title: Site-specific modification of calmodulin Ca²(+) affinity tunes the skeletal muscle ryanodine receptor activation profile.
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.
authors.affiliation: Department of Chemistry, Georgia State University, Atlanta, 30303, USA.
authors.forename: Jie
authors.initials: J
authors.lastname: Jiang
authors.forename: Yubin
authors.initials: Y
authors.lastname: Zhou
authors.forename: Jin
authors.initials: J
authors.lastname: Zou
authors.forename: Yanyi
authors.initials: Y
authors.lastname: Chen
authors.forename: Priya
authors.initials: P
authors.lastname: Patel
authors.forename: Jenny J
authors.initials: JJ
authors.lastname: Yang
authors.forename: Edward M
authors.initials: EM
authors.lastname: Balog
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism*; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012433:Ryanodine / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013552:Swine; D014316:Tritium
chemical_list: D002147:Calmodulin; D019837:Ryanodine Receptor Calcium Release Channel; D014316:Tritium; D012433:Ryanodine; D002118:Calcium