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BACKGROUND: This study was designed to determine whether the cardiac ryanodine receptor (RyR2) central domain, a region associated with catecholamine polymorphic ventricular tachycardia (CPVT) mutations, interacts with the RyR2 regulators, ATP and the FK506-binding protein 12.6 (FKBP12.6). METHODS: Wild-type (WT) RyR2 central domain constructs (G(2236)to G(2491)) and those containing the CPVT mutations P2328S and N2386I, were expressed as recombinant proteins. Folding and stability of the proteins were examined by circular dichroism (CD) spectroscopy and guanidine hydrochloride chemical denaturation. RESULTS: The far-UV CD spectra showed a soluble stably-folded protein with WT and mutant proteins exhibiting a similar secondary structure. Chemical denaturation analysis also confirmed a stable protein for both WT and mutant constructs with similar two-state unfolding. ATP and caffeine binding was measured by fluorescence spectroscopy. Both ATP and caffeine bound with an EC50 of ~200-400μM, and the affinity was the same for WT and mutant constructs. Sequence alignment with other ATP binding proteins indicated the RyR2 central domain contains the signature of an ATP binding pocket. Interaction of the central domain with FKBP12.6 was tested by glutaraldehyde cross-linking and no association was found. CONCLUSIONS: The RyR2 central domain, expressed as a 'correctly' folded recombinant protein, bound ATP in accord with bioinformatics evidence of conserved ATP binding sequence motifs. An interaction with FKBP12.6 was not evident. CPVT mutations did not disrupt the secondary structure nor binding to ATP. GENERAL SIGNIFICANCE: Part of the RyR2 central domain CPVT mutation cluster, can be expressed independently with retention of ATP binding. more...

Aims Our objective was to explore the functional interdependence of protein kinase A (PKA) phosphorylation with binding of modulatory FK506 binding proteins (FKBP12/12.6) to the ryanodine receptor (RyR). RyR type 1 or type 2 was prepared from rabbit skeletal muscle or pig cardiac muscle, respectively. In heart failure, RyR2 dysfunction is implicated in fatal arrhythmia and RyR1 dysfunction is associated with muscle fatigue. A controversial underlying mechanism of RyR1/2 dysfunction is proposed to be hyperphosphorylation of RyR1/2 by PKA, causing loss of FKBP12/12.6 binding that is reversible by the experimental inhibitory drug K201 (JTV519). Phosphorylation is also a trigger for fatal arrhythmia in catecholaminergic polymorphic ventricular tachycardia associated with point mutations in RyR2. Methods and results Equilibrium binding kinetics of RyR1/2 to FKBP12/12.6 were measured using surface plasmon resonance (Biacore). Free Ca2+ concentration was used to modulate the open/closed conformation of RyR1/2 channels measured using [3H]ryanodine binding assays. The affinity constant— K A, for RyR1/2 binding to FKBP12/12.6, was significantly greater for the closed compared with the open conformation. The effect of phosphorylation or K201 was to reduce the K A of the closed conformation by increasing the rate of dissociation k d. K201 reduced [3H]ryanodine binding to RyR1/2 at all free Ca2+ concentrations including PKA phosphorylated preparations. Conclusion The results are explained through a model proposing that phosphorylation and K201 acted similarly to change the conformation of RyR1/2 and regulate FKBP12/12.6 binding. K201 stabilized the conformation, whereas phosphorylation facilitated a subsequent molecular event that might increase the rate of an open/closed conformational transition. more...

The cardiac ryanodine receptor (RyR2) Ca2+ release channel plays a central role in the rapid calcium release from the sarcoplasmic reticulum that is essential for muscle excitation-contraction coupling. Discrete mutations that have been discovered within the RyR2 associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) are found to cluster in distinct regions of RyR2. These regions may represent important regulatory domains of the molecule that either directly or indirectly can affect channel gating. In response to a physiological trigger, RyR2 mutations are believed to cause diastolic Ca2+ leak which results in arrhythmia, making them an important potential therapeutic target.We have expressed the RyR2 central domain CPVT-associated region and examined parameters which contribute to the structural and functional stability. A wild-type construct was compared with three constructs each containing a different CPVT mutation (P2328S, N2368I or A2387P). Circular dichroism spectroscopy revealed that none of the mutations significantly altered the percentage ellipticity of the protein, indicating that the overall conformation of the polypeptide backbone is only slightly affected by the mutations. Chemical denaturation using guanidine hydrochloride and monitoring tryptophan fluorescence suggested that the P2328S mutation was less stable than the wild-type or the other two mutations, indicated by a lower free energy change upon unfolding. A predicted ATP binding motif has previously been proposed in this domain and using fluorescence spectroscopy for the wild-type construct, ATP binding was observed with a Kd ∼0.03mM. more...

The sarcoplasmic reticulum (SR) of cardiomyocytes contains the cardiac ryanodine receptor (RyR2) - a calcium release channel that plays a pivotal role in mediating the SR calcium release essential for cardiac excitation-contraction coupling. Discrete clusters of point mutations, that may comprise important regulatory regions within RyR2, have been associated with catecholaminergic polymorphic ventricular tachycardia (CPVT). In response to a physiological trigger, RyR2 bearing CPVT mutations are believed to cause diastolic calcium leak, which can result in a fatal arrhythmia. A RyR2 central domain, which encompasses both a CPVT-associated region and a predicted ATP binding motif, has been expressed as a soluble recombinant protein. The wild-type protein was compared with three constructs each containing a different CPVT mutation (P2328S, F2331S and N2368I). ATP is a physiological activator of RyR2 activity and caffeine has a similar effect. Fluorescence spectroscopy was used to record changes in the intrinsic fluorescence of the single tryptophan residue present within the expressed domain, and we found evidence for protein conformational changes upon ATP or caffeine binding. We observed a similar binding affinity of the expressed RyR2 central domain for both compounds, with an EC50 ∼100μM. The presence of the individual mutations made no apparent difference to the EC50. However, there was an increased initial fluorescence value observed for each of the mutant constructs compared to WT. Analysis of the central domain sequence revealed homology with conserved motifs typical of P-loop kinases. Interestingly, many of the CPVT mutations present in this region are coincident with sites corresponding to the Walker A, Walker B and LID motifs in ATP binding proteins. more...