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Functional Analysis of the Human Concentrative Nucleoside Transporter-1 Variant Hcnt1s546p Provides Insight Into the Sodium-Binding Pocket

Autores: Cano-Soldado, P.; Gorraitz Eusa, Edurne; Errasti-Murugarren, E.; Casado, F. J.; Lostao Crespo, María del Pilar; Pastor-Anglada, M.
ISSN: 0363-6143
Volumen: 302
Número: 1
Páginas: C257-C266
Fecha de publicación: 2012
Cano-Soldado P, Gorraitz E, Errasti-Murugarren E, Casado FJ, Lostao MP, Pastor-Anglada M. Functional analysis of the human concentrative nucleoside transporter-1 variant hCNT1S546P provides insight into the sodium-binding pocket. Am J Physiol Cell Physiol 302: C257-C266, 2012. First published October 12, 2011; doi: 10.1152/ajpcell.00198.2011.-SLC28 genes, encoding concentrative nucleoside transporter proteins (CNT), show little genetic variability, although a few single nucleotide polymorphisms (SNPs) have been associated with marked functional disturbances. In particular, human CNT1S546P had been reported to result in negligible thymidine uptake. In this study we have characterized the molecular mechanisms responsible for this apparent loss of function. The hCNT1S546P variant showed an appropriate endoplasmic reticulum export and insertion into the plasma membrane, whereas loss of nucleoside translocation ability affected all tested nucleoside and nucleoside-derived drugs. Site-directed mutagenesis analysis revealed that it is the lack of the serine residue itself responsible for the loss of hCNT1 function. This serine residue is highly conserved, and mutation of the analogous serine in hCNT2 (Ser541) and hCNT3 (Ser568) resulted in total and partial loss of function, respectively. Moreover, hCNT3, the only member that shows a 2Na(+)/1 nucleoside stoichiometry, showed altered Na(+) binding properties associated with a shift in the Hill coefficient, consistent with one Na(+) binding site being affected by the mutation. Two-electrode voltage-clamp studies using the hCNT1S546P mutant revealed the occurrence of Na(+) leak, which was dependent on the concentration of extracellular Na(+) indicating that, although the variant is unable to transport nucleosides, there is an uncoupled sodium transport.