A conformationally mobile cysteine residue (Cys-561) modulates Na+ and H+ activation of human CNT3

In humans, the SLC28 concentrative nucleoside transporter (CNT) protein family is represented by three Na(+)-coupled members; human CNT1 (hCNT1) and hCNT2 are pyrimidine and purine nucleoside-selective, respectively, whereas hCNT3 transports both purine and pyrimidine nucleosides and nucleoside drugs. Belonging to a phylogenetic CNT subfamily distinct from hCNT1/2, hCNT3 also mediates H(+)/nucleoside cotransport. Using heterologous expression in Xenopus oocytes, we have characterized a cysteineless version of hCNT3 (hCNT3C-). Processed normally to the cell surface, hCNT3C-exhibited hCNT3-like transport properties, but displayed a decrease in apparent affinity specific for Na(+) and not H(+). Site-directed mutagenesis experiments in wild-type and hCNT3C-backgrounds identified intramembranous Cys-561 as the residue responsible for this altered Na(+)-binding phenotype. Alanine at this position restored Na(+) binding affinity, whereas substitution with larger neutral amino acids (threonine, valine, and isoleucine) abolished hCNT3 H(+)-dependent nucleoside transport activity. Independent of these findings, we have established that Cys-561 is located in a mobile region of the hCNT3 translocation pore adjacent to the nucleoside binding pocket and that access of p-chloromercuribenzene sulfonate to this residue reports a specific H(+)-induced conformational state of the protein ( Slugoski, M. D., Ng, A. M. L., Yao, S. Y. M., Smith, K. M., Lin, C. C., Zhang, J., Karpinski, E., Cass, C. E., Baldwin, S. A., and Young, J. D. (2008) J. Biol. Chem. 283, 8496-8507 ). The present investigation validates hCNT3C- as a template for substituted cysteine accessibility method studies of CNTs and reveals a pivotal functional role for Cys-561 in Na(+)- as well as H(+)-coupled modes of hCNT3 nucleoside transport.