterozygous knock out mouse model, 22 most human heterozygous individuals carriers of premature truncations of CASQ2 are asymptomatic.…”

“…Terentyev et al 13 linked the observed decrease in free calcium to the impairment of CASQ2 to act as the intraluminal calcium sensor of the RyR2 macromolecular complex 20 and to the impairment of the physiological deactivation of the RyR2 at low intraluminal calcium levels. Furthermore, based on single channel analysis, 21 we have shown that R33Q failed to inactivate RyR2 as intra-SR [Ca 2ϩ ] decreased. Thus, despite the leaky RyR2, normal calcium transients were recorded because of the compensatory effect of the high calcium binding provided by overexpressed CASQ2-R33Q and endogenous CASQ2-WT.…”

Calcium alternans in a couplon network model of ventricular myocytes: role of sarcoplasmic reticulum load

“…In addition, no luminal Ca regulation of RyR is present in this model, the existence of which has been demonstrated in experimental studies (23,29,43,65). However, the regulation of RyRs is still an issue in debate, which is summarized in a recent review article by Sobie and Lederer (60).…”

Calsequestrin Accumulation in Rough Endoplasmic Reticulum Promotes Perinuclear Ca2+ Release

“…First, the RyR2 luminal Ca 2ϩ -sensing mechanism in the rough ER may be different from that in the junctional SR. The junctional SR is a specialized subcellular compartment where CSQ2 co-localizes with and acts in concert with triadin-1 and junctin to regulate the luminal Ca 2ϩ sensing of RyR2 (6,7,13,36,37). However, this complex has not been shown to exist in the rough ER.…”