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

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.

Keywords: calmodulin; intracellular calcium; ryanodine receptors.