Anionic Modulation of the Calcium-Sensing Receptor

  • Sheherzad Salman

Student thesis: Phd

Abstract

The calcium-sensing receptor (CaR) maintains extracellular calcium (Ca2+o) homeostasis by controlling parathyroid hormone (PTH) secretion and renal Ca2+ reabsorption. Activating CaR mutations can cause autosomal dominant hypocalcaemia type-1 (ADH1) which is characterised by chronic PTH suppression and hypocalcaemia, but also by hyperphosphataemia in some patients due to the loss of PTH-induced renal phosphate (Pi) excretion. We reported recently that Pi represents a non-competitive antagonist for wild-type CaR (CaRWT). Therefore, here I investigated whether the hyperphosphataemia of ADH1 might potentially blunt the gain-of-function in such mutant receptors. Indeed, in two ADH1-associated mutants, CaRA840V and CaRE228K, (expressed stably in HEK293 cells) 1.8 mM Pi (equivalent to the serum Pi concentration observed in those patients) significantly inhibited CaR-induced intracellular Ca2+ (Ca2+i) mobilisation and extracellular-regulated kinase (ERK) phosphorylation. A similar inhibitory effect of Pi was also seen in CaRWT further validating our previous work. Therefore, in ADH1, the PTH suppression and hypocalcaemia induced by the gain-of-function CaR might be even more severe but for the associated hyperphosphataemia. Next, secondary hyperparathyroidism (SHPT) is a serious complication of advanced CKD and is characterised by hyperphosphataemia, increased PTH secretion, parathyroid hyperplasia and decreased parathyroid CaR expression. I investigated the inhibition of CaR by Pi and specifically whether Pi inhibits CaR gene expression and protein abundance. HEK293 cells transfected with CaRWT and CaRR62A (a mutant lacking the putative Pi-binding site), in addition to human medullary thyroid cancer (TT) cells (which express CaR endogenously) were cultured in a novel, modified Dulbecco’s modified Eagle medium (DMEM) containing either physiological or pathophysiological concentrations of Pi to measure CaR expression. The cells were processed for western blotting to observe CaR protein abundance, as well as by qualitative polymerase chain reaction (qPCR) and RNA-seq to determine relative gene expression changes. CaR protein abundance and relative gene expression was not apparently sensitive to Pi in CaR-transfected HEK293 cells, though R568 did increase CaR abundance in CaRWT-transfected cells, but interestingly not in CaRR62A-transfected cells. In TT cells, CaR protein abundance decreased significantly in cells co-stimulated with R568 and spermine (RS; CaR positive allosteric & orthosteric modulators, respectively) but this was not affected by Pi. RNA-seq analysis also found significant genomic effects of the RS treatment but no modulating effect of Pi. Interestingly, the gene for calcitonin, a hormone that is secreted in response to CaR activation, exhibited significant downregulation in the RS-treated TT cells. Pathologic acidosis can inhibit CaR and thus increase PTH secretion (with alkalosis doing the opposite) but the molecular site of this pH effect on the CaR is unknown. Our preliminary data using molecular dynamic (MD) simulations suggests that the bicarbonate (BCT) ion (HCO3-) might bind to the CaR ECD at Arg-66 to stabilise its active conformation. Since the protonation state of BCT is pH-dependent this might explain the CaR’s pH sensitivity. Here I found that BCT significantly enhanced CaR-induced Ca2+i mobilisation and ERK phosphorylation, effects not seen with CaRR66A in which the putative BCT site is missing. Neither did BCT modulate carbachol-induced signalling, or, elicit responses in wild-type HEK cells lacking the CaR, further implicating CaR as BCT’s target. Finally, the pH sensitivity seen with CaRWT was attenuated with the CaRR66A mutant. These data confirm and extend our recent unpublished investigations of CaR pH sensitivity and further implicate Arg-66 as the site of pH / BCT action. Together, this project enhances our understanding of the Pi- and pH-mediated modulation of CaR activity which is of specifi
Date of Award1 Aug 2023
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorDonald Ward (Supervisor)

Cite this

'