Journal
BIOCHEMICAL JOURNAL
Volume 476, Issue -, Pages 193-209Publisher
PORTLAND PRESS LTD
DOI: 10.1042/BCJ20180545
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Funding
- Obelske Family Foundation
- Novo Nordic Foundation [NNF15OC0012345, NNF16OC0023344]
- Lundbeck Foundation [R151-2013-14432]
- Danish Council for Independent Research [DFF-4093-00242, DFF-4181-00447]
- Canadian Institutes of Health Research (CIHR)
- Heart and Stroke Foundation of Alberta, N.W.T. and Nunavut
- Canada Foundation for Innovation (CFI)
- Heart and Stroke Foundation Chair in Cardiovascular Research
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The Ca2+ sensor calmodulin (CaM) regulates cardiac ryanodine receptor (RyR2)-mediated Ca(2+)release from the sarcoplasmic reticulum. CaM inhibits RyR2 in a Ca2+-dependent manner and aberrant CaM-dependent inhibition results in life-threatening cardiac arrhythmias. However, the molecular details of the CaM-RyR2 interaction remain unclear. Four CaM-binding domains (CaMBD1a, -1b, -2, and -3) in RyR2 have been proposed. Here, we investigated the Ca(2+)dependent interactions between CaM and these CaMBDs by monitoring changes in the fluorescence anisotropy of carboxytetramethylrhodamine (TAMRA)-labeled CaMBD peptides during titration with CaM at a wide range of Ca(2+)concentrations. We showed that CaM bound to all four CaMBDs with affinities that increased with Ca2+ concentration. CaM bound to CaMBD2 and -3 with high affinities across all Ca(2+)concentrations tested, but bound to CaMBD1 a and -lb only at Ca(2+)concentrations above 0.2 mu M. Binding experiments using individual CaM domains revealed that the CaM C-domain preferentially bound to CaMBD2, and the N-domain to CaMBD3. Moreover, the Ca2+ affinity of the CaM C-domain in complex with CaMBD2 or -3 was so high that these complexes are essentially Ca2+ saturated under resting Ca2+ conditions. Conversely, the N-domain senses Ca2+ exactly in the transition from resting to activating Ca(2+)when complexed to either CaMBD2 or -3. Altogether, our results support a binding model where the CaM C-domain is anchored to RyR2 CaMBD2 and saturated with Ca2+ during Ca2+ oscillations, while the CaM N-domain functions as a dynamic Ca(2+)sensor that can bridge noncontiguous regions of RyR2 or clamp down onto CaMBD2.
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