ATP and Caffeine Binding to the Human RyR2 Central Domain Encompassing a CPVT Mutation Cluster

Abstract

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.