Elevated serum phosphate is associated with increased parathyroid hormone (PTH) levels, particularly in chronic kidney disease (CKD), where impaired renal phosphate excretion causes hyperphosphataemia. We previously reported that extracellular inorganic phosphate (Pi) acts as a non-competitive antagonist of the calcium-sensing receptor (CaSR) through direct receptor inhibition rather than simply chelating ionised Ca²⁺. Mutation of the CaSR anion-binding residue Arg-62, but not Arg-66, reduced phosphate sensitivity. Structural modelling suggested that a salt bridge between Arg-62 and Glu-277 stabilises closure of the Venus Flytrap domain, whereas phosphate binding to Arg-62 may destabilise the active receptor conformation.
To investigate this mechanism, we generated a CaSRR62A knock-in mouse using CRISPR/Cas9. Homozygous CaSRR62A mice exhibited approximately two-fold higher serum PTH concentrations than wild-type littermates, particularly after 10 weeks of age. Mutant mice were smaller after 20 weeks and displayed relative hypercalcaemia, hypophosphataemia, reduced vitamin D and increased urinary Ca/creatinine ratios. Heterozygous mice showed intermediate phenotypes.
Ex vivo, wild-type parathyroid glands exhibited increased PTH secretion in response to elevated phosphate (1.2-1.6 mM vs. 0.8 mM control), despite correction for Ca²⁺/Pi binding. Pi-induced PTH release was attenuated, but not abolished, in glands from CaSRR62A homozygotes. These findings indicate that Arg-62 contributes to Pi sensing by the CaSR and that disruption of this residue causes moderate hyperparathyroidism, although additional mechanisms must account for the full inhibitory effect of Pi.
In human calcitonin-secreting TT cells expressing endogenous CaSR, co-stimulation with R568 and spermine induced transient intracellular Ca²⁺ mobilisation that was blocked by NPS-2143, confirming CaSR dependence. Increasing phosphate concentrations (0.8-2.0 mM) significantly suppressed Ca²⁺ mobilisation (IC₅₀ 1.1 mM), and 2 mM phosphate markedly inhibited calcitonin secretion, providing further evidence of direct inhibition of CaSR signalling by phosphate.