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.
Slugoski
Melissa D
MD
Membrane Protein Research Group, Departments of Physiology, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada.
Smith
Kyla M
KM
Mulinta
Ras
R
Ng
Amy M L
AM
Yao
Sylvia Y M
SY
Morrison
Ellen L
EL
Lee
Queenie O T
QO
Zhang
Jing
J
Karpinski
Edward
E
Cass
Carol E
CE
Baldwin
Stephen A
SA
Young
James D
JD
D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D006801:Humans; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000166:cytology; D017354:Point Mutation; D017434:Protein Structure, Tertiary / Q000502:physiology; D011522:Protons; D012964:Sodium / Q000378:metabolism*; D014981:Xenopus
D026901:Membrane Transport Proteins; D011522:Protons; C065171:cif nucleoside transporter; D012964:Sodium; D003545:Cysteine