A proton-mediated conformational shift identifies a mobile pore-lining cysteine residue (Cys-561) in human concentrative nucleoside transporter 3.
The concentrative nucleoside transporter (CNT) protein family in humans is represented by three members, hCNT1, hCNT2, and hCNT3. Belonging to a CNT subfamily phylogenetically distinct from hCNT1/2, hCNT3 mediates transport of a broad range of purine and pyrimidine nucleosides and nucleoside drugs, whereas hCNT1 and hCNT2 are pyrimidine and purine nucleoside-selective, respectively. All three hCNTs are Na(+)-coupled. Unlike hCNT1/2, however, hCNT3 is also capable of H(+)-mediated nucleoside cotransport. Using site-directed mutagenesis in combination with heterologous expression in Xenopus oocytes, we have identified a C-terminal intramembranous cysteine residue of hCNT3 (Cys-561) that reversibly binds the hydrophilic thiol-reactive reagent p-chloromercuribenzene sulfonate (PCMBS). Access of this membrane-impermeant probe to Cys-561, as determined by inhibition of hCNT3 transport activity, required H(+), but not Na(+), and was blocked by extracellular uridine. Although this cysteine residue is also present in hCNT1 and hCNT2, neither transporter was affected by PCMBS. We conclude that Cys-561 is located in the translocation pore in a mobile region within or closely adjacent to the nucleoside binding pocket and that access of PCMBS to this residue reports a specific H(+)-induced conformational state of the protein.
Slugoski
Melissa D
MD
Membrane Protein Research Group, Department of Physiology, University of Alberta, Edmonton, Alberta, Canada.
Ng
Amy M L
AM
Yao
Sylvia Y M
SY
Smith
Kyla M
KM
Lin
Colin C
CC
Zhang
Jing
J
Karpinski
Edward
E
Cass
Carol E
CE
Baldwin
Stephen A
SA
Young
James D
JD
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004594:Electrophysiology; D006801:Humans; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008698:Mesylates / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D018408:Patch-Clamp Techniques; D017434:Protein Structure, Tertiary; D011522:Protons; D016415:Sequence Alignment; D014529:Uridine / Q000378:metabolism; D014982:Xenopus laevis
D026901:Membrane Transport Proteins; D008698:Mesylates; D011522:Protons; C065171:cif nucleoside transporter; C074502:methanethiosulfonate; D002731:4-Chloromercuribenzenesulfonate; D003545:Cysteine; D014529:Uridine