Inorganic phosphate homeostasis is dependent, in part, on phosphate-induced PTH secretion and PTH-dependent inhibition of renal phosphate reabsorption. Renal phosphate reabsorption is mediated by the Npt2a and Npt2c transporters. However, the mechanism by which phosphate stimulates PTH secretion is unknown.
The calcium-sensing receptor (CaSR) is a master inhibitory controller of PTH secretion, which is activated by extracellular Ca2+. Whether phosphate promotes PTH secretion by inhibiting the CaSR or via some other mechanism has not been clear. Recently, structures of the extracellular domain of the CaSR have been determined for both its inactive and active conformations by Xray crystallography (Geng et al., 2016). Comparison of these structures supports the hypothesis that inorganic phosphate binding at two key sites (site 1 and site 3) stabilises the CaSR in its inactive conformation and may thus inhibit receptor function.
In the current study we tested this hypothesis and found that inorganic phosphate is a non-competitive inhibitor of the CaSR that stimulates PTH secretion. Firstly, we demonstrated that inorganic phosphate acutely suppresses extracellular Ca2+-stimulated Ca2+i mobilization in HEK-293 cells that stably express the CaSR (CaSR-HEK cells), acting to lower the receptor’s maximal response by around 30-40% in a phosphate concentration-dependent manner (0.5-3.0 mM). Inorganic phosphate also acutely stimulated PTH secretion from both human parathyroid cells perifused in vitro and mouse parathyroid glands incubated ex vivo, and parathyroid glands from CaSR-null mice, which exhibited hypersecretion of PTH due to loss of CaSR-mediated inhibitory control, were insensitive to changes in phosphate concentration. Finally, we undertook mutational analysis of residues that support phosphate binding to sites 1 and 3. In particular, the mutation R62A (site-1) but not R66A (site-3) disabled phosphate sensing in CaSR-HEK cells.
The results indicate that phosphate inhibits the CaSR by disabling a key salt bridge between R62 and E277 that stabilises the active form of the receptor and provide mechanisms for phosphate-dependent stimulation of PTH secretion under both physiological and pathophysiological conditions. The inhibitory effect of markedly elevated plasma inorganic phosphate concentrations on the CaSR together with low or low-normal levels of calcium may drive the high levels of plasma PTH observed in the context of chronic kidney disease.
Geng Y et al., (2016) Structural mechanism of ligand activation in human calcium-sensing receptor. Elife 5:pii: e13662.