Ca2+-dependent calmodulin binding to cardiac ryanodine receptor (RyR2) calmodulin-binding domains.

Malene Brohus,Filip Van Petegem,Sui Rong Wayne Chen,Mads T Søndergaard,Michael T Overgaard

doi:10.1042/bcj20180545

Abstract

The Ca2+ sensor calmodulin (CaM) regulates cardiac ryanodine receptor (RyR2)-mediated Ca2+ 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 Ca2+-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 Ca2+ 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 Ca2+ concentrations tested, but bound to CaMBD1a and -1b only at Ca2+ concentrations above 0.2 µM. 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 Ca2+ 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 Ca2+ sensor that can bridge noncontiguous regions of RyR2 or clamp down onto CaMBD2.

Highlights

Cardiac ryanodine receptors (RyR2s) are large intracellular Ca2+ channels that control the release of Ca2+ from the sarcoplasmic reticulum (SR) in cardiomyocytes [1]
The ability of CaM to interact with the four suggested CaM-binding domain (CaMBD) was evaluated by comparing the locations of each CaMBD in the rabbit RyR1 cryo-EM structure with the demonstrated CaM-binding site around CaMBD2 (Figure 2)
The size of CaM relative to the RyR channel is indicated in Figure 2 by juxtaposing the channel and the crystal structure of Ca2+-saturated CaM in complex with the RyR1 CaMBD2 (CaM/ RyR1CaMBD2)

Summary

Introduction

Cardiac ryanodine receptors (RyR2s) are large intracellular Ca2+ channels that control the release of Ca2+ from the sarcoplasmic reticulum (SR) in cardiomyocytes [1]. Excitation of cardiomyocytes causes a small influx of extracellular Ca2+ through voltage-gated Ca2+ channels (CaV1.2s). This initial rise in cytosolic Ca2+ activates RyR2 and results in a Ca2+-induced Ca2+ release (CICR) sufficient to cause myofilament contraction [1,2]. RyR2 forms homotetrameric channels that arrange in spatially well-defined Ca2+ release units [1]. This organization means that the concentration of free Ca2+ in the cytosol near RyR2 oscillates between ∼0.2 mM at diastole and 200–400 mM during CICR and systole [2,4,5]. The two CaM domains display distinct Ca2+ affinities and -kinetics, and are connected by a flexible linker region that allows them to make independent interactions [10,11,12,13,14]

Results

Discussion

Conclusion