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

Le Xu, Venkat R. Chirasani, Jordan S. Carter, Daniel A. Pasek, Nikolay V. Dokholyan, Naohiro Yamaguchi, Gerhard Meissner

Research output: Contribution to journalArticle

6 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)19501-19509
Number of pages9
JournalJournal of Biological Chemistry
Volume293
Issue number50
DOIs
StatePublished - Dec 14 2018

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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