Inactivating mutations of the calcium-sensing receptor (CaSR) gene, present in homozygous or heterozygous forms, cause neonatal severe hyperparathyroidism or familial hypocalciuric hypercalcemia. The R-568 binds to the transmembrane region of the CaSR thereby enhancing its sensitivity to extracellular calcium ([Ca2+]o) and inhibiting parathyroid hormone (PTH) secretion. The therapeutic potential of calcimimetics like R-568 has been demonstrated in patients with primary hyperparathyroidism as well as in patients exhibiting secondary hyperparathyroidism. In the present study we tested, whether R-568 could improve transmembrane signaling of naturally occurring inactivating mutants of the CaSR.
Mutant cDNAs of seven CaSR mutations (W530G, C568Y, W718X, M734R, L849P, Q926R and Q1005N) were generated and transfected into HEK293 cells. Functional characterization was performed by measuring intracellular calcium in response to varying [Ca2+]o. The transfected HEK293 cells were stimulated with 3 or 10 mM [Ca2+]o with or without R-568 (1 μM). R-568 significantly enhanced the intracellular Ca2+ response to 3 mM [Ca2+]o in cells expressing the wild-type (WT) CaSR, and the W530G, Q926R and Q1005N mutants (P<0.01). The C568Y mutant did not respond to [Ca2+]o up to 30 mM. In the presence R-568 (1 μM), however, 10 mM [Ca2+]o caused an increase in intracellular Ca2+ by 30 nM (n=10). By contrast, in cells expressing the W718X, M734R, and L849P mutant no [Ca2+]o-induced cytosolic Ca2+ response could be observed even in the presence of R-568 (1 μM).
In conclusion, the allosteric calcimimetic R-568 could enhance transmembrane signaling in certain inactivating mutants of the CaSR. This might offer medical treatment to severely affected individuals (homozygotes) harbouring a calcimimetic-sensitive CaSR mutant. The sensitizing action of R-568 appears to be restricted to mutants, that have mutations in the extra- or intracellular part of the CaSR, since all non-responsive mutants were either truncated (W718X) or mutated in the transmembrane domain (M734R, L849P).