The skeletal muscle isoform of the ryanodine receptor Ca2+-release channel (RyR1) is regulated by Ca2+ and CaM (calmodulin). CaM shifts the biphasic Ca2+-dependence of RyR1 activation leftward, effectively increasing channel opening at low Ca2+ and decreasing channel opening at high Ca2+. The conversion of CaM from a RyR1 activator into an inhibitor is due to the binding of Ca2+ to CaM; however, which of CaM's four Ca2+-binding sites serves as the switch for this conversion is unclear. We engineered a series of mutant CaMs designed to individually increase the Ca2+ affinity of each of CaM's EF-hands by increasing the number of acidic residues in Ca2+-chelating positions. Domain-specific Ca2+ affinities of each CaM variant were determined by equilibrium fluorescence titration. Mutations in sites I (T26D) or II (N60D) in CaM's N-terminal domain had little effect on CaM Ca2+ affinity and regulation of RyR1. However, the site III mutation N97D increased the Ca2+-binding affinity of CaM's C-terminal domain and caused CaM to inhibit RyR1 at a lower Ca2+ concentration than wild-type CaM. Conversely, the site IV mutation Q135D decreased the Ca2+-binding affinity of CaM's C-terminal domain and caused CaM to inhibit RyR1 at higher Ca2+ concentrations. These results support the hypothesis that Ca2+ binding to CaM's C-terminal acts as the switch converting CaM from a RyR1 activator into a channel inhibitor. These results indicate further that targeting CaM's Ca2+ affinity may be a valid strategy to tune the activation profile of CaM-regulated ion channels.
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Research Article|
October 25 2010
Site-specific modification of calmodulin Ca2+ affinity tunes the skeletal muscle ryanodine receptor activation profile
Jie Jiang;
Jie Jiang
*Department of Chemistry, Georgia State University, Atlanta, GA 30303, U.S.A.
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Yubin Zhou;
Yubin Zhou
*Department of Chemistry, Georgia State University, Atlanta, GA 30303, U.S.A.
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Jin Zou;
Jin Zou
*Department of Chemistry, Georgia State University, Atlanta, GA 30303, U.S.A.
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Yanyi Chen;
Yanyi Chen
*Department of Chemistry, Georgia State University, Atlanta, GA 30303, U.S.A.
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Priya Patel;
Priya Patel
†School of Applied Physiology, Georgia Institute of Technology, Atlanta, GA 30332, U.S.A.
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Jenny J. Yang;
Jenny J. Yang
1
*Department of Chemistry, Georgia State University, Atlanta, GA 30303, U.S.A.
1Correspondence may be addressed to either of these authors (email chejjy@langate.gsu.edu or ed.balog@ap.gatech.edu).
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Edward M. Balog
Edward M. Balog
1
†School of Applied Physiology, Georgia Institute of Technology, Atlanta, GA 30332, U.S.A.
1Correspondence may be addressed to either of these authors (email chejjy@langate.gsu.edu or ed.balog@ap.gatech.edu).
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Publisher: Portland Press Ltd
Received:
April 05 2010
Revision Received:
August 03 2010
Accepted:
September 03 2010
Accepted Manuscript online:
September 03 2010
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2010 Biochemical Society
2010
Biochem J (2010) 432 (1): 89–99.
Article history
Received:
April 05 2010
Revision Received:
August 03 2010
Accepted:
September 03 2010
Accepted Manuscript online:
September 03 2010
Citation
Jie Jiang, Yubin Zhou, Jin Zou, Yanyi Chen, Priya Patel, Jenny J. Yang, Edward M. Balog; Site-specific modification of calmodulin Ca2+ affinity tunes the skeletal muscle ryanodine receptor activation profile. Biochem J 15 November 2010; 432 (1): 89–99. doi: https://doi.org/10.1042/BJ20100505
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