Site-specific modification of calmodulin Ca²(+) affinity tunes the skeletal muscle ryanodine receptor activation profile

Biochem J. 2010 Nov 15;432(1):89-99. doi: 10.1042/BJ20100505.

Abstract

The skeletal muscle isoform of the ryanodine receptor Ca²(+)-release channel (RyR1) is regulated by Ca²(+) and CaM (calmodulin). CaM shifts the biphasic Ca²(+)-dependence of RyR1 activation leftward, effectively increasing channel opening at low Ca²(+) and decreasing channel opening at high Ca²(+). The conversion of CaM from a RyR1 activator into an inhibitor is due to the binding of Ca²(+) to CaM; however, which of CaM's four Ca²(+)-binding sites serves as the switch for this conversion is unclear. We engineered a series of mutant CaMs designed to individually increase the Ca²(+) affinity of each of CaM's EF-hands by increasing the number of acidic residues in Ca²(+)-chelating positions. Domain-specific Ca²(+) 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 Ca²(+) affinity and regulation of RyR1. However, the site III mutation N97D increased the Ca²(+)-binding affinity of CaM's C-terminal domain and caused CaM to inhibit RyR1 at a lower Ca²(+) concentration than wild-type CaM. Conversely, the site IV mutation Q135D decreased the Ca²(+)-binding affinity of CaM's C-terminal domain and caused CaM to inhibit RyR1 at higher Ca²(+) concentrations. These results support the hypothesis that Ca²(+) 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 Ca²(+) affinity may be a valid strategy to tune the activation profile of CaM-regulated ion channels.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Animals
  • Binding Sites / genetics
  • Binding, Competitive
  • Calcium / chemistry
  • Calcium / metabolism*
  • Calmodulin / chemistry
  • Calmodulin / genetics
  • Calmodulin / metabolism*
  • Circular Dichroism
  • Kinetics
  • Models, Molecular
  • Molecular Sequence Data
  • Muscle, Skeletal / metabolism*
  • Mutation
  • Protein Binding
  • Protein Conformation
  • Protein Structure, Tertiary
  • Ryanodine / metabolism
  • Ryanodine Receptor Calcium Release Channel / genetics
  • Ryanodine Receptor Calcium Release Channel / metabolism*
  • Ryanodine Receptor Calcium Release Channel / physiology
  • Sarcoplasmic Reticulum / metabolism
  • Sequence Homology, Amino Acid
  • Swine
  • Tritium

Substances

  • Calmodulin
  • Ryanodine Receptor Calcium Release Channel
  • Tritium
  • Ryanodine
  • Calcium