Ca 2 -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

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Abstract

Cryo-electron micrograph studies recently have identified a Ca 2 -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 Ca 2 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 Ca 2 release in response to caffeine, which indicated that they retained functionality as caffeine-sensitive, Ca 2 -conducting channels in the HEK293 cell system. Using [ 3 H]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 Ca 2 . 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 Mg 2 and elevated Ca 2 . RyR1-T5001A exhibited decreased Ca 2 sensitivity compared with WT-RyR1 in single-channel measurements. Computational methods suggested that electrostatic interactions between Ca 2 and negatively charged glutamate residues have a critical role in transducing the functional effects of Ca 2 on RyR1. We conclude that the removal of negative charges in the recently identified RyR1 Ca 2 -binding site impairs RyR1 activation by physiological Ca 2 concentrations and results in loss of binding to Ca 2 or reduced Ca 2 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 - Jan 1 2018

Fingerprint

Ryanodine Receptor Calcium Release Channel
Ion Channels
Muscle
Skeletal Muscle
Chemical activation
Caffeine
HEK293 Cells
Binding Sites
Computational methods
Ryanodine
Coulomb interactions
Static Electricity

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Xu, L., Chirasani, V. R., Carter, J. S., Pasek, D. A., Dokholyan, N. V., Yamaguchi, N., & Meissner, G. (2018). Ca 2 -mediated activation of the skeletal-muscle ryanodine receptor ion channel Journal of Biological Chemistry, 293(50), 19501-19509. https://doi.org/10.1074/jbc.RA118.004453
Xu, Le ; Chirasani, Venkat R. ; Carter, Jordan S. ; Pasek, Daniel A. ; Dokholyan, Nikolay V. ; Yamaguchi, Naohiro ; Meissner, Gerhard. / Ca 2 -mediated activation of the skeletal-muscle ryanodine receptor ion channel In: Journal of Biological Chemistry. 2018 ; Vol. 293, No. 50. pp. 19501-19509.
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Xu, L, Chirasani, VR, Carter, JS, Pasek, DA, Dokholyan, NV, Yamaguchi, N & Meissner, G 2018, ' Ca 2 -mediated activation of the skeletal-muscle ryanodine receptor ion channel ', Journal of Biological Chemistry, vol. 293, no. 50, pp. 19501-19509. https://doi.org/10.1074/jbc.RA118.004453

Ca 2 -mediated activation of the skeletal-muscle ryanodine receptor ion channel . / Xu, Le; Chirasani, Venkat R.; Carter, Jordan S.; Pasek, Daniel A.; Dokholyan, Nikolay V.; Yamaguchi, Naohiro; Meissner, Gerhard.

In: Journal of Biological Chemistry, Vol. 293, No. 50, 01.01.2018, p. 19501-19509.

Research output: Contribution to journalArticle

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T1 - Ca 2 -mediated activation of the skeletal-muscle ryanodine receptor ion channel

AU - Xu, Le

AU - Chirasani, Venkat R.

AU - Carter, Jordan S.

AU - Pasek, Daniel A.

AU - Dokholyan, Nikolay V.

AU - Yamaguchi, Naohiro

AU - Meissner, Gerhard

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