Exp Clin Endocrinol Diabetes 2014; 122 - OP8_41
DOI: 10.1055/s-0034-1372016

Activating CaSR mutations causing Bartter syndrome type 5 are characterized by a distinctly diminished inhibitory phosphorylation on amino acid T888

S Letz 1, C Haag 2, E Schulze 2, K Frank-Raue 2, F Raue 2, B Mayr 1, C Schöfl 1
  • 1Universitätsklinikum Erlangen, Division of Endocrinology and Diabetes, Department of Medicine I, Erlangen, Germany
  • 2Endocrine Practice, Heidelberg, Germany

Introduction: Activating mutations of the calcium sensing receptor (CaSR) cause autosomal dominant hypocalcemia (ADH) characterized by low serum calcium, low PTH and hypercalciuria. 4 activating CaSR mutations cause additional renal wasting of sodium, potassium and other salts called Bartter syndrome type 5 (BS5). The cause of this additional phenotype is unknown. Mutations causing BS5 lack the typical decrease in signaling response after repeated stimulation that is seen in wild type CaSR and ADH mutants. Phosphorylation of CaSR on amino acid T888 by PKC inhibits CaSR signaling. We studied whether this negative feedback mechanism is involved in the absence of signaling desensitization in BS5 mutants.

Methods: ADH and BS5 mutants were expressed in human embryonic kidney 293T cells and receptor signaling was studied by measurement of intracellular free calcium and determination of receptor phosphorylation on amino acid T888 by Western Blot.

Results: All activating mutants showed left shifted dose response curves to [Ca2+]o. Repeated stimulation with 3mM [Ca2+]o elicited decreased responses in ADH mutants and wild type CaSR but increased responses in BS5. ADH and wildtype CaSR showed a robust increase in T888 phosphorylation in response to increasing concentrations of [Ca2+]o, while in BS5 mutants this increase was much weaker. Activation of PKC by PMA caused a marked increase in T888 phosphorylation and a significant decrease in CaSR signaling in all samples. Inhibition of PKC by GF109203X in contrast decreased T888 phosphorylation and increased signaling in ADH and wild type CaSR but did not further augment signaling in BS5 mutants.

Conclusion: The inhibitory feedback via PKC appears to be intact in ADH mutants. Although PKC can phosphorylate T888 in BS5 mutants, this inhibitory feedback mechanism is not activated after stimulation with physiological concentrations of [Ca2+]o in BS5 mutants which may contribute to the different clinical phenotypes of BS5 mutants.