The calcium-sensing receptor as a mineral sensor

Mammalian extracellular calcium concentration is tightly maintained by parathyroid hormone (PTH) secretion under the control of the calcium-sensing receptor (CaSR). Chronically elevated PTH secretion is a complication of chronic kidney disease (CKD), as are hyperphosphataemia and metabolic acidosis. The extracellular domain of the CaSR was crystallised simultaneously by two research groups, revealing four different anion binding sites that were predicted to be occupied by PO4/SO4 and/or HCO3. It has been long appreciated that increased serum phosphate (Pi) levels are associated with elevated PTH secretion via an unknown molecular mechanism. Here, I have tested whether pathophysiological changes in extracellular Pi concentration cause significant changes in CaSR activity sufficient to alter PTH secretion levels. Raising extracellular Pi levels from 0.8 mM (physiological) to 2 mM (as seen in patients with end-stage renal disease) negatively modulated CaSR signalling, as shown by Ca2+ mobilisation and extracellular signal-regulated kinase (ERK) activation, in CaSR-transfected HEK-293 cells. The inhibitory effect of Pi was lost in the mutant CaSRR62A, suggesting that Pi binding at residue Arg62 breaks a salt bridge interaction believed to be crucial for stabilising the CaSR active conformation. Next, pathophysiologic Pi concentrations were sufficient to induce rapid and reversible increases in PTH secretion from human and murine parathyroid tissue. This effect was not seen in glands from mice with a parathyroid gland-specific CaSR knock out. Together, these results indicate that the CaSR can sense moderate changes in extracellular Pi, which explain its stimulatory effect on PTH secretion and suggest a new mechanism for the aetiology of secondary hyperparathyroidism. Next, blood acidosis and alkalosis are also known to be associated with increased or suppressed PTH secretion respectively. Previous work from this laboratory established that pathophysiological pH changes (up to 0.2 units) can modulate CaSR activity via a histidine residue-independent mechanism. The CaSR ECD crystal model predicts the presence of a bicarbonate binding site. Interestingly, bicarbonate protonation equilibrium (pKa 6.1 at 37°C) is altered by pathophysiologic changes in pH. Thus, the binding of the most abundant protonation states of bicarbonate, HCO3 and H2CO3, to the CaSR ECD were studied in silico. Molecular dynamics simulations determined that while HCO3 effectively stabilises CaSR structure by supporting a complex hydrogen bond network, H2CO3 does not bind to the receptor and impairs several hydrogen bond interactions. In addition, I disrupted the bicarbonate binding site in vitro with CaSRR66A mutant, which partially abolishes CaSR's pH sensitivity. These results suggest a pH sensing mechanism mediated by HCO3 bound at the CaSR that has not been described in any other GPCR. Together, these data show that the newly identified anion binding sites in the CaSR ECD crystal structures are crucial for regulating CaSR response in different environ- ments. The CaSR acts as a sophisticated global mineral sensor able to integrate multiple physiological signal inputs from Ca2+, Pi and pH (HCO3), to fine-tune a modulated PTH response. These findings have significant implications for patients with CKD and secondary hyperparathyroidism, and open new research possibilities in the CaSR field.