L-proline and L-pipecolate induce enkephalin-sensitive currents in human embryonic kidney 293 cells transfected with the high-affinity mammalian brain L-proline transporter.
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The high-affinity mammalian brain L-proline transporter (PROT) belongs to the GAT1 gene family, which includes Na- and Cl-dependent plasma membrane carriers for neurotransmitters, osmolites, and metabolites. These transporters couple substrate flux to transmembrane electrochemical gradients, particularly the Na gradient. In the nervous system, transporters clear synapses and help to replenish transmitters in nerve terminals. The localization of PROT to specific excitatory terminals in rat forebrain suggests a role for this carrier in excitatory transmission (). We investigated the voltage regulation and electrogenicity of this novel transporter, using human embryonic kidney (HEK) 293 cells stably transfected with rat PROT cDNA. In physiological solutions between -140 and -40 mV, L-proline (PRO) and its six-member ring congener L-pipecolate (PIP) induced inward current. The current-voltage relationship and the variance of current fluctuations were similar for PRO- and PIP-induced current, and the ratio of induced variance to the mean current ranged from 20 to 60 fA. Des-Tyr-Leu-enkephalin (GGFL), a competitive peptide inhibitor of PROT, reduced the rat PROT-associated current to control levels. GGFL alone did not elicit currents, and the GGFL-sensitive substrate-induced current was absent in nontransfected cells. Finally, GGFL inhibited PROT-mediated transport only when applied to the extracellular face of PROT. These data suggest that (1) PROT uptake is electrogenic, (2) individual transporter currents are voltage-independent, and (3) GGFL is a nonsubstrate inhibitor that interacts either with an extracellular domain of PROT or in an externally accessible pore. (+info)
Molecular characterization of CTNS deletions in nephropathic cystinosis: development of a PCR-based detection assay.
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Nephropathic cystinosis is an autosomal recessive disorder that is characterized by accumulation of intralysosomal cystine and is caused by a defect in the transport of cystine across the lysosomal membrane. Using a positional cloning strategy, we recently cloned the causative gene, CTNS, and identified pathogenic mutations, including deletions, that span the cystinosis locus. Two types of deletions were detected-one of 9.5-16 kb, which was seen in a single family, and one of approximately 65 kb, which is the most frequent mutation found in the homozygous state in nearly one-third of cystinotic individuals. We present here characterization of the deletion breakpoints and demonstrate that, although both deletions occur in regions of repetitive sequences, they are the result of nonhomologous recombination. This type of mechanism suggests that the approximately 65-kb deletion is not a recurrent mutation, and our results confirm that it is identical in all patients. Haplotype analysis shows that this large deletion is due to a founder effect that occurred in a white individual and that probably arose in the middle of the first millenium. We also describe a rapid PCR-based assay that will accurately detect both homozygous and heterozygous deletions, and we use it to show that the approximately 65-kb deletion is present in either the homozygous or the heterozygous state in 76% of cystinotic patients of European origin. (+info)
Molecular analysis of cystinosis: probable Irish origin of the most common French Canadian mutation.
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Infantile nephropathic cystinosis, an autosomal recessive disease characterized by a lysosomal accumulation of cystine, presents as failure to thrive, rickets and proximal renal tubular acidosis. The cystinosis gene, CTNS, which maps to chromosome 17p13, encodes a predicted 55 kDa protein with characteristics of a lysosomal membrane protein. We have conducted extensive linkage analysis in a French Canadian cystinosis cohort identifying a founding haplotype present in approximately half (21/40) of the chromosomes studied. Subsequent mutational analysis, in addition to identifying two novel mutations, has unexpectedly revealed a mutation which has been previously found in Irish (but not French) cystinotic families on these 21 French Canadian chromosomes. Haplotype analysis of two Irish families with this mutation supports the hypothesis that Celtic chromosomes represent an extensive portion of cystinosis chromosomes in French Canada. Our analysis underlines the genetic heterogeneity of the French Canadian population, reflecting a frequently unrecognized contribution from non-Gallic sources including the Irish. (+info)
Interactions between the thiol-group reagent N-ethylmaleimide and neutral and basic amino acid transporter-related amino acid transport.
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The neutral and basic amino acid transport protein (NBAT) expressed in renal and jejunal brush-border membranes is involved in amino acid and cystine absorption. NBAT mutations result in Type 1 cystinuria. A C-terminal myc-tagged NBAT (NBATmyc) retains the amino acid transport and protein-protein interaction properties of NBAT when expressed in Xenopus oocytes. Neutral amino acid (Ala, Phe)-cationic amino acid (Arg) heteroexchanges related to NBATmyc expression in oocytes are inactivated by treatment with the thiol-group reagent N-ethylmaleimide (NEM), although significant Arg-Arg and Ala-Ala homoexchanges persist. Inactivation of heteroexchange activity by NEM is accompanied by loss of >85% of alanine and cystine uptake, with smaller (<50%) inhibition of arginine and phenylalanine uptake. NEM-sensitive cystine uptake and arginine-alanine heteroexchange (system b(0,+) activity) are not expressed by an NBAT truncation mutant (NBATmyc-Sph1) lacking the 13 C-terminal amino acid residues, but the mutant expresses NEM-resistant transport activity (system y(+)L-like) equivalent to that of full-length NBATmyc. The deleted region of NBATmyc-Sph1 contains two cysteine residues (671/683) which may be the targets of NEM action. The synthetic amino acid 2-trifluoromethylhistidine (TFMH) stimulated alanine efflux at pH 7.5 and arginine at pH 5.5, but not vice versa, establishing the existence of distinct pathways for cationic and neutral amino acid homoexchange (TFMH is zwitterionic at pH 7.5 and cationic at pH 5.5). We suggest that NBAT expresses a combination of system b(0,+) and y(+)L-like activities, possibly by interacting with different light-chain subunits endogenous to oocytes (as does the homologous 4F2hc protein). The C-terminus of NBAT may also have an additional, direct role in the mechanism of System b(0,+) transport (the major transport activity that is defective in Type 1 cystinuria). (+info)
Severity of phenotype in cystinosis varies with mutations in the CTNS gene: predicted effect on the model of cystinosin.
