FRET-based mapping of calmodulin bound to the RyR1 Ca 2+ release channel

Razvan L. Cornea, Florentin Nitu, Simon Gruber, Katherine Kohler, Michael Satzer, David D. Thomas, Bradley R. Fruen

Research output: Contribution to journalArticle

44 Scopus citations

Abstract

Calmodulin (CaM) functions as a regulatory subunit of ryanodine receptor (RyR) channels, modulating channel activity in response to changing [Ca 2+]i. To investigate the structural basis of CaM regulation of the RyR1 isoform, we used site-directed labeling of channel regulatory subunits and fluorescence resonance energy transfer (FRET). Donor fluorophore was targeted to the RyR1 cytoplasmic assembly by preincubating sarcoplasmic reticulum membranes with a fluorescent FK506-binding protein (FKBP), and FRET was determined following incubations in the presence of fluorescent CaMs in which acceptor fluorophore was attached within the N lobe, central linker, or C lobe. Results demonstrated strong FRET to acceptors attached within CaM's N lobe, whereas substantially weaker FRET was observed when acceptor was attached within CaM's central linker or C lobe. Surprisingly, Ca 2+ evoked little change in FRET to any of the 3 CaM domains. Donor-acceptor distances derived from our FRET measurements provide insights into CaM's location and orientation within the RyR1 3D architecture and the conformational switching that underlies CaM regulation of the channel. These results establish a powerful new approach to resolving the structure and function of RyR channels.

Original languageEnglish (US)
Pages (from-to)6128-6133
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume106
Issue number15
DOIs
Publication statusPublished - Apr 14 2009
Externally publishedYes

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Keywords

  • Calcium
  • Excitation-contraction coupling
  • FKBP
  • Fluorescence
  • Ryanodine receptor
  • Sarcoplasmic reticulum

ASJC Scopus subject areas

  • General

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