Functional Characterization of a Calcium-Sensing Receptor Mutation in Severe Autosomal Dominant Hypocalcemia with a Bartter-Like Syndrome ROSA VARGAS-POUSSOU,* † CHUNFA HUANG, ‡ PHILIPPE HULIN, § PASCAL HOUILLIER, ¶ XAVIER JEUNEMAı ˆTRE, # MICHEL PAILLARD, ¶ GABRIELLE PLANELLES, § MICHE ` LE DE ´ CHAUX, † R. TYLER MILLER, ‡ and CORINNE ANTIGNAC* *Inserm U423, † Department of Physiology, Universite ´ Rene ´ Descartes, Ho ˆpital Necker-Enfants Malades, Paris, France; ‡ Case-Western Reserve University, Nephrology Section, Cleveland, Ohio; § Inserm U467, Universite ´ Rene ´ Descartes, ¶ Department of Physiology and Inserm U356, and # Department of Genetics, Ho ˆpital Europe ´en Georges Pompidou, Universite ´ Pierre et Marie Curie, Paris, France. Abstract. The extracellular Ca 2 -sensing receptor (CaSR) plays an essential role in extracellular Ca 2 homeostasis by regulating the rate of parathyroid hormone (PTH) secretion and the rate of calcium reabsorption by the kidney. Activation of the renal CaSR is thought to inhibit paracellular divalent cation reabsorption in the cortical ascending limb (cTAL) both di- rectly and indirectly via a decrease in NaCl transport. How- ever, in patients with autosomal dominant hypocalcemia (ADH), caused by CaSR gain-of-function mutations, a defect in tubular NaCl reabsorption with renal loss of NaCl has not been described so far. This article describes a patient with ADH due to a gain-of-function mutation in the CaSR, L125P, associated with a Bartter-like syndrome that is characterized by a decrease in distal tubular fractional chloride reabsorption rate and negative NaCl balance with secondary hyperaldosteronism and hypokalemia. The kinetics of activation of the L125P mutant receptor expressed in HEK-293 cells, assessed by mea- suring CaSR-stimulated changes in intracellular Ca 2 and ERK activity, showed a dramatic reduction in the EC 50 for extracellular Ca 2 compared with the wild-type and a loss-of- function mutant CaSR (I40F). This study describes the first case of ADH associated with a Bartter-like syndrome. It is herein proposed that the L125P mutation of the CaSR, which represents the most potent gain-of-function mutation reported so far, may reduce NaCl reabsorption in the cTAL sufficiently to result in renal loss of NaCl with secondary hyperaldoste- ronism and hypokalemia. The extracellular Ca 2 -sensing receptor (CaSR) plays an es- sential role in extracellular Ca 2 homeostasis by regulating the rate of parathyroid hormone (PTH) secretion and the rate of calcium reabsorption by the kidney (1,2). The physiologic importance of this receptor in determining the extracellular calcium concentration has been documented by the character- ization of human syndromes resulting from gain or loss-of- function mutations in the CaSR gene. When present in the heterozygous state, loss-of-function mutations cause familial hypocalciuric hypercalcemia (FHH, OMIM 145980), whereas in the homozygous state, these mutations cause neonatal severe hyperparathyroidism (NSHPT, OMIM 239200) (3,4). In con- trast, gain-of-function mutations are responsible for a form of autosomal dominant hypocalcemia (ADH, OMIM 601198) (5). The CaSR is a member of the G protein– coupled receptor (GPCR) family and belongs to subfamily 3, which is charac- terized by a large amino-terminal extracellular domain (6). The CaSR functions as a dimer, with dimerization occurring through interactions of the cysteines of the extracellular do- main (7). The CaSR signals via pertussis toxin-sensitive and -insensitive G proteins (G i and G q/11 families) to regulate second messengers that include cAMP, inositol triphosphate, diacylglycerol, intracellular Ca 2 , and arachidonic acid (AA) metabolites (1). These second messengers presumably regulate kinases, phosphatases, and other signaling molecules. Like many GPCR that act through G i and G q/11 , the CaSR stimulates mitogen-activated protein kinase (MAPK) signaling cascades, particularly the extracellular signal-regulated kinases (ERK) subfamily (8). In the kidney, the CaSR is primarily expressed on the basolateral cell surface in the cortical thick ascending limb (cTAL) but is also expressed in most tubule segments (9 –11). Activation of the CaSR by hypercalcemia, hypermag- nesemia, or gain-of-function mutations inhibits divalent cation reabsorption in the renal tubule, which results in urinary loss of Received January 22, 2002. Accepted May 20, 2002. Correspondence to Dr. Rosa Vargas-Poussou, INSERM U. 423, Ho ˆpital Necker-Enfants Malades, Tour Lavoisier, 6 e etage, 149, rue de Se `vres, 75743 Paris Cedex 15. Phone: 33-1-44-49-54-24; Fax: 33-1-44-49-02-90; E-mail: vargas@necker.fr 1046-6673/1309-2259 Journal of the American Society of Nephrology Copyright © 2002 by the American Society of Nephrology DOI: 10.1097/01.ASN.0000025781.16723.68 J Am Soc Nephrol 13: 2259–2266, 2002