Calcium-dependent energetics of calmodulin domain interactions with regulatory regions of the Ryanodine Receptor Type 1 (RyR1)

Abstract

Calmodulin (CaM) allosterically regulates the homo-tetrameric human Ryanodine Receptor Type 1 (hRyR1): apo CaM activates the channel, while (Ca2+)4–CaM inhibits it. CaM-binding RyR1 residues 1975–1999 and 3614–3643 were proposed to allow CaM to bridge adjacent RyR1 subunits. Fluorescence anisotropy titrations monitored the binding of CaM and its domains to peptides encompassing hRyR11975–1999 or hRyR13614-3643. Both CaM and its C-domain associated in a calcium-independent manner with hRyR13614–3643 while N-domain required calcium and bound ~250-fold more weakly. Association with hRyR111975–1999 was weak. Both hRyR1 peptides increased the calcium-binding affinity of both CaM domains, while maintaining differences between them. These energetics support the CaM C-domain association with hRyR13614–3643 at low calcium, positioning CaM to respond to calcium efflux. However, the CaM N-domain affinity for hRyR11975–1999 alone was insufficient to support CaM bridging adjacent RyR1 subunits. Other proteins or elements of the hRyR1 structure must contribute to the energetics of CaM-mediated regulation.