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.

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@article{Ozawa1997AnOM, title={An optical method for evaluating ion selectivity for calcium signaling pathways in the cell.}, author={Takuya Ozawa and Mariko Kakuta and Mitsuru Sugawara and Yōji Umezawa and Mitsuhiko Ikura}, journal={Analytical chemistry}, year={1997}, volume={69 15}, pages={3081-5} }