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

Ca 2+ 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 ( RYR 2) by cytosolic Ca 2+ elevations. Although this process is not critical for skeletal E–C coupling, the activation and inhibition of the skeletal ryanodine receptor ( RYR 1) seem to be important for overall muscle function. The RYR 1 and RYR 2 channels fall within the large category of Ca 2+ ‐binding proteins that harbour highly selective Ca 2+ ‐binding sites to receive and translate the various Ca 2+ 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 Ca 2+ 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 Ca 2+ 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 Ca 2+ ‐binding sites responsible for activation and inhibition in the RYR 1 channel, as much less work has been conducted on the RYR 2 channel.