Abstract
IntroductionActivating calcium sensing receptor (CaSR) mutations cause autosomal dominant hypocalcemia (ADH) characterized by low serum calcium, inappropriately low PTH and relative hypercalciuria. Four activating CaSR mutations cause additional renal wasting of sodium, chloride and other salts, a condition called Bartter syndrome (BS) type 5. Until today there is no specific medical treatment for BS type 5 and ADH. We investigated the effects of different allosteric CaSR antagonists (calcilytics) on activating CaSR mutants.MethodsAll 4 known mutations causing BS type 5 and five ADH mutations were expressed in HEK 293T cells and receptor signalling was studied by measurement of intracellular free calcium in response to extracellular calcium ([Ca2+]o). To investigate the effect of calcilytics, cells were stimulated with 3 mM [Ca2+]o in the presence or absence of NPS-2143, ATF936 or AXT914.ResultsAll BS type 5 and ADH mutants showed enhanced signalling activity to [Ca2+]o with left shifted dose response curves. In contrast to the amino alcohol NPS-2143, which was only partially effective, the quinazolinone calcilytics ATF936 and AXT914 significantly mitigated excessive cytosolic calcium signalling of all BS type 5 and ADH mutants studied. When these mutants were co-expressed with wild-type CaSR to approximate heterozygosity in patients, ATF936 and AXT914 were also effective on all mutants.ConclusionThe calcilytics ATF936 and AXT914 are capable of attenuating enhanced cytosolic calcium signalling activity of CaSR mutations causing BS type 5 and ADH. Quinazolinone calcilytics might therefore offer a novel treatment option for patients with activating CaSR mutations.
Highlights
Activating calcium sensing receptor (CaSR) mutations cause autosomal dominant hypocalcemia (ADH) characterized by low serum calcium, inappropriately low PTH and relative hypercalciuria
Since the synthesis of the first calcilytic compound NPS-2143 [17, 18] several allosteric antagonists of the CaSR from different chemical classes such as amino alcohols, diaminocyclohexanes, quinazolinones and benzimidazoles have been developed [19]. These calcilytics have the potential to directly correct the molecular cause of ADH and Bartter syndrome (BS) type 5
Expression vectors for wild-type and mutant CaSR were generated by sitedirected mutagenesis and transfected in human embryonic kidney (HEK) 293T cells cultured on glass coverslips [20,21,22]
Summary
The calcium-sensing receptor (CaSR) is a key regulator of calcium homeostasis and is expressed in parathyroid, kidney, bone and other organs involved in calcium metabolism [1]. Activation of the receptor by rising serum calcium inhibits PTH secretion from parathyroid cells and increases calcium excretion by the kidney. Since the synthesis of the first calcilytic compound NPS-2143 [17, 18] several allosteric antagonists of the CaSR from different chemical classes such as amino alcohols, diaminocyclohexanes, quinazolinones and benzimidazoles have been developed [19]. These calcilytics have the potential to directly correct the molecular cause of ADH and BS type 5. In this study we tested whether amino alcohol and quinazolinone calcilytics could mitigate excessive cytosolic calcium signal transduction of CaSR mutants leading to BS type 5 or ADH potentially providing a novel treatment option for these patients [19]
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