Resolved Structural States of Calmodulin in Regulation of Skeletal Muscle Calcium Release

Megan R. McCarthy, Yahor Savich, Razvan L. Cornea, David D. Thomas

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

Calmodulin (CaM) is proposed to modulate activity of the skeletal muscle sarcoplasmic reticulum (SR) calcium release channel (ryanodine receptor, RyR1 isoform) via a mechanism dependent on the conformation of RyR1-bound CaM. However, the correlation between CaM structure and functional regulation of RyR in physiologically relevant conditions is largely unknown. Here, we have used time-resolved fluorescence resonance energy transfer (TR-FRET) to study structural changes in CaM that may play a role in the regulation of RyR1. We covalently labeled each lobe of CaM (N and C) with fluorescent probes and used intramolecular TR-FRET to assess interlobe distances when CaM is bound to RyR1 in SR membranes, purified RyR1, or a peptide corresponding to the CaM-binding domain of RyR (RyRp). TR-FRET resolved an equilibrium between two distinct structural states (conformations) of CaM, each characterized by an interlobe distance and Gaussian distribution width (disorder). In isolated CaM, at low Ca2+, the two conformations of CaM are resolved, centered at 5 nm (closed) and 7 nm (open). At high Ca2+, the equilibrium shifts to favor the open conformation. In the presence of RyRp at high Ca2+, the closed conformation shifts to a more compact conformation and is the major component. When CaM is bound to full-length RyR1, either purified or in SR membranes, strikingly different results were obtained: 1) the two conformations are resolved and more ordered, 2) the open state is the major component, and 3) Ca2+ stabilized the closed conformation by a factor of two. We conclude that the Ca2+-dependent structural distribution of CaM bound to RyR1 is distinct from that of CaM bound to RyRp. We propose that the function of RyR1 is tuned to the Ca2+-dependent structural dynamics of bound CaM.

Original languageEnglish (US)
Pages (from-to)1090-1100
Number of pages11
JournalBiophysical journal
Volume118
Issue number5
DOIs
StatePublished - Mar 10 2020

Bibliographical note

Funding Information:
Spectroscopy was performed at the Biophysical Technology Center, University of Minnesota. Mass spectrometry was carried out at the University of Minnesota Chemistry Department Mass Spectrometry Laboratory. This work was supported in part by National Institutes of Health (NIH) Fellowship 1F31AG052329-01A1 and American Heart Association Predoctoral Fellowship 15PRE25700131 to M.R.M., NIH training grant T32AR007612 and University of Minnesota Interdisciplinary Doctoral Fellowship to Y.S., NIH grants R37AG026160 to D.D.T., and R01HL092097 to R.L.C.

Funding Information:
We thank Dr. John Lipscomb for helpful feedback and discussions on the binding assays, Mykola Dimura for modeling discussion, Dr. Jingyan Zhang and Dr. Margaret Titus for critical feedback on the manuscript, Claire Haskin and Opoku Akyeampong for assistance with HSR preparations, Dr. Robyn Rebbeck for technical advice, and Destiny Ziebol for assistance with manuscript preparation. Spectroscopy was performed at the Biophysical Technology Center, University of Minnesota. Mass spectrometry was carried out at the University of Minnesota Chemistry Department Mass Spectrometry Laboratory. This work was supported in part by National Institutes of Health (NIH) Fellowship 1F31AG052329-01A1 and American Heart Association Predoctoral Fellowship 15PRE25700131 to M.R.M. NIH training grant T32AR007612 and University of Minnesota Interdisciplinary Doctoral Fellowship to Y.S. NIH grants R37AG026160 to D.D.T. and R01HL092097 to R.L.C.

Publisher Copyright:
© 2020

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

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