RESUMEN
The human malaria parasite Plasmodium falciparum is capable of adapting to vastly different extracellular Ca(2+) environments while maintaining tight control of its intracellular Ca(2+) concentration. The mechanisms underpinning Ca(2+) homeostasis in this important pathogen are only partly understood. Here we have functionally expressed the putative Ca(2+)/H(+) antiporter PfCHA in Xenopus laevis oocytes. Our data suggest that PfCHA mediates H(+)-coupled Ca(2+) and Mn(2+) exchange. The apparent dissociation constant K(M) for Ca(2+) of 2.2 +/- 0.7 mM and the maximal velocity V(max) of 0.6 +/- 0.1 nmol per oocyte per hour are consistent with PfCHA being a low-affinity high-capacity Ca(2+) carrier. In the parasite, PfCHA was found to localize to the mitochondrion. Physiological studies conducted with live parasitized erythrocytes, and using Fluo-4 and Rhod-2 to monitor cytoplasmic and mitochondrial Ca(2+) dynamics, suggest that the mitochondrion serves as a dynamic Ca(2+) store and that PfCHA functions as a Ca(2+) efflux system expelling excess Ca(2+) from the mitochondrion. PfCHA lacks appreciable homologies to the human mitochondrial Ca(2+)/H(+) exchanger and might represent an evolutionary divergent class of mitochondrial cation antiporter, which, in turn, might provide novel opportunities for intervention.
Asunto(s)
Antiportadores/metabolismo , Cationes Bivalentes/metabolismo , Proteínas Mitocondriales/metabolismo , Plasmodium falciparum/metabolismo , Protones , Proteínas Protozoarias/metabolismo , Animales , Antiportadores/genética , Calcio/metabolismo , Expresión Génica , Cinética , Manganeso/metabolismo , Mitocondrias/química , Proteínas Mitocondriales/genética , Modelos Biológicos , Modelos Moleculares , Oocitos , Plasmodium falciparum/genética , Unión Proteica , Proteínas Protozoarias/genética , Xenopus laevisRESUMEN
Resistance to several anti-malarial drugs has been associated with polymorphisms within the P-glycoprotein homologue (Pgh-1, PfMDR1) of the human malaria parasite Plasmodium falciparum. Pgh-1, coded for by the gene pfmdr1, is predominately located at the membrane of the parasite's digestive vacuole. How polymorphisms within this transporter mediate alter anti-malarial drug responsiveness has remained obscure. Here we have functionally expressed pfmdr1 in Xenopus laevis oocytes. Our data demonstrate that Pgh-1 transports vinblastine, an established substrate of mammalian MDR1, and the anti-malarial drugs halofantrine, quinine and chloroquine. Importantly, polymorphisms within Pgh-1 alter the substrate specificity for the anti-malarial drugs. Wild-type Pgh-1 transports quinine and chloroquine, but not halofantrine, whereas polymorphic Pgh-1 variants, associated with altered drug responsivenesses, transport halofantrine but not quinine and chloroquine. Our data further suggest that quinine acts as an inhibitor of Pgh-1. Our data are discussed in terms of the model that Pgh-1-mediates, in a variant-specific manner, import of certain drugs into the P. falciparum digestive vacuole, and that this contributes to accumulation of, and susceptibility to, the drug in question.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Antimaláricos/farmacología , Resistencia a Medicamentos/genética , Plasmodium falciparum/genética , Proteínas Protozoarias/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Animales , Genes Protozoarios , Concentración de Iones de Hidrógeno , Potenciales de la Membrana , Oocitos/metabolismo , Oocitos/fisiología , Pruebas de Sensibilidad Parasitaria , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/metabolismo , Polimorfismo Genético , Proteínas Protozoarias/genética , ARN Protozoario/genética , Especificidad por Sustrato , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMEN
