Site-specific modification of calmodulin Ca(+) affinity tunes the skeletal muscle ryanodine receptor activation profile. Academic Article uri icon

abstract

  • The skeletal muscle isoform of the ryanodine receptor Ca(+)-release channel (RyR1) is regulated by Ca(+) and CaM (calmodulin). CaM shifts the biphasic Ca(+)-dependence of RyR1 activation leftward, effectively increasing channel opening at low Ca(+) and decreasing channel opening at high Ca(+). The conversion of CaM from a RyR1 activator into an inhibitor is due to the binding of Ca(+) to CaM; however, which of CaM's four Ca(+)-binding sites serves as the switch for this conversion is unclear. We engineered a series of mutant CaMs designed to individually increase the Ca(+) affinity of each of CaM's EF-hands by increasing the number of acidic residues in Ca(+)-chelating positions. Domain-specific Ca(+) affinities of each CaM variant were determined by equilibrium fluorescence titration. Mutations in sites I (T26D) or II (N60D) in CaM's N-terminal domain had little effect on CaM Ca(+) affinity and regulation of RyR1. However, the site III mutation N97D increased the Ca(+)-binding affinity of CaM's C-terminal domain and caused CaM to inhibit RyR1 at a lower Ca(+) concentration than wild-type CaM. Conversely, the site IV mutation Q135D decreased the Ca(+)-binding affinity of CaM's C-terminal domain and caused CaM to inhibit RyR1 at higher Ca(+) concentrations. These results support the hypothesis that Ca(+) binding to CaM's C-terminal acts as the switch converting CaM from a RyR1 activator into a channel inhibitor. These results indicate further that targeting CaM's Ca(+) affinity may be a valid strategy to tune the activation profile of CaM-regulated ion channels.

published proceedings

  • Biochem J

author list (cited authors)

  • Jiang, J., Zhou, Y., Zou, J., Chen, Y., Patel, P., Yang, J. J., & Balog, E. M.

citation count

  • 18

complete list of authors

  • Jiang, Jie||Zhou, Yubin||Zou, Jin||Chen, Yanyi||Patel, Priya||Yang, Jenny J||Balog, Edward M

publication date

  • November 2010