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1.
Pharmacogenet Genomics ; 17(9): 773-82, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17700366

RESUMO

BACKGROUND: OCTN1 is a multispecific transporter of organic cations and zwitterions, including several clinically important drugs as well as the antioxidant ergothioneine. OCTN1 is highly expressed in the kidney, where it is thought to aid in active secretion of organic cations, and may facilitate the active reabsorption of ergothioneine. Genetic variation in OCTN1 may help to explain interindividual variability in the pharmacokinetics of many cationic or zwitterionic drugs. METHODS: We screened for human genetic variants in the OCTN1 coding region by direct sequencing in a large sample (n=270) of ethnically diverse healthy volunteers. RESULTS: Six protein sequence-altering variants were identified, including five-amino-acid substitutions and one nonsense mutation. Two of the variants, T306I and L503F, were polymorphic, occurring at frequencies of 37 and 19%, respectively, in the total sample. Allele frequencies are varied by ethnicity. In biochemical assays, two of the variants (D165G and R282X) resulted in complete loss of transport function, and one variant (M205I) caused a reduction in activity to approximately 50% of the reference sequence protein. One variant, L503F, showed altered substrate specificity; this variant occurred at particularly high allele frequency (42%) in the European-American participants in our sample. Subcellular localization and ergothioneine inhibition kinetics were similar among the common amino-acid sequence variants of OCTN1. CONCLUSIONS: The common OCTN1-L503F variant may explain a significant amount of population variation in the pharmacokinetics of OCTN1 substrate drugs. The rare loss-of-function variants provide a rational tool for studying the importance of ergothioneine in humans in vivo.


Assuntos
Proteínas de Transporte de Cátions Orgânicos/genética , Substituição de Aminoácidos , Linhagem Celular , Códon sem Sentido , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Modelos Moleculares , Proteínas de Transporte de Cátions Orgânicos/química , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Farmacogenética , Polimorfismo Genético , Estrutura Secundária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Simportadores , Transfecção
2.
J Pharmacol Exp Ther ; 318(2): 521-9, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16702441

RESUMO

Methotrexate (MTX) is used in patients with malignant and autoimmune diseases. This drug is primarily excreted unchanged in the urine, and its net excretion occurs via active secretory and reabsorptive processes. We characterized the interaction of MTX with human organic-anion transporting polypeptide transporter (OATP) 1A2, which is expressed in tissues important for MTX disposition and toxicity, such as the intestine, kidney, liver, and endothelial cells of the blood-brain barrier. In Xenopus laevis oocytes expressing OATP1A2, the uptake of the model substrate, estrone-3-sulfate (ES), was enhanced 30-fold compared with uninjected oocytes. MTX uptake in oocytes expressing OATP1A2 was saturable (Km = 457 +/- 118 microM; Vmax = 17.5 +/- 4.9 pmol/oocyte/60 min) and sensitive to extracellular pH. That is, acidic pHs stimulated MTX uptake by as much as 7-fold. Seven novel protein-altering variants were identified in 270 ethnically diverse DNA samples. Four protein-altering variants in OATP1A2 exhibited altered transport of ES and/or MTX. The common variant, protein reference sequence (p.) Ile13Thr, was hyperfunctional for ES and MTX and showed a 2-fold increase in the V(max) for ES. The common variant, p. Glu172Asp, exhibited reduced maximal transport capacity for ES and MTX. p. Arg168Cys was hypofunctional, and p. Asn277DEL was nonfunctional. Because of its expression on the apical membrane of the distal tubule and in tissues relevant to MTX disposition and toxicity, these findings suggest that OATP1A2 may play a role in active tubular reabsorption of MTX and in MTX-induced toxicities. Furthermore, genetic variation in OATP1A2 may contribute to variation in MTX disposition and response.


Assuntos
Antagonistas do Ácido Fólico/farmacologia , Metotrexato/farmacologia , Transportadores de Ânions Orgânicos/efeitos dos fármacos , Transportadores de Ânions Orgânicos/genética , Alelos , Sequência de Aminoácidos , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Antagonistas do Ácido Fólico/farmacocinética , Variação Genética , Humanos , Concentração de Íons de Hidrogênio , Metotrexato/análogos & derivados , Metotrexato/farmacocinética , Dados de Sequência Molecular , Oócitos/metabolismo , Xenopus laevis
3.
Am J Physiol Renal Physiol ; 290(4): F905-12, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16291576

RESUMO

The human organic anion transporter, OAT3 (SLC22A8), plays a critical role in renal drug elimination, by mediating the entry of a wide variety of organic anions, including a number of commonly used pharmaceuticals, into the renal proximal tubular cells. To understand the nature and extent of genetic variation in OAT3, and to determine whether such variation affects its function, we identified OAT3 variants in a large, ethnically diverse sample population and studied their transport activities in cellular assays. We identified a total of 10 distinct coding-region variants, which altered the encoded amino acid sequence, in DNA samples from 270 individuals (80 African-Americans, 80 European-Americans, 60 Asian-Americans, and 50 Mexican-Americans). The overall prevalence of these OAT3 variants was relatively low among the screened population, with only three variants having allele frequencies of >1% in a particular ethnic group. Clones of each variant were created by site-directed mutagenesis, expressed in HEK-293 cells, and tested for function using the model substrates, estrone sulfate (ES) and cimetidine (CIM). The results revealed a high degree of functional heterogeneity among OAT3 variants, with three variants (p. Arg149Ser, p. Gln239Stop, and p. Ile260Arg) that resulted in complete loss of function, and several others with significantly reduced function. One of the more common variants (p. Ile305Phe), found in 3.5% of Asian-Americans, appeared to have altered substrate specificity. This variant exhibited a reduced ability to transport ES, but a preserved ability to transport CIM. These data suggest that genetic variation in OAT3 may contribute to variation in the disposition of drugs.


