title: Calcium binding to calmodulin mutants having domain-specific effects on the regulation of ion channels.
abstract: Calmodulin (CaM) is an essential, eukaryotic protein comprised of two highly homologous domains (N and C). CaM binds four calcium ions cooperatively, regulating a wide array of target proteins. A genetic screen of Paramecia by Kung [Kung, C. et al. (1992) Cell Calcium 13, 413-425] demonstrated that the domains of CaM have separable physiological roles: "under-reactive" mutations affecting calcium-dependent sodium currents mapped to the N-domain, while "over-reactive" mutations affecting calcium-dependent potassium currents localized to the C-domain of CaM. To determine whether and how these mutations affected intrinsic calcium-binding properties of CaM domains, phenylalanine fluorescence was used to monitor calcium binding to sites I and II (N-domain) and tyrosine fluorescence was used to monitor sites III and IV (C-domain). To explore interdomain interactions, binding properties of each full-length mutant were compared to those of its corresponding domain fragments. The calcium-binding properties of six under-reactive mutants (V35I/D50N, G40E, G40E/D50N, D50G, E54K, and G59S) and one over-reactive mutant (M145V) were indistinguishable from those of wild-type CaM, despite their deleterious physiological effects on ion-channel regulation. Four over-reactive mutants (D95G, S101F, E104K, and H135R) significantly decreased the calcium affinity of the C-domain. Of these, one (E104K) also increased the calcium affinity of the N-domain, demonstrating that the magnitude and direction of wild-type interdomain coupling had been perturbed. This suggests that, while some of these mutations alter calcium-binding directly, others probably alter CaM-channel association or calcium-triggered conformational change in the context of a ternary complex with the affected ion channel.
authors.affiliation: Department of Biochemistry, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242-1109, USA.
authors.forename: Wendy S
authors.initials: WS
authors.lastname: VanScyoc
authors.forename: Rhonda A
authors.initials: RA
authors.lastname: Newman
authors.forename: Brenda R
authors.initials: BR
authors.lastname: Sorensen
authors.forename: Madeline A
authors.initials: MA
authors.lastname: Shea
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007473:Ion Channels / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D010247:Paramecium / Q000201:enzymology / Q000235:genetics; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012694:Serine / Q000235:genetics / Q000378:metabolism; D017186:Titrimetry
chemical_list: D002147:Calmodulin; D007473:Ion Channels; D012694:Serine; D010649:Phenylalanine; D002118:Calcium