Characterisation of Calcium-Sensing Receptor Extracellular pH Sensitivity and Intracellular Signal Integration

UoM administered thesis: Phd

  • Authors:
  • Katherine Campion

Abstract

Parathyroid hormone (PTH) secretion maintains free-ionised extracellular calcium (Ca2+o) homeostasis under the control of the calcium-sensing receptor (CaR). In humans and dogs, blood acidosis and alkalosis is associated with increased or suppressed PTH secretion respectively. Furthermore, large (1.0 pH unit) changes in extracellular pH (pHo) alter Ca2+o sensitivity of the CaR in CaR-transfected HEK-293 cells (CaR-HEK). Indeed, it has been found in this laboratory that even pathophysiological acidosis (pH 7.2) renders CaR less sensitive to Ca2+o while pathophysiological alkalosis (pH 7.6) increases its Ca2+o sensitivity, both in CaR-HEK and parathyroid cells. If true in vivo, then CaR's pHo sensitivity might represent a mechanistic link between metabolic acidosis and hyperparathyroidism in ageing and renal disease. However, in acidosis one might speculate that the additional H+ could displace Ca2+ bound to plasma albumin, thus increasing free-Ca2+ concentration and so compensating for the decreased CaR responsiveness. Therefore, I first demonstrated that a physiologically-relevant concentration of albumin (5% w/v) failed to overcome the inhibitory effect of pH 7.2 or stimulatory effect of pH 7.6 on CaR-induced intracellular Ca2+ (Ca2+i) mobilisation.Determining the molecular basis of CaR pHo sensitivity would help explain cationic activation of CaR and permit the generation of experimental CaR models that specifically lack pHo sensitivity. With extracellular histidine and free cysteine residues the most likely candidates for pHo sensing (given their sidechains' pK values), all 17 such CaR residues were mutated to non-ionisable residues. However, none of the resulting CaR mutants exhibited significantly decreased CaR pHo sensitivity. Even co-mutation of the two residues whose individual mutation appeared to elicit modest reductions (CaRH429V and CaRH495V) failed to exhibit any change in CaR pHo sensitivity. I conclude therefore, that neither extracellular histidine nor free cysteine residues account for CaR pHo sensitivity. Next, it is known that cytosolic cAMP drives PTH secretion in vivo and that cAMP potentiates Ca2+o-induced Ca2+i mobilisation in CaR-HEK cells. Given the physiological importance of tightly controlled PTH secretion and Ca2+o homeostasis, here I investigated the influence of cAMP on CaR signalling in CaR-HEK cells. Agents that increase cytosolic cAMP levels such as forskolin and isoproterenol potentiated Ca2+o-induced Ca2+i mobilisation and lowered the Ca2+o threshold for Ca2+i mobilisation. Indeed, forskolin lowered the EC50 for Ca2+o on CaR (2.3 ± 0.1 vs. 3.0 ± 0.1 mM control, P

Details

Original languageEnglish
Awarding Institution
Supervisors/Advisors
Award date31 Dec 2013