Specific Mutations in Transmembrane Helix 8 of Human Concentrative Na+/Nucleoside Cotransporter hCNT1 Affect Permeant Selectivity and Cation Coupling
| Autor: | Edward Karpinski, James D. Young, Amy M. L. Ng, Sylvia Y.M. Yao, Stephen A. Baldwin, Carol E. Cass, Shaun K. Loewen, Kyla M. Smith, Melissa D. Slugoski |
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| Rok vydání: | 2007 |
| Předmět: |
Stereochemistry
Cytidine transport Cytidine Lithium Biochemistry Protein Structure Secondary Substrate Specificity Xenopus laevis chemistry.chemical_compound Leucine Serine medicine Animals Humans Amino Acid Sequence Uridine Peptide sequence Sodium Mutagenesis Membrane Transport Proteins Cations Monovalent Adenosine Kinetics Transmembrane domain chemistry Cotransporter 4-Chloromercuribenzenesulfonate Nucleoside Thymidine medicine.drug |
| Zdroj: | Biochemistry. 46:1684-1693 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi061692s |
| Popis: | The Na + /nudeoside cotransporters hCNT1 (650 residues) and hCNT2 (658 residues) are 72% identical in amino acid sequence and contain 13 putative transmembrane helices (TMs). Both transport uridine and adenosine but are otherwise selective for pyrimidine (system cit) and purine (system cif) nucleosides, respectively. Previously, we used site-directed mutagenesis and functional expression in Xenopus oocytes to identify two pairs of adjacent residues in TMs 7 and 8 of hCNT1 (Ser 319 -Gln 320 and Ser 353 -Leu 354 ) that, when converted to the corresponding residues in hCNT2 (Gly-Met and Thr-Val, respectively), changed the permeant selectivity of the transporter from cit to cif. We now report an investigation of the effects of corresponding mutations in TM 8 alone and demonstrate unique S353T-and L354V-induced changes in nucleoside specificity and cation coupling, respectively. hCNTI mutation S353T produced a profound decrease in cytidine transport efficiency (V max /K m ratio) and, in combination with L354V (S353T/L354V), resulted in a novel uridine-preferring transport phenotype. In addition, the L354V mutation markedly increased the apparent affinity of hCNTI for Na + and Li + . Both hCNT1 TM 8 residues exhibited uridine-protectable inhibition by p-chloromercuribenzene sulfonate when converted to Cys, suggesting that they occupy positions within or closely adjacent to a common cation/nucleoside translocation pore. |
| Databáze: | OpenAIRE |
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