title: 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 (Ca(2+))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 hRyR(11975-1999) or hRyR1(3614-3643). Both CaM and its C-domain associated in a calcium-independent manner with hRyR1(3614-3643) while N-domain required calcium and bound ~250-fold more weakly. Association with hRyR1(11975-1999) 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 hRyR1(3614-3643) at low calcium, positioning CaM to respond to calcium efflux. However, the CaM N-domain affinity for hRyR(11975-1999) 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.
authors.affiliation: Dept. of Biochemistry, Univ. of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA 52242-1109, United States.
authors.forename: Rhonda A
authors.initials: RA
authors.lastname: Newman
authors.affiliation: Dept. of Biochemistry, Univ. of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA 52242-1109, United States.
authors.forename: Brenda R
authors.initials: BR
authors.lastname: Sorensen
authors.affiliation: Department of Physics and Astronomy, Drake University, Des Moines, IA 50311-4516, United States.
authors.forename: Adina M
authors.initials: AM
authors.lastname: Kilpatrick
authors.affiliation: Dept. of Biochemistry, Univ. of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA 52242-1109, United States. Electronic address: madeline-shea@uiowa.edu.
authors.forename: Madeline A
authors.initials: MA
authors.lastname: Shea
mesh_terms: D000595:Amino Acid Sequence; D001059:Apoproteins / Q000737:chemistry* / Q000235:genetics; D002118:Calcium / Q000737:chemistry*; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D019837:Ryanodine Receptor Calcium Release Channel / Q000138:chemical synthesis / Q000737:chemistry*; D013194:Staining and Labeling; D013816:Thermodynamics
chemical_list: D001059:Apoproteins; D002147:Calmodulin; D005452:Fluoresceins; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel; C024098:6-carboxyfluorescein; D002118:Calcium
keywords: Anisotropy; Fluorescence; Intracellular calcium channel; Thermodynamics; Titration