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.

Diminished inhibition and facilitated activation of RyR2‐mediated Ca2+ release is a common defect of arrhythmogenic calmodulin mutations

“…This is consistent with CaM binding to CaMBD and Ca 2+ binding to CaM being thermodynamically coupled [5,32,[35][36][37], and this enabled an estimation of CaM's affinity for binding to Ca 2+ when complexed with RyR2(R3581-L3611), i.e. estimating CaM's sensitivity to Ca 2+ when already bound to RyR2 CaMBD [5,6,31]. To this end, the FA measurements for the titration of CaM and peptide at constant concentrations (fixed ratio) with Ca 2+ were extracted from the larger titration data sets (Fig.…”

“…These serial titration curve analyses showed that CaM‐WT even without Ca 2+ (< 0.3 n m [Ca 2+ ] free ), bound to RyR2(R3581–L3611) with moderate affinity ( K D 1.4 μ m ) and that the CaM‐RyR2(R3581–L3611) interaction was extremely [Ca 2+ ] free ‐dependent (very steep decline in K D as a function of [Ca 2+ ] free ) with the K D decreasing approximately 1200‐fold to 1.1 n m over the [Ca 2+ ] free range 0.01–10 μ m . In the presence of RyR2(R3581–L3611) both the N‐ and C‐domain of CaM‐WT are fully Ca 2+ ‐bound at 10 μ m [Ca 2+ ] free and above, and no further increase in affinity with increasing [Ca 2+ ] free was observed above 10 μ m [Ca 2+ ] free . All arrhythmogenic CaM mutants retained a moderate affinity for binding to the RyR2 CaMBD both without Ca 2+ ( K D 1–2 μ m ), and also showed high affinity binding with 10 μ m [Ca 2+ ] free ( K D 1–8 n m ).…”

Role of cardiac ryanodine receptor calmodulin‐binding domains in mediating the action of arrhythmogenic calmodulin N‐domain mutation N54I

“…To investigate whether CaM‐N54I binding to RyR2 CaMBDs differs from that of the CaM‐WT, we measured the affinity of CaM‐WT and ‐N54I for binding to peptides representing each of the RyR2 CaMBDs (Table ). Because these interactions are strongly dependent on the [Ca 2+ ] free , we measured CaM’s affinity for binding to each peptide in a physiologically relevant [Ca 2+ ] free range (~ 0.05–400 µ m ) . This was done by titrating fluorescently labeled peptides with CaM in [Ca 2+ ] free ‐buffered solutions and monitoring the extent of CaM binding to the peptides using the change in peptide fluorescence anisotropy (FA).…”

“…By fitting these FA titration curves to a stoichiometric binding model, we obtained the affinity of CaM for binding to each peptide expressed as that interaction’s dissociation constant ( K D ) at each [Ca 2+ ] free (Fig. ) . A low K D equates to extensive protein–peptide complex formation and thus a high‐affinity interaction (see ‘Materials and methods’).…”

“…Our current model for the CaM‐RyR2 interaction is that under cardiomyocyte resting conditions (~ 100 n m [Ca 2+ ] cyt ), the CaM C‐domain is nearly saturated with Ca 2+ and constitutively anchored to CaMBD2 around Trp‐3587, whereas the CaM N‐domain is not bound to Ca 2+ or CaMBD2 and may interact with other RyR2 CaMBDs. Upon cardiomyocyte excitation, the CaM N‐domain binds Ca 2+ as [Ca 2+ ] cyt increases and this changes its interaction with RyR2 CaMBDs, and with one possibility being that the Ca 2+ ‐saturated CaM N‐domain binds to RyR2 CaMBD2 around Phe‐3603, as observed in cryo‐EM structures with Ca 2+ present . Structures of RyR2 in the physiological resting state (closed RyR2 and only the CaM C‐domain Ca 2+ ‐loaded) are not available, and the effect of the CaM C‐domain’s shift from Phe‐3607 to Trp‐3587 on the Ca 2+ ‐free CaM N‐domain remains unknown.…”