ABSTRACT
CONTEXT AND OBJECTIVE: Activating mutations in the calcium-sensing receptor (CaSR) gene cause autosomal dominant hypocalcemia (ADH). The aims of the present study were the functional characterization of novel mutations of the CaSR found in patients, the comparison of in vitro receptor function with clinical parameters, and the effect of the allosteric calcilytic NPS-2143 on the signaling of mutant receptors as a potential new treatment for ADH patients. METHODS: Wild-type and mutant CaSR (T151R, P221L, E767Q, G830S, and A844T) were expressed in human embryonic kidney cells (HEK 293T). Receptor signaling was studied by measuring intracellular free calcium in response to different concentrations of extracellular calcium ([Ca(2+)](o)) in the presence or absence of NPS-2143. RESULTS: All ADH patients had lowered serum calcium ranging from 1.7 to 2.0 mm and inadequate intact PTH and urinary calcium excretion. In vitro testing of CaSR mutations from these patients revealed exaggerated [Ca(2+)](o)-induced cytosolic Ca(2+) responses with EC(50) values for [Ca(2+)](o) ranging from 1.56 to 3.15 mM, which was lower than for the wild-type receptor (4.27 mM). The calcilytic NPS-2143 diminished the responsiveness to [Ca(2+)](o) in the CaSR mutants T151R, E767Q, G830S, and A844T. The mutant P221L, however, was only responsive when coexpressed with the wild-type CaSR. CONCLUSION: Calcilytics might offer medical treatment for patients with autosomal dominant hypocalcemia caused by calcilytic-sensitive CaSR mutants.
Subject(s)
Mutation , Naphthalenes/pharmacology , Receptors, Calcium-Sensing/antagonists & inhibitors , Receptors, Calcium-Sensing/genetics , Calcium/metabolism , Cells, Cultured , Drug Evaluation, Preclinical , Humans , Hypocalcemia/genetics , Mutation/physiology , Receptors, Calcium-Sensing/physiology , Signal Transduction/drug effects , Signal Transduction/genetics , TransfectionABSTRACT
CONTEXT AND OBJECTIVE: Inactivating mutations in the calcium-sensing receptor (CaSR) gene cause neonatal severe hyperparathyroidism and familial hypocalciuric hypercalcemia (FHH). The aims of the present study were the functional characterization of novel mutations of the CaSR found in FHH patients, the comparison of in vitro receptor function with clinical parameters, and the effect of the allosteric calcimimetic NPS R-568 on the signaling of mutant receptors. METHODS: Wild-type and mutant CaSRs (W530G, C568Y, W718X, M734R, L849P, Q926R, and D1005N) were expressed in human embryonic kidney 293 cells. Receptor signaling was studied by measuring intracellular free calcium in response to different concentrations of extracellular calcium ([Ca(2+)](o)). RESULTS: Four CaSR mutations (C568Y, W718X, M734R, and L849P) demonstrated a complete lack of a [Ca(2+)](o)-induced cytosolic Ca(2+) response up to 30 mm [Ca(2+)](o), whereas the CaSR mutants W530G, Q926R, and D1005N retained some sensitivity to [Ca(2+)](o). There was no significant relation between the in vitro calcium sensitivity, serum calcium, and intact PTH levels in the patients. Patients with C-terminal CaSR mutations had a calcium to creatine ratio above the established diagnostic threshold of 0.01 for FHH. The calcimimetic NPS R-568 enhanced the responsiveness to [Ca(2+)](o) in CaSR mutants of the extracellular domain (W530G and C568Y) as well as the intracellular C-terminal domain (Q926R and D1005N). CONCLUSION: Therefore, calcimimetics might offer medical treatment for symptomatic FHH patients, and more important, for patients with neonatal severe hyperparathyroidism that harbor calcimimetic-sensitive CaSR mutants.
