Ca²-mediated activation of the skeletal-muscle ryanodine receptor ion channel

Cryo-electron micrograph studies recently have identified a Ca²-binding site in the 2,200-kDa ryanodine receptor ion channel (RyR1) in skeletal muscle. To clarify the role of this site in regulating RyR1 activity, here we applied mutational, electro-physiological, and computational methods. Three amino acid residues that interact directly with Ca² were replaced, and these RyR1 variants were expressed in HEK293 cells. Single-site RyR1-E3893Q, -E3893V, -E3967Q, -E3967V, and -T5001A variants and double-site RyR1-E3893Q/E3967Q and -E3893V/ E3967V variants displayed cellular Ca² release in response to caffeine, which indicated that they retained functionality as caffeine-sensitive, Ca²-conducting channels in the HEK293 cell system. Using [³H]ryanodine binding and single-channel measurements of membrane isolates, we found that single- and double-site RyR1-E3893 and -E3967 variants are not activated by Ca². We also noted that RyR1-E3893Q/E3967Q and -E3893V/ E3967V variants maintain caffeine- and ATP-induced activation and that RyR1-E3893Q/E3967Q is inhibited by Mg² and elevated Ca². RyR1-T5001A exhibited decreased Ca² sensitivity compared with WT-RyR1 in single-channel measurements. Computational methods suggested that electrostatic interactions between Ca² and negatively charged glutamate residues have a critical role in transducing the functional effects of Ca² on RyR1. We conclude that the removal of negative charges in the recently identified RyR1 Ca²-binding site impairs RyR1 activation by physiological Ca² concentrations and results in loss of binding to Ca² or reduced Ca² affinity of the binding site.