Activation of protein kinase C (PKC) downregulates the human cationic amino acid transporters hCAT-1 (SLC7A1) and hCAT-3 (SLC7A3) (Rotmann A, Strand D, Martiné U, Closs EI. J Biol Chem 279: 54185-54192, 2004; Rotmann A, Vekony N, Gassner D, Niegisch G, Strand D, Martine U, Closs EI. Biochem J 395: 117-123, 2006). However, others found that PKC increased arginine transport in various mammalian cell types, suggesting that the expression of different arginine transporters might be responsible for the opposite PKC effects. We thus investigated the consequence of PKC activation by phorbol-12-myristate-13-acetate (PMA) in various human cell lines expressing leucine-insensitive system y(+) [hCAT-1, hCAT-2B (SLC7A2), or hCAT-3] as well as leucine-sensitive system y(+)L [y(+)LAT1 (SLC7A7) or y(+)LAT2 (SLC7A6)] arginine transporters. PMA reduced system y(+) activity in all cell lines tested, independent of the hCAT isoform expressed, while mRNAs encoding the individual hCAT isoforms were either unchanged or increased. System y(+)L activity was also inhibited by PMA. The extent and onset of inhibition varied between cell lines; however, a PMA-induced increase in arginine transport was never observed. In addition, when expressed in Xenopus laevis oocytes, y(+)LAT1 and y(+)LAT2 activity was reduced by PMA, and this inhibition could be prevented by the PKC inhibitor bisindolylmaleimide I. In ECV304 cells, PMA-induced inhibition of systems y(+) and y(+)L could be prevented by Gö6976, a specific inhibitor of conventional PKCs. Thymelea toxin, which activates preferentially classical PKC, had a similar inhibitory effect as PMA. In contrast, phosphatidylinositol-3,4,5-triphosphate-dipalmitoyl, an activator of atypical PKC, had no effect. These data demonstrate that systems y(+) and y(+)L are both downregulated by classical PKC.
Asunto(s)
Sistema de Transporte de Aminoácidos y+L/metabolismo , Sistema de Transporte de Aminoácidos y+/metabolismo , Proteína Quinasa C/metabolismo , Arginina/metabolismo , Secuencia de Bases , Transporte Biológico Activo/efectos de los fármacos , Transporte Biológico Activo/fisiología , Línea Celular Tumoral , Activación Enzimática , Regulación de la Expresión Génica , Humanos , Leucina/metabolismo , ARN Mensajero/metabolismo , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
We have previously shown that activation of PKC (protein kinase C) results in internalization of hCAT-1 [human CAT-1 (cationic amino acid transporter 1)] and a decrease in arginine transport [Rotmann, Strand, Martiné and Closs (2004) J. Biol. Chem. 279, 54185-54192]. However, others found increased transport rates for arginine in response to PKC activation, suggesting a differential effect of PKC on different CAT isoforms. Therefore we investigated the effect of PKC on hCAT-3, an isoform expressed in thymus, brain, ovary, uterus and mammary gland. In Xenopus laevis oocytes and human U373MG glioblastoma cells, hCAT-3-mediated L-arginine transport was significantly reduced upon treatment with compounds that activate classical PKC. In contrast, inactive phorbol esters and an activator of novel PKC isoforms had no effect. PKC inhibitors (including the PKCalpha-preferring Ro 31-8280) reduced the inhibitory effect of the PKC-activating compounds. Microscopic analyses revealed a PMA-induced reduction in the cell-surface expression of fusion proteins between hCAT-3 and enhanced green fluorescent protein expressed in X. laevis oocytes and glioblastoma cells. Western-blot analysis of biotinylated surface proteins demonstrated a PMA-induced decrease in hCAT-3 in the plasma membrane, but not in total protein lysates. Pretreatment with a PKC inhibitor also reduced this PMA effect. It is concluded that similar to hCAT-1, hCAT-3 activity is decreased by PKC via reduction of transporter molecules in the plasma membrane. Classical PKC isoforms seem to be responsible for this effect.
