Kidney Week

Abstract: TH-OR048

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

Session Information

• Bone and Mineral Metabolism: Basic Research
  November 07, 2019 | Location: 145, Walter E. Washington Convention Center
  Abstract Time: 05:54 PM - 06:06 PM

Category: Bone and Mineral Metabolism

• 401 Bone and Mineral Metabolism: Basic

Authors

• Pacios centeno, Patricia, The University of Manchester , Manchester, United Kingdom

Patricia Pacios centeno, MS



I have been researching the calcium-sensing receptor (CaSR) since 1996, initially at Boston Children's Hospital and now at The University of Manchester (UK). Since 2016, I have been part of the EU-funded CaSR-Biomedicine consortium and in conjunction with this team I have very recently reported that the CaSR also represents a phosphate sensor in the parathyroid gland, capable of eliciting increased PTH secretion. This finding was made possible by the characterisation of the CaSR's crystal structure, a structure that has additionally permitted identification of the CaSR's pH-sensitive site, as reported here.

• Ward, Donald T,, The University of Manchester , Manchester, United Kingdom

Donald T, Ward,

Background

The calcium-sensing receptor (CaR) is the principal controller of parathyroid hormone (PTH) secretion. Mild acidosis (pH_o 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 pH_o sensitivity remains unknown [1]. The crystal structure of the CaR reveals that CaR^R62 and CaR^R66 stabilize its active conformation. Therefore, here we investivated whether these residues mediate CaR pH_o 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) CaR^WT or CaR^R66A. CaR stimulated with HEPES buffer containing the EC₅₀ concentration for Ca²⁺ (3.5mM CaR^WT, 5 CaR^R66A) at either pH 7, 7.4 or 7.6.

Results

The CaR crystal structure predicts that following activation, CaR^R66 creates a hydrogen bond with CaR^S301 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 CaR^R66, impairing the hydrogen bond and inhibiting CaR activity. Indeed we found that lowering pH_o from 7.4 to 7.0 inhibited CaR-induced Ca²⁺_i mobilisation in CaR^WT (-40 ±5%; P<0.001 ANOVA) whereas in CaR^R66A there was no significant effect (-14 ±9%; ns). Similarly, pH_o 7.0 inhibited CaR-induced ERK phosphorylation in CaR^WT (-86 ±5%; P<0.01) but not significantly in CaR^R66A (-30 ±12%; ns). Then in alkalosis, higher pH_o renders bicarbonate more negative which we hypothsize will better support the R66-S301 hydrogen bond, and thus enhance receptor activity. As predicted, raising pH_o from 7.4 to 7.6 stimulated CaR-induced Ca²⁺_i mobilisation in CaR^WT (+25 ±11%; P<0.001) but not in CaR^R66A (+3 ±12%; ns). Similarly, pH_o 7.6 enhanced CaR-induced ERK phosphorylation in CaR^WT (24 ±17%; P<0.05) but not in CaR^R66A (+9 ±12%; ns). Unlike for CaR^R66A, the CaR^R62A mutant retained its CaR^WT-like pH_o senstivity.

Conclusion

Together, these data identify CaR^R66 as a site of pH_o 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.