Assuntos
Variação Genética , Transportadores de Ânions Orgânicos Sódio-Independentes/genética , Transportadores de Ânions Orgânicos Sódio-Independentes/fisiologia , Alelos , Análise Mutacional de DNA , Etnicidade , Genética Populacional , Humanos , Preparações Farmacêuticas/metabolismo , Farmacocinética
4.
Mol Pharmacol ; 65(3): 512-9, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-14978229

RESUMO

The concentrative nucleoside transporter, CNT1 (SLC28A1), mediates the cellular uptake of naturally occurring pyrimidine nucleosides and many structurally diverse anticancer and antiviral nucleoside analogs. As a first step toward understanding whether genetic variation in CNT1 contributes to variation in the uptake and disposition of clinically used nucleoside analogs, we determined the haplotype structure and functionally analyzed all coding region variants of CNT1 identified in ethnically diverse populations (100 African Americans, 100 European Americans, 30 Asians, 10 Mexican Americans, and 7 Pacific Islanders) (Leabman et al., 2003). A total of 58 coding region haplotypes were identified using PHASE analysis, 44 of which contained at least one amino acid variant. More than half of the coding region haplotypes were population-specific. Using site-directed mutagenesis, 15 protein-altering CNT1 variants, including one amino acid insertion and one base pair (bp) deletion, were constructed and expressed in Xenopus laevis oocytes. All variant transporters took up [3H]thymidine with the exception of CNT1-Ser546Pro, a rare variant, and CNT1-1153del, a single bp deletion found at a frequency of 3% in the African American population. The bp deletion results in a frame-shift followed by a stop-codon. The anticancer nucleoside analog gemcitabine had a reduced affinity for CNT1-Val189Ile (a common CNT1 variant found at a frequency of 26%) compared with reference CNT1 (IC50=13.8 +/- 0.60 microM for CNT1-reference and 23.3 +/- 1.5 microM for CNT1-Val189Ile, p<0.05). These data suggest that common genetic variants of CNT1 may contribute to variation in systemic and intracellular levels of anti-cancer nucleoside analogs.


Assuntos
Variação Genética , Proteínas de Transporte de Nucleosídeos/genética , Sequência de Aminoácidos , Transporte Biológico , DNA Complementar/análise , Haplótipos , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Dados de Sequência Molecular , Proteínas de Transporte de Nucleosídeos/química , Proteínas de Transporte de Nucleosídeos/metabolismo , Estrutura Secundária de Proteína
5.
Pharmacogenetics ; 12(5): 395-405, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12142729

RESUMO

The completion of the Human Genome Project and the development of high-throughput polymorphism identification methods have allowed researchers to carry out full genetic analyses of many clinically relevant genes. However, few studies have combined genetic analysis with in vitro phenotyping to better understand the relationship between genetic variation and protein function. Many transporters in the kidney are thought to play key roles in defense against a variety of foreign substances. The goal of this study was to understand the relationship between variation in a gene encoding a major renal xenobiotic transporter, OCT2, and transporter function. We report a comprehensive genetic analysis and functional characterization of variants of OCT2. Twenty-eight variable sites in the OCT2 gene were identified in a collection of 247 ethnically diverse DNA samples. Eight caused non-synonymous amino acid changes, of which four were present at >/= 1% in an ethnic population. All four of these altered transporter function assayed in Xenopus laevis oocytes. Analysis of nucleotide diversity (pi) revealed a higher prevalence of synonymous (pi = 22.4 x 10-4) versus non-synonymous (pi = 2.1 x 10-4) changes in OCT2 than in other genes. In addition, the non-synonymous sites had a significant tendency to exhibit more skewed allele frequencies (more negative Tajima's D-values) compared to synonymous sites. The population-genetic analysis, together with the functional characterization, suggests that selection has acted against amino acid changes in OCT2. This selection may be due to a necessary role of OCT2 in the renal elimination of endogenous amines or xenobiotics, including environmental toxins, neurotoxic amines and therapeutic drugs.


Assuntos
Rim/fisiologia , Proteínas de Transporte de Cátions Orgânicos/genética , Polimorfismo Genético , Alelos , Sequência de Aminoácidos , Animais , Primers do DNA , Feminino , Variação Genética , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Oócitos/fisiologia , Proteínas de Transporte de Cátions Orgânicos/química , Proteínas de Transporte de Cátions Orgânicos/fisiologia , Transportador 2 de Cátion Orgânico , Reação em Cadeia da Polimerase , Conformação Proteica , Xenobióticos/farmacocinética , Xenopus laevis
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