Intracellular Ca2+ oscillations generated via the extracellular Ca2+-sensing receptor (CaSR) in response to extracellular Ca2+ or l-phenylalanine: Impact of the highly conservative mutation Ser170Thr

Abstract

The extracellular Ca2+-sensing receptor (CaSR) is an allosteric protein that responds to changes in the extracellular concentration of Ca2+ ([Ca2+]e) and aromatic amino acids with the production of different patterns of oscillations in intracellular Ca2+ concentration ([Ca2+]i). An increase in [Ca2+]e stimulates sinusoidal oscillations in [Ca2+]i whereas aromatic amino acid-induced CaR activation in the presence of a threshold [Ca2+]e promotes transient oscillations in [Ca2+]i. Here, we examined spontaneous and ligand-evoked [Ca2+]i oscillations in single HEK-293 cells transfected with the wild type CaSR or with a mutant CaSR in which Ser170 was converted to Thr (CaSRS170T). Our analysis demonstrates that cells expressing CaSRS170T display [Ca2+]i oscillations in the presence of low concentrations of extracellular Ca2+ and respond to L-Phe with robust transient [Ca2+]i oscillations. Our results indicate that the S170T mutation induces a marked increase in CaSR sensitivity to [Ca2+]e and imply that the allosteric regulation of the CaSR by aromatic amino acids is not only mediated by an heterotropic positive effect on Ca2+ binding cooperativity but, as biased agonists, aromatic amino acids stabilize a CaSR conformation that couples to a different signaling pathway leading to transient [Ca2+]i oscillations.