title: An optical method for evaluating ion selectivity for calcium signaling pathways in the cell.
abstract: A method for evaluating a physiologically relevant ion selectivity of Ca2+ signaling pathways in biological cells based on a Ca(2+)-dependent on/off switch for cellular processes via calmodulin (CaM) chemistry is described. CaM serves as a primary ion receptor for Ca2+ and a given CaM-binding peptide as a target for a CaM-Ca2+ complex. Upon accommodating four Ca2+ ions in its binding sites, CaM undergoes a conformational change to form a CaM-Ca(2+)-target peptide ternary complex. This Ca(2+)-induced selective binding of the Ca(2+)-CaM complex to the target peptide was monitored by a surface plasmon resonance (SPR) technique. As a target peptide, a 26-amino acid residue of M13 derived from skeletal muscle myosin light-chain kinase was used. The target peptide was covalently immobilized in the dextran matrix on top of gold, over which sample solutions containing Ca2+ and CaM were injected in a flow system. Ca(2+)-dependent SPR signals were observed for Ca2+ concentrations from 3.2 x 10(-8) to 1.1 x 10(-5) M and it leveled off. The observed SPR signals were explained as due to an increase in the refractive indexes caused by a Ca2+ ion-switched protein/ peptide interaction, i.e., Ca2+ ion to CaM and subsequent additional binding of the thus formed complex with immobilized M13. No SPR signals were however, induced by Mg2+, K+, and Li+ at concentrations as high as 1.0 x 10(-1) M; these results and previous spectroscopic data taken together conclude that these ions do not induce CaM/peptide interaction. Large changes in SPR signals were observed with a Sr2+ ion concentration over 5.1 x 10(-4) M; Sr2+ ion behaved in this case as a strong agonist toward the Ca(2+)-dependent on/off switch of CaM. The present system thus exhibited "physiologically more relevant" ion selectivity in that relevant metal ions could switch on the CaM/peptide or -protein interaction rather than merely be bound to CaM causing no further signal transduction. The potential use of this finding for more widely evaluating cation selectivity toward the Ca2+ signaling process was discussed.
authors.affiliation: Department of Chemistry, School of Science, University of Tokyo, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Ozawa
authors.forename: M
authors.initials: M
authors.lastname: Kakuta
authors.forename: M
authors.initials: M
authors.lastname: Sugawara
authors.forename: Y
authors.initials: Y
authors.lastname: Umezawa
authors.forename: M
authors.initials: M
authors.lastname: Ikura
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015374:Biosensing Techniques; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D002413:Cations, Divalent; D002417:Cattle; D008969:Molecular Sequence Data; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry / Q000378:metabolism*; D055095:Optics and Photonics; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D012680:Sensitivity and Specificity; D015398:Signal Transduction; D013499:Surface Properties
chemical_list: D002147:Calmodulin; D002413:Cations, Divalent; C107660:M13 protein (myosin light-chain kinase); D010446:Peptide Fragments; D009219:Myosin-Light-Chain Kinase; D002118:Calcium