Abstract
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.
Highlights
Membranes [1,2,3]
Five of the hCNT3 cysteine residues are located in putative transmembrane helix (TM) (TMs 1, 11, 12, and 13), and the remainder hCNT1 LALSKLVYPEVEESKFRREEGVKLTYGDAQNLIEAASTGAAISVKVVANI hCNT2 LASSKLAYPEVEESKFKSEEGVKLPRGKERNVLEAASNGAVDAIGLATNV hCNT3 LAAAKLFWPETEKPKITLKNAMKMESGDSGNLLEAATQGASSSISLVANI 450 reside within the extramembranous N- and C-terminal regions of the protein
TM 13 hCNT1 VSLVNACMAGILYMPRGAEVDCMSLLN - - - - TTLSSSSFEIYQCCREAFQ hCNT2 VSLISACMAGILYVPRGAEADCVSFPN - - - - TSFTNRTYETYMCCRGLFQ hCNT3 ACFMTACIAGILSSTP - VDINCHHVLENAFNSTFPGNTTKVIACCQSLLS 649 viously established that wild-type hCNT1 is not inhibited by p-chloromercuribenzene sulfonate (PCMBS) [21]
Summary
Site-directed Mutagenesis and DNA Sequencing— hCNT3 cDNA (GenBankTM accession number AF305210) in the Xenopus expression vector pGEM-HE [23] provided the template for construction of hCNT3 mutants by the oligonucleotide-directed technique [24] using reagents from the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer’s directions. Groups of 12 oocytes were incubated at room temperature (20 °C) in 200 l of transport medium containing either 100 mM NaCl or choline chloride (ChCl) and 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES, pH 7.5 and 8.5, or MES, pH 5.5. In PCMBS inhibition studies oocytes were pretreated with PCMBS on ice for 10 min and washed 5 times with ice-cold transport medium to remove excess organomercurial before the assay of transport activity. Electrophysiology—Steady-state and presteady-state currents in hCNT3-producing oocytes were measured at room temperature (20 °C) using the whole-cell, two-electrode voltage clamp (GeneClamp 500B, Molecular Devices Corp.) as previously described (16 –18). After microelectrode penetration, resting membrane potential was measured over a 10-min period before the start of the experiment.
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