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Abstract: TH-OR048

Identification of an Extracellular pH-Sensitive Residue in the Calcium-Sensing Receptor

Session Information

Category: Bone and Mineral Metabolism

  • 401 Bone and Mineral Metabolism: Basic

Authors

  • Pacios centeno, Patricia, The University of Manchester , Manchester, United Kingdom
  • Ward, Donald T,, The University of Manchester , Manchester, United Kingdom
Background

The calcium-sensing receptor (CaR) is the principal controller of parathyroid hormone (PTH) secretion. Mild acidosis (pHo 7.2) inhibits CaR signalling in HEK-293 and bovine parathyroid cells, so permitting increased PTH secretion from human parathyroid cells [1]. Thus, acidosis could contribute to the CaR underactivation and secondary hyperparathyroidism of CKD but where the molecular site of the pHo sensitivity remains unknown [1]. The crystal structure of the CaR reveals that CaRR62 and CaRR66 stabilize its active conformation. Therefore, here we investivated whether these residues mediate CaR pHo sensitivity.

Methods

CaR activity was measured as Ca2+i mobilization (Fura-2) and extracellular signal-regulated kinase (ERK) phosphorylation in HEK-293 cells transfected with (wild-type) CaRWT or CaRR66A. CaR stimulated with HEPES buffer containing the EC50 concentration for Ca2+ (3.5mM CaRWT, 5 CaRR66A) at either pH 7, 7.4 or 7.6.

Results

The CaR crystal structure predicts that following activation, CaRR66 creates a hydrogen bond with CaRS301 that is supported by a bound, negatively-charged bicarbonate ion (pKa 6.1). We hypothesize therefore that in pathophysiological acidosis, the more neutral bicarbonates will no longer bind CaRR66, impairing the hydrogen bond and inhibiting CaR activity. Indeed we found that lowering pHo from 7.4 to 7.0 inhibited CaR-induced Ca2+i mobilisation in CaRWT (-40 ±5%; P<0.001 ANOVA) whereas in CaRR66A there was no significant effect (-14 ±9%; ns). Similarly, pHo 7.0 inhibited CaR-induced ERK phosphorylation in CaRWT (-86 ±5%; P<0.01) but not significantly in CaRR66A (-30 ±12%; ns). Then in alkalosis, higher pHo renders bicarbonate more negative which we hypothsize will better support the R66-S301 hydrogen bond, and thus enhance receptor activity. As predicted, raising pHo from 7.4 to 7.6 stimulated CaR-induced Ca2+i mobilisation in CaRWT (+25 ±11%; P<0.001) but not in CaRR66A (+3 ±12%; ns). Similarly, pHo 7.6 enhanced CaR-induced ERK phosphorylation in CaRWT (24 ±17%; P<0.05) but not in CaRR66A (+9 ±12%; ns). Unlike for CaRR66A, the CaRR62A mutant retained its CaRWT-like pHo senstivity.

Conclusion

Together, these data identify CaRR66 as a site of pHo sensitivity in CaR representing another potential contributor to the secondary hyperparathyroidism of CKD where acidosis is present.

[1] Campion et al (2015) J Am Soc Nephrol 26, 2163-2171.

Funding

  • Government Support - Non-U.S.