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Infantile nephropathic cystinosis is a rare, autosomal recessive disease caused by a defect in the transport of cystine across the lysosomal membrane and characterized by early onset of renal proximal tubular dysfunction. Late-onset cystinosis, a rarer form of the disorder, is characterized by onset of symptoms between 12 and 15 years of age. We previously characterized the cystinosis gene, CTNS, and identified pathogenic mutations in patients with infantile nephropathic cystinosis, including a common, approximately 65 kb deletion which encompasses exons 1-10. Structure predictions suggested that the gene product, cystinosin, is a novel integral lysosomal membrane protein. We now examine the predicted effect of mutations on this model of cystinosin. In this study, we screened patients with infantile nephropathic cystinosis, those with late-onset cystinosis and patients whose phenotype does not fit the classical definitions. We found 23 different mutations in CTNS; 14 are novel mutations. Out of 25 patients with infantile nephropathic cystinosis, 12 have two severely truncating mutations, which is consistent with a loss of functional protein, and 13 have missense or in-frame deletions, which would result in disruption of transmembrane domains and loss of protein function. Mutations found in two late-onset patients affect functionally unimportant regions of cystinosin, which accounts for their milder phenotype. For three patients, the age of onset of cystinosis was <7 years but the course of the disease was milder than the infantile nephropathic form. This suggests that the missense mutations found in these individuals allow production of functional protein and may also indicate regions of cystinosin which are not functionally important. (+info)
Intracellular assembly of Kell and XK blood group proteins.
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Kell, a 93 kDa type II membrane glycoprotein, and XK, a 444 amino acid multi-pass membrane protein, are blood group proteins that exist as a disulfide-bonded complex on human red cells. The mechanism of Kell/XK assembly was studied in transfected COS cells co-expressing Kell and XK proteins. Time course studies combined with endonuclease-H treatment and cell fractionation showed that Kell and XK are assembled in the endoplasmic reticulum. At later times the Kell component of the complex was not cleaved by endonuclease-H, indicating N-linked oligosaccharide processing and transport of the complex to a Golgi and/or a post-Golgi cell fraction. Surface-labeling of transfected COS cells, expressing both Kell and XK, demonstrated that the Kell/XK complex travels to the plasma membrane. XK expressed in the absence of Kell was also transported to the cell surface indicating that linkage of Kell and XK is not obligatory for cell surface expression. (+info)
Cloning, functional characterisation and population analysis of a variant form of the human glycine type 2 transporter.
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Two forms of glycine transporter have been described to date, GlyT-1 and GlyT-2. The GlyT-2 form is expressed mainly in the spinal cord, brainstem and cerebellum. Here we describe the identification of a variant form of the human GlyT-2 (SC6), showing three amino acid changes to the previously reported protein. Population analysis identified the allele causing one of the polymorphisms, D463N, at 10% within the population with 3% being homozygous for the change. We also transfected our new variant into mammalian cells and compared it to the published cDNA, showing that the three amino acid changes present have no major effect on the biochemical properties of the transporter. (+info)
Proline catabolism by Pseudomonas putida: cloning, characterization, and expression of the put genes in the presence of root exudates.
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Pseudomonas putida KT2442 is a root-colonizing strain which can use proline, one of the major components in root exudates, as its sole carbon and nitrogen source. A P. putida mutant unable to grow with proline as the sole carbon and nitrogen source was isolated after random mini-Tn5-Km mutagenesis. The mini-Tn5 insertion was located at the putA gene, which is adjacent to and divergent from the putP gene. The putA gene codes for a protein of 1,315 amino acid residues which is homologous to the PutA protein of Escherichia coli, Salmonella enterica serovar Typhimurium, Rhodobacter capsulatus, and several Rhizobium strains. The central part of P. putida PutA showed homology to the proline dehydrogenase of Saccharomyces cerevisiae and Drosophila melanogaster, whereas the C-terminal end was homologous to the pyrroline-5-carboxylate dehydrogenase of S. cerevisiae and a number of aldehyde dehydrogenases. This suggests that in P. putida, both enzymatic steps for proline conversion to glutamic acid are catalyzed by a single polypeptide. The putP gene was homologous to the putP genes of several prokaryotic microorganisms, and its gene product is an integral inner-membrane protein involved in the uptake of proline. The expression of both genes was induced by proline added in the culture medium and was regulated by PutA. In a P. putida putA-deficient background, expression of both putA and putP genes was maximal and proline independent. Corn root exudates collected during 7 days also strongly induced the P. putida put genes, as determined by using fusions of the put promoters to 'lacZ. The induction ratio for the putA promoter (about 20-fold) was 6-fold higher than the induction ratio for the putP promoter. (+info)