Insight towards the identification of cytosolic Ca²⁺ -binding sites in ryanodine receptors from skeletal and cardiac muscle

Ca²⁺ plays a critical role in several processes involved in skeletal and cardiac muscle contraction. One key step in cardiac excitation-contraction (E-C) coupling is the activation of the cardiac ryanodine receptor (RYR2) by cytosolic Ca²⁺ elevations. Although this process is not critical for skeletal E-C coupling, the activation and inhibition of the skeletal ryanodine receptor (RYR1) seem to be important for overall muscle function. The RYR1 and RYR2 channels fall within the large category of Ca²⁺ -binding proteins that harbour highly selective Ca²⁺ -binding sites to receive and translate the various Ca²⁺ signals into specific functional responses. However, little is known about the precise localization of these sites within the cytosolic assembly of both RYR isoforms, although several experimental lines of evidence have highlighted their EF-hand nature. EF-hand proteins share a common helix-loop-helix structural motif with highly conserved residues involved in Ca²⁺ coordination. The first step in predicting EF-hand positive regions is to compare the primary protein structure with the EF-hand motif by employing available bioinformatics tools. Although this simple method narrows down search regions, it does not provide solid evidence regarding which regions bind Ca²⁺ in both RYR isoforms. In this review, we seek to highlight the key findings and experimental approaches that should strengthen our future efforts to identify the cytosolic Ca²⁺ -binding sites responsible for activation and inhibition in the RYR1 channel, as much less work has been conducted on the RYR2 channel.