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

Some G protein-coupled receptors (GPCRs) exert biological functions and pharmacological actions by interacting with metal ions directly and allosterically; however, it is not easy to measure the binding parameters between them to gain insights into their mechanisms of action. In this study, a mini separation column packed with calcium sensing receptor (CaSR) immobilized on 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 of this study indicate that the CaSR-immobilized column could recognize different metal ions in the following order of affinities: Gd³⁺ > Ca²⁺ > Na⁺, affording association equilibrium constants and the total binding sites of the orthosteric ligands Ca²⁺ and Gd³⁺ and sensitively probing the binding affinities of the two metal ions in response to CaSR-targeted specific allosteric modulators. Molecular docking demonstrated that the larger binding affinity of Gd³⁺ than that of Ca²⁺ is attributable to the stronger electrostatic forces of Gd³⁺ with the residues ASN189, GLU191 and HIS192 of CaSR owing to the more positive charges. Unlike current methods, the 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 important information about their mechanisms of action.