An easy and sensitive approach to measure the binding parameters of metal ionic ligands and calcium sensing receptor in ion chromatography

Abstract

Some of G protein-coupled receptors (GPCRs) exerted the biological function and pharmacologic actions by interacting with metal ions directly and allosterically, however, it is not easy to measure the binding parameters between them for gaining insights into their action mechanisms. In this work, a mini separation column, packed with calcium sensing receptor (CaSR) immobilized solid matrix particles (9.37 mg CaSR / per gram silica, 0.8 g), was prepared to reveal the binding action of metal ionic ligands in ion chromatography. The results indicated that CaSR-immobilized column was capable to recognize the different metal ions in the rank of the affinities Gd3+ > Ca2+ > Na+, to afford association equilibrium constants and the total binding sites of orthosteric ligands Ca2+ and Gd3+, and to probe sensitively the binding affinities of the two metal ions in response to CaSR-targeted specific allosteric modulators. Molecular docking gave the explanation that the larger binding affinity of Gd3+ than Ca2+ was attributable to the stronger electrostatic forces of Gd3+ with the residues ASN189, GLU191 and HIS192 of CaSR owing to the more positive charges. Different from the current methods, CaSR-immobilized column represents an easy and sensitive approach to estimate the binding parameters of metal ions and GPCRs in solution using relatively small quantities of materials, providing the important information about their action mechanisms.