RESUMEN
Of 12 naturally occurring, activating mutations in the seven-transmembrane (7TM) domain of the human Ca2+ receptor (CaR) identified previously in subjects with autosomal dominant hypocalcemia (ADH), five appear at the junction of TM helices 6 and 7 between residue Ile819 and Glu837. After identifying a sixth activating mutation in this region, V836L, in an ADH patient, we studied the remaining residues in this region to determine whether they are potential sites for activating mutations. Alanine-scanning mutagenesis revealed five additional residues in this region that when substituted by alanine led to CaR activation. We also found that, whereas E837A did not activate the receptor, E837D and E837K mutations did. Thus, region Ile819-Glu837 of the 7TM domain represents a "hot spot" for naturally occurring, activating mutations of the receptor, and most of the residues in this region apparently maintain the 7TM domain in its inactive configuration. Unique among the residues in this region, Pro823, which is highly conserved in family 3 of the G protein-coupled receptors, when mutated to either alanine or glycine, despite good expression severely impaired CaR activation by Ca2+. Both the P823A mutation and NPS 2143, a negative allosteric modulator that acts on the 7TM through a critical interaction with Glu837, blocked activation of the CaR by various ADH mutations. These results suggest that the 7TM domain region Ile819-Glu837 plays a key role in CaR activation by Ca2+. The implications of our finding that NPS 2143 corrects the molecular defect of ADH mutations for treatment of this disease are also discussed.
Asunto(s)
Calcio/metabolismo , Receptores Sensibles al Calcio/química , Alanina/química , Sitio Alostérico , Secuencia de Bases , Línea Celular , Membrana Celular/metabolismo , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Glutamina/química , Humanos , Hidrólisis , Immunoblotting , Iones , Isoleucina/química , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Fosfatidilinositoles/química , Prolina/química , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , TransfecciónRESUMEN
BACKGROUND: We have previously shown that the rat kidney reabsorbs metabolically significant amounts of iron and that it expresses the divalent metal transporter 1, DMT1. The Belgrade (b) rat carries a mutation in DMT1 gene, which causes hypochromic, microcytic anemia due to impaired intestinal iron absorption and transport of iron out of the transferrin cycle endosome. In the duodenum of b/b rats, expression of DMT1 mRNA and protein is increased, suggesting a feedback regulation by iron stores. The aim of this study was to investigate iron handling and DMT1 expression in the kidneys of Belgrade rats. METHODS: Animals were maintained for 3 weeks on a synthetic diet containing 185 mg/kg iron (FeSO4), after which functional and molecular parameters were analyzed in male heterozygous (+/b) and homozygous (b/b) rats (N = 4 to 6 for each group). RESULTS: Serum iron concentration was significantly higher in b/b compared to +/b rats while urinary iron excretion rates were unchanged in b/b compared to +/b rats. Northern analysis using a rat DMT1 probe showed comparable mRNA levels between +/b and b/b animals. Western analysis and immunofluorescence microscopy performed using a polyclonal antibody against rat DMT1 showed that DMT1-specific immunoreactivity was almost absent in the kidneys of b/b rats compared to that seen in +/b animals. CONCLUSION: Our results indicate that the G185R mutation of DMT1 causes protein instability in the kidneys of b/b rats. Given that +/b and b/b rats excrete comparable amounts of iron, the lack of DMT1 protein is compensated by an alternative, yet to be identified, mechanism.
Asunto(s)
Anemia/metabolismo , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Proteínas de Unión a Hierro/genética , Proteínas de Unión a Hierro/metabolismo , Hierro/sangre , Hierro/orina , Riñón/metabolismo , Anemia/patología , Animales , Ingestión de Líquidos , Ingestión de Alimentos , Heces , Femenino , Riñón/patología , Magnesio/sangre , Masculino , Mutación Puntual , Potasio/sangre , ARN Mensajero/análisis , Ratas , Ratas Mutantes , OrinaRESUMEN
Extracellular Ca(2+) concentration [Ca(2+)](o) is vital for a number of processes varying from blood clotting to regulation membrane permeability and excitability. For this reason [Ca(2+)](o) is under strict control of a complex homeostatic system that includes parathyroid glands, kidneys, bones and intestine. The extracellular Ca(2+)-sensing receptor is an essential component of this system, regulating parathyroid hormone secretion, Ca(2+) (and Mg(2+)) excretion by the kidney, bone remodeling and Ca(2+) reabsorption by the gastrointestinal tract. The Ca(2+)-sensing receptor is also present in organs without an obvious link with mineral ion metabolism. This review will describe the discovery of a novel class of ion-sensing receptor(s), receptor-effector coupling and the roles of the Ca(2+)-sensing receptor inside and outside the Ca(2+)(o) homeostatic system.
Asunto(s)
Receptores Sensibles al Calcio/fisiología , Compuestos de Anilina/uso terapéutico , Animales , Calcio/metabolismo , Humanos , Magnesio/metabolismo , Concentración Osmolar , Fenetilaminas , Propilaminas , Receptores Sensibles al Calcio/agonistas , Receptores Sensibles al Calcio/efectos de los fármacosRESUMEN
Aminoglycoside antibiotics (AGAs) are nephrotoxic, with most of the damage confined to the proximal tubule, but the mechanism for cellular toxicity is not clear. It has been previously shown that the extracellular-calcium sensing receptor (CaR) is expressed in intact rat proximal tubule and can be stimulated by the AGA neomycin. To investigate whether CaR could contribute to AGA-induced nephrotoxicity, the acute responses to various AGAs in the proximal tubule-derived opossum kidney (OK) cell line were examined. The presence in OK cells of CaR-related transcripts and protein was demonstrated by northern analyses, reverse transcriptase-PCR, immunocytochemistry, and immunoblotting. OK cells responded to elevated extracellular calcium (Ca(2+)(o)) and neomycin but also to gentamicin and tobramycin with an increase in cytosolic [Ca(2+)]. Ca(2+)(o), neomycin, and gentamicin also activated the extracellular signal-regulated kinases, ERK1 and ERK2. Neomycin-induced ERK activation was both dose- and time-dependent and was attenuated by inhibitors of phosphatidylinositol 3-kinase, phosphatidylinositol bisphosphate (PIP(2))-specific phospholipase C, and MEK1, but not of protein kinase C. Thus, proximal tubular OK cells express a CaR that mediates Ca(2+)(i) mobilization and PIP(2)-PLC-dependent ERK activation in response to AGAs and thus could play a role in AGA-induced nephrotoxicity.