Activating mutations of the calcium-sensing receptor: Calcium oscillations and negative feedback via PKC in mutants causing autosomal dominant hypocalcemia and Bartter syndrome type 5

Introduction: Activating mutations of the calcium sensing receptor (CaSR) cause autosomal dominant hypocalcemia (ADH) characterized by low serum calcium, low PTH and relative hypercalciuria. 4 activating CaSR mutations also cause renal wasting of sodium, potassium and other salts (Bartter syndrome type 5). It is not clear why these 4 activating mutations have this additional phenotype. Phosphorylation of CaSR T888 by PKC constitutes an inhibitory feedback mechanism that leads to oscillations of intracellular calcium ([Ca²⁺]_i) at 2 – 4 mM extracellular calcium ([Ca²⁺]_o) but to a more sustained elevation of [Ca²⁺]_i at higher concentrations. We tested for functional differences between wildtype CaSR and mutants causing ADH or Bartter Syndrome Type 5 by investigating oscillatory responses of [Ca²⁺]_i

Methods: 4 ADH mutations and all 4 known mutations causing Bartter syndrome type 5 were expressed in HEK 293T cells and receptor signalling was studied by counting the frequency of [Ca²⁺]_i oscillations in response to rising concentrations of [Ca²⁺]_o and PKC activation with PMA.

Results: Wildtype CaSR and all mutants showed no regular oscillations under resting conditions and stimulation with 1 mM [Ca²⁺]_o. Stimulation with 2 mM [Ca²⁺]_o elicited mostly regular oscillations between 0.4 and 2 oscillations/minute in wildtype and all mutants. At 3 mM and 4 mM [Ca²⁺]_o, the frequency of oscillations in Bartter Syndrome Type 5 mutants was significantly lower than in ADH mutants and wildtype (0.9/min vs. 1.9/min, p < 0.05). PKC activation appears to reduce oscillations in both wildtype and mutants.

Conclusion: The inhibitory feedback via PKC appears to be intact in activating mutations, the oscillation frequencies of intracellular calcium however differ between activating mutants causing ADH and Bartter Syndrome Type 5. This may indicate differences in intracellular signalling mechanisms possibly contributing to the different clinical phenotypes of these mutants.