Asunto(s)
Transportador de Aminoácidos Catiónicos 1/metabolismo , Membrana Celular/metabolismo , Regulación hacia Abajo , Proteína Quinasa C/metabolismo , Animales , Especificidad de Anticuerpos , Arginina/metabolismo , Transporte Biológico/efectos de los fármacos , Línea Celular Tumoral , Regulación hacia Abajo/genética , Activación Enzimática , Glioblastoma/metabolismo , Humanos , Oocitos/metabolismo , Teratocarcinoma/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales Cultivadas , XenopusRESUMEN
The human cationic amino acid transporter hCAT-1 is almost ubiquitously expressed and probably the most important entity for supplying cells with extracellular arginine, lysine, and ornithine. We have previously shown that hCAT-1-mediated transport is decreased after protein kinase C (PKC) activation by phorbol 12-myristate 13-acetate (PMA) (Gräf, P., Forstermann, U., and Closs, E. I. (2001) Br. J. Pharmacol. 132, 1193-1200). In the present study, we examined the mechanism of this down-regulation. In both Xenopus laevis oocytes and U373MG glioblastoma cells, PMA treatment promoted the internalization of hCAT-1 (fused to the enhanced green fluorescence protein (EGFP)) as visualized by fluorescence microscopy. Biotinylation of cell surface proteins and subsequent Western blot analyses confirmed that the cell surface expression of hCAT-1.EGFP was significantly reduced upon PMA treatment. Pretreatment with the PKC inhibitor bisindolylmaleimide I prevented the reduction by PMA of both hCAT-1.EGFP-induced arginine transport and the internalization of the transporter. Similar results were obtained with hCAT-1 expressed endogenously in DLD-1 colon carcinoma cells. Inhibition of protein synthesis did not augment the PMA effect. In addition, the PMA effect was reverted in washout experiments without changing the hCAT-1 protein expression, suggesting that the PMA effect is reversible in these cells. PKC did not phosphorylate hCAT-1 directly as evidenced by in vivo phosphorylation experiments and mutational analysis, indicating an indirect action of PKC on hCAT-1.
Asunto(s)
Transportador de Aminoácidos Catiónicos 1/metabolismo , Homeostasis , Proteína Quinasa C/metabolismo , Animales , Biotinilación , Transportador de Aminoácidos Catiónicos 1/análisis , Transportador de Aminoácidos Catiónicos 1/genética , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos , Expresión Génica , Glioblastoma , Proteínas Fluorescentes Verdes/genética , Humanos , Indoles/farmacología , Maleimidas/farmacología , Microscopía Fluorescente , Mutagénesis Sitio-Dirigida , Oocitos/química , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Proteínas Recombinantes de Fusión , Acetato de Tetradecanoilforbol/farmacología , Transfección , Células Tumorales Cultivadas , Xenopus laevisRESUMEN
The supply of arginine may become rate limiting for enzymatic reactions that use this semiessential amino acid as a substrate (e.g., nitric oxide, agmatine, creatine, and urea synthesis), particularly under conditions of high demand such as growth, sepsis, or wound healing. In addition, arginine acts as a signaling molecule that regulates essential cellular functions such as protein synthesis, apoptosis, and growth. In the past decade, a number of carrier proteins for amino acids have been identified on the molecular level. They belong to different gene families, exhibit overlapping but distinctive substrate specificities, and can further be distinguished by their requirement for the cotransport or countertransport of inorganic ions. A number of these transporters function as exchangers rather than uniporters. Uptake of amino acids by these transporters therefore depends largely on the intracellular substrate composition. Hence, there is a complex crosstalk between transporters for cationic and neutral amino acids as well as for peptides. This article briefly reviews current knowledge regarding mammalian plasma membrane transporters that accept arginine as a substrate.
Asunto(s)
Sistemas de Transporte de Aminoácidos/metabolismo , Arginina/metabolismo , Membrana Celular/metabolismo , Sistemas de Transporte de Aminoácidos/química , Animales , HumanosRESUMEN
Cationic amino acid transporters play an important role in the intracellular supply of L-Arg and the generation of nitric oxide. Since the transport of L-Arg is voltage-dependent, we aimed at determining the intracellular L-Arg concentration and describing the transport of L-Arg in terms of Michaelis-Menten kinetics, taking into account membrane voltage. The human isoforms of the cationic amino acid transporters, hCAT-1, hCAT-2A, and hCAT-2B, were expressed in oocytes from Xenopus laevis and studied with the voltage clamp technique and in tracer experiments. We found that L-Arg was concentrated intracellularly by all hCAT isoforms and that influx and efflux, in the steady state of exchange, were nearly mirror images. Conductance measurements at symmetric concentrations of L-Arg (inside/outside) allowed us to determine KM and Vmax. The empty transporter of hCAT-2B featured an unexpected potassium conductance, which was inhibited by L-Arg.