RESUMEN
BACKGROUND/AIMS: The transmembrane Klotho protein contributes to inhibition of 1,25(OH)2D3 formation. The extracellular domain of Klotho protein could function as an enzyme with e.g. ß-glucuronidase activity, be cleaved off and be released into blood and cerebrospinal fluid. Klotho regulates several cellular transporters. Klotho protein deficiency accelerates the appearance of age related disorders including neurodegeneration and muscle wasting and eventually leads to premature death. The main site of Klotho protein expression is the kidney. Klotho protein is also appreciably expressed in other tissues including chorioid plexus. The present study explored the effect of Klotho protein on the creatine transporter CreaT (Slc6A8), which participates in the maintenance of neuronal function and survival. METHODS: To this end cRNA encoding Slc6A8 was injected into Xenopus oocytes with and without additional injection of cRNA encoding Klotho protein. Creatine transporter CreaT (Slc6A8) activity was estimated from creatine induced current determined by two-electrode voltage-clamp. RESULTS: Coexpression of Klotho protein significantly increased creatine-induced current in Slc6A8 expressing Xenopus oocytes. Coexpression of Klotho protein delayed the decline of creatine induced current following inhibition of carrier insertion into the cell membrane by brefeldin A (5 µM). The increase of creatine induced current by coexpression of Klotho protein in Slc6A8 expressing Xenopus oocytes was reversed by ß-glucuronidase inhibitor (DSAL). Similarly, treatment of Slc6A8 expressing Xenopus oocytes with recombinant human alpha Klotho protein significantly increased creatine induced current. CONCLUSION: Klotho protein up-regulates the activity of creatine transporter CreaT (Slc6A8) by stabilizing the carrier protein in the cell membrane, an effect requiring ß-glucuronidase activity of Klotho protein.
Asunto(s)
Glucuronidasa/fisiología , Proteínas del Tejido Nervioso/biosíntesis , Proteínas de Transporte de Neurotransmisores en la Membrana Plasmática/biosíntesis , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Supervivencia Celular/genética , Inhibidores Enzimáticos/uso terapéutico , Glucuronidasa/antagonistas & inhibidores , Glucuronidasa/genética , Glicoproteínas , Humanos , Proteínas Klotho , Proteínas del Tejido Nervioso/genética , Neuronas , Oocitos , Técnicas de Placa-Clamp , Proteínas de Transporte de Neurotransmisores en la Membrana Plasmática/genética , ARN Complementario/biosíntesis , ARN Complementario/genética , Regulación hacia Arriba , XenopusRESUMEN
BACKGROUND/AIMS: Transport regulation involves several kinases including SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1), which are under control of WNK (with-no-K[Lys]) kinases. The present study explored whether SPAK and/or OSR1 participate in the regulation of the creatine transporter CreaT (SLC6A8), which accomplishes Na+ coupled cellular uptake of creatine in several tissues including kidney, intestine, heart, skeletal muscle and brain. METHODS: cRNA encoding SLC6A8 was injected into Xenopus laevis oocytes with or without additional injection of cRNA encoding wild-type SPAK, constitutively active (T233E)SPAK, WNK insensitive (T233A)SPAK, catalytically inactive (D212A)SPAK, wild-type OSR1, constitutively active (T185E)OSR1, WNK insensitive (T185A)OSR1 and catalytically inactive (D164A)OSR1. Transporter activity was determined from creatine (1 mM) induced current utilizing dual electrode voltage clamp. RESULTS: Coexpression of wild-type SPAK and of (T233E)SPAK, but not of (T233A)SPAK or of (D212A)SPAK was followed by a significant decrease of creatine induced current in SLC6A8 expressing oocytes. Coexpression of SPAK significantly decreased maximal transport rate. Coexpression of wild-type OSR1, (T185E)OSR1 and (T185A)OSR1 but not of (D164A)OSR1 significantly negatively regulated SLC6A8 activity. OSR1 again decreased significantly maximal transport rate. CONCLUSIONS: Both, SPAK and OSR1, are negative regulators of the creatine transporter SLC6A8.
Asunto(s)
Proteínas del Tejido Nervioso/biosíntesis , Proteínas de Transporte de Neurotransmisores en la Membrana Plasmática/biosíntesis , Proteínas Serina-Treonina Quinasas/fisiología , Animales , Creatina/metabolismo , Regulación de la Expresión Génica , Humanos , Proteínas del Tejido Nervioso/genética , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Técnicas de Placa-Clamp , Proteínas de Transporte de Neurotransmisores en la Membrana Plasmática/genética , Distribución Tisular , Xenopus laevisRESUMEN
X-linked intellectual disability (XLID), also known as X-linked mental retardation, is a highly genetically heterogeneous condition for which mutations in >90 different genes have been identified. In this study, we used a custom-made sequencing array based on the Affymetrix 50k platform for mutation screening in 17 known XLID genes in patients from 135 families and found eight single-nucleotide changes that were absent in controls. For four mutations affecting ATRX (p.1761M>T), PQBP1 (p.155R>X) and SLC6A8 (p.390P>L and p.477S>L), we provide evidence for a functional involvement of these changes in the aetiology of intellectual disability.
Asunto(s)
Proteínas Portadoras/genética , ADN Helicasas/genética , Discapacidad Intelectual Ligada al Cromosoma X/genética , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Proteínas de Transporte de Neurotransmisores en la Membrana Plasmática/genética , Polimorfismo de Nucleótido Simple , Proteínas Portadoras/biosíntesis , Cromosomas Humanos X/química , ADN Helicasas/biosíntesis , Proteínas de Unión al ADN , Femenino , Genes Ligados a X , Estudios de Asociación Genética , Pruebas Genéticas , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Hibridación Genética , Masculino , Proteínas del Tejido Nervioso/biosíntesis , Proteínas Nucleares/biosíntesis , Linaje , Proteínas de Transporte de Neurotransmisores en la Membrana Plasmática/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína Nuclear Ligada al Cromosoma XRESUMEN
Lithium is an efficacious drug for the treatment of mood disorders, and its application is also considered a potential therapy for brain damage. However, the mechanisms underlying lithium's therapeutic action and toxic effects in the nervous system remain largely elusive. Here we report on the use of a versatile genetic model, the fruit fly Drosophila melanogaster, to discover novel molecular components involved in the lithium-responsive neurobiological process. We previously identified CG15088, which encodes a putative nutrient amino acid transporter of the solute carrier 6 (SLC6) family, as one of the genes most significantly upregulated in response to lithium treatment. This gene was the only SLC6 gene induced by lithium, and was thus designated as Lithium-inducible SLC6 transporter or List. Either RNA interference (RNAi)-mediated knockdown or complete deletion of List resulted in a remarkable increase in the susceptibility of adult flies to lithium's toxic effects, whereas transgenic expression of wild-type List significantly suppressed the lithium hypersensitive phenotype of List-deficient flies. Other ions such as sodium, potassium and chloride did not induce List upregulation, nor did they affect the viability of flies with suppressed List expression. These results indicate that lithium's biochemical or physical properties, rather than general osmotic responses, are responsible for the lithium-induced upregulation of List, as well as for the lithium-susceptible phenotype observed in List knockdown flies. Interestingly, flies became significantly more susceptible to lithium toxicity when List RNAi was specifically expressed in glia than when it was expressed in neurons or muscles, which is consistent with potential glial expression of List. These results show that the List transporter confers resistance to lithium toxicity, possibly as a consequence of its amino acid transporter activity in CNS glia. Our results have provided a new avenue of investigation toward a better understanding of the molecular and cellular mechanisms that underlie lithium-responsive neurobiological process.