RESUMO
Yeast amino acid transporters of the APC superfamily are responsible for the proton motive force-driven uptake of amino acids into the cell, which for most secondary transporters is a reversible process. The l-lysine proton symporter Lyp1 of Saccharomyces cerevisiae is special in that the Michaelis constant from out-to-in transport ( Kmoutâin ) is much lower than Kminâout , which allows accumulation of l-lysine to submolar concentration. It has been proposed that high intracellular lysine is part of the antioxidant mechanism of the cell. The molecular basis for the unique kinetic properties of Lyp1 is unknown. We compared the sequence of Lyp1 with APC para- and orthologues and find structural features that set Lyp1 apart, including differences in extracellular loop regions. We screened the extracellular loops by alanine mutagenesis and determined Lyp1 localization and activity and find positions that affect either the localization or activity of Lyp1. Half of the affected mutants are located in the extension of extracellular loop 3 or in a predicted α-helix in extracellular loop 4. Our data indicate that extracellular loops not only connect the transmembrane helices but also serve functionally important roles.
Assuntos
Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sistemas de Transporte de Aminoácidos Básicos/análise , Sistemas de Transporte de Aminoácidos Básicos/genética , Biologia Computacional , Cinética , Lisina/metabolismo , Modelos Moleculares , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/análise , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
ABSTRACT The aim of the present study was to investigate the effect of donor pH on the transdermal permeability of the model drugs across rat skin and also to determine the major route of transport of the drugs. Weakly acidic drugs (partition coefficient) ibuprofen (3.6), aceclofenac (3.9), glipizide (1.9) and weakly basic drugs olanzapine (3.6), telmisartan (6.0), and sildenafil citrate (1.9) were selected for the study. The ex vivo permeation studies of these drugs at different donor pH (pH - 1.2, 4, 5, 6.8, 7.4, and 8) using Franz diffusion cell (area, 7.54 cm2) has shown a pH-dependent permeability. Among these drugs the weakly acidic drugs has shown higher permeation rates compared to the weakly basic drugs. The permeability coefficient and the distribution coefficient of the weakly basic drugs increased on increasing the pH whereas the weakly acidic drugs showed an inverse relation. The weakly basic drugs also showed an increase in permeation with increase in the fraction of unionized species indicating dominance of transcellular route of permeation. With an exception of sildenafil citrate, a weakly basic salt form of the drug which showed a high permeation value at pH 7.4 where 57% of the drug was unionized, indicating the involvement of both paracellular and transcellular route in its permeation.
Assuntos
Animais , Masculino , Ratos , Aminoácidos Acídicos/análise , Sistemas de Transporte de Aminoácidos Básicos/análise , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Pele , Solubilidade , Preparações Farmacêuticas/análiseRESUMO
To test the hypothesis that AA transporter transcripts are present in the large intestine and similarly expressed along the intestinal tract, mRNA abundance of candidate AA transporter genes solute carrier (SLC) family 7, member 9 (SLC7A9), SLC7A1, SLC7A8, and SLC43A1 encoding for b(0,+)-type AA transporter (b(0,+)AT), cationic AA transporter-1 (CAT-1), L-type AA transporter-2 (LAT-2), and L-type AA transporter-3 (LAT-3), respectively, was determined in small and large intestinal segments of the horse. Mucosa was collected from the equine small (jejunum and ileum) and large intestine (cecum, left ventral colon, and left dorsal colon), flash frozen in liquid nitrogen, and stored at -80 degrees C. Messenger RNA was isolated from tissue samples, followed by manufacture of cDNA. Relative quantitative reverse transcription-PCR was conducted using the 2(-DeltaDeltaCT) method, with glyceraldehyde-3-phosphate dehydrogenase serving as the housekeeping gene. Compared with the jejunum, cationic and neutral AA transporter SLC7A9 mRNA abundance was similar in the ileum, cecum, and large intestinal segments. Compared with the jejunum, cationic AA transporter SLC7A1 mRNA abundance was similar in the ileum and decreased in the cecum, left ventral colon, and left dorsal colon (P < 0.001). Neutral AA transporter SLC7A8 mRNA abundance decreased from the cranial to caudal end of the intestinal tract (P < 0.001). Neutral AA transporter SLC43A1 mRNA abundance was similar in the ileum and left dorsal colon and increased in the cecum (P < 0.01) and left ventral colon (P < 0.1) compared with the jejunum. Cationic and neutral AA transporter SLC7A9 mRNA abundance was similarly expressed in the large compared with small intestine, whereas cationic AA transporter SLC7A1 was of low abundance in the large intestine; neutral AA transporters SLC7A8 and SLC43A1 were differentially expressed with decreased abundance of SLC7A8 and increased abundance of SLC43A1 in the large intestine. Results indicate that the large intestine might contribute to both cationic and neutral AA uptake and absorption predominantly via transporters LAT-3 and b(0,+)AT.
Assuntos
Sistemas de Transporte de Aminoácidos Básicos/análise , Sistemas de Transporte de Aminoácidos Neutros/análise , Cavalos/metabolismo , Intestinos/química , Sistemas de Transporte de Aminoácidos Básicos/biossíntese , Sistemas de Transporte de Aminoácidos Neutros/biossíntese , Animais , Transportador 1 de Aminoácidos Catiônicos/análise , Transportador 1 de Aminoácidos Catiônicos/biossíntese , Ceco/química , Ceco/metabolismo , Colo/química , Colo/metabolismo , Cavalos/fisiologia , Íleo/química , Íleo/metabolismo , Mucosa Intestinal/metabolismo , Jejuno/química , Jejuno/metabolismo , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
System L amino acid transporters mediate the movement of bulky neutral amino acids across cell membranes. Until now three proteins that induce system L activity have been identified: LAT1, LAT2, and LAT3. The former two proteins belong to the solute carrier family 7 (SLC7), whereas the latter belongs to SLC43. In the present study we present a new cDNA, designated LAT4, which also mediates system L activity when expressed in Xenopus laevis oocytes. Human LAT4 exhibits 57% identity to human LAT3. Like LAT3, the amino acid transport activity induced by LAT4 is sodium-, chloride- and pH-independent, is not trans-stimulated, and shows two kinetic components. The low affinity component of LAT4 induced activity is sensitive to the sulfhydryl-specific reagent N-ethylmaleimide but not that with high affinity. Mutation in LAT4 of the SLC43 conserved serine 297 to alanine abolishes sensitivity to N-ethylmaleimide. LAT4 activity is detected at the basolateral membrane of PCT kidney cells. In situ hybridization experiments show that LAT4 mRNA is restricted to the epithelial cells of the distal tubule and the collecting duct in the kidney. In the intestine, LAT4 is mainly present in the cells of the crypt.