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

• Published in: The Journal of biological chemistry Vol. 293; no. 50; pp. 19501 - 19509
• Main Authors: Xu, Le, Chirasani, Venkat R., Carter, Jordan S., Pasek, Daniel A., Dokholyan, Nikolay V., Yamaguchi, Naohiro, Meissner, Gerhard
• Format: Journal Article
• Language: English
• Published: 11200 Rockville Pike, Suite 302, Rockville, MD 20852-3110, U.S.A Elsevier Inc 14.12.2018; American Society for Biochemistry and Molecular Biology
• Subjects: Ca2+ activation; Ca2+ release channel; calcium homeostasis; computation; ion gating; Membrane Biology; mutagenesis; ryanodine receptor; sarcoplasmic reticulum (SR); skeletal muscle; structure-function analysis; Ca2+ release channel; ion gating; structure-function analysis; Ca2+ activation; calcium homeostasis; ryanodine receptor; skeletal muscle; sarcoplasmic reticulum (SR); mutagenesis; computation
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.RA118.004453

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, electrophysiological, 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.