Substituted cysteine accessibility method analysis of human concentrative nucleoside transporter hCNT3 reveals a novel discontinuous region of functional importance within the CNT family motif (G/A)XKX3NEFVA(Y/M/F).

The human SLC28 family of integral membrane CNT (concentrative nucleoside transporter) proteins has three members, hCNT1, hCNT2, and hCNT3. Na(+)-coupled hCNT1 and hCNT2 transport pyrimidine and purine nucleosides, respectively, whereas hCNT3 mediates transport of both pyrimidine and purine nucleosides utilizing Na(+) and/or H(+) electrochemical gradients. These and other eukaryote CNTs are currently defined by a putative 13-transmembrane helix (TM) topology model with an intracellular N terminus and a glycosylated extracellular C terminus. Recent mutagenesis studies, however, have provided evidence supporting an alternative 15-TM membrane architecture. In the absence of CNT crystal structures, valuable information can be gained about residue localization and function using substituted cysteine accessibility method analysis with thiol-reactive reagents, such as p-chloromercuribenzene sulfonate. Using heterologous expression in Xenopus oocytes and the cysteineless hCNT3 protein hCNT3C-, substituted cysteine accessibility method analysis with p-chloromercuribenzene sulfonate was performed on the TM 11-13 region, including bridging extramembranous loops. The results identified residues of functional importance and, consistent with a new revised 15-TM CNT membrane architecture, suggest a novel membrane-associated topology for a region of the protein (TM 11A) that includes the highly conserved CNT family motif (G/A)XKX(3)NEFVA(Y/M/F).

Conserved glutamate residues Glu-343 and Glu-519 provide mechanistic insights into cation/nucleoside cotransport by human concentrative nucleoside transporter hCNT3.

Human concentrative nucleoside transporter 3 (hCNT3) utilizes electrochemical gradients of both Na(+) and H(+) to accumulate pyrimidine and purine nucleosides within cells. We have employed radioisotope flux and electrophysiological techniques in combination with site-directed mutagenesis and heterologous expression in Xenopus oocytes to identify two conserved pore-lining glutamate residues (Glu-343 and Glu-519) with essential roles in hCNT3 Na(+)/nucleoside and H(+)/nucleoside cotransport. Mutation of Glu-343 and Glu-519 to aspartate, glutamine, and cysteine severely compromised hCNT3 transport function, and changes included altered nucleoside and cation activation kinetics (all mutants), loss or impairment of H(+) dependence (all mutants), shift in Na(+):nucleoside stoichiometry from 2:1 to 1:1 (E519C), complete loss of catalytic activity (E519Q) and, similar to the corresponding mutant in Na(+)-specific hCNT1, uncoupled Na(+) currents (E343Q). Consistent with close-proximity integration of cation/solute-binding sites within a common cation/permeant translocation pore, mutation of Glu-343 and Glu-519 also altered hCNT3 nucleoside transport selectivity. Both residues were accessible to the external medium and inhibited by p-chloromercuribenzene sulfonate when converted to cysteine.

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.

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.

Conserved glutamate residues are critically involved in Na+/nucleoside cotransport by human concentrative nucleoside transporter 1 (hCNT1).

Human concentrative nucleoside transporter 1 (hCNT1), the first discovered of three human members of the SLC28 (CNT) protein family, is a Na+/nucleoside cotransporter with 650 amino acids. The potential functional roles of 10 conserved aspartate and glutamate residues in hCNT1 were investigated by site-directed mutagenesis and heterologous expression in Xenopus oocytes. Initially, each of the 10 residues was replaced by the corresponding neutral amino acid (asparagine or glutamine). Five of the resulting mutants showed unchanged Na+-dependent uridine transport activity (D172N, E338Q, E389Q, E413Q, and D565N) and were not investigated further. Three were retained in intracellular membranes (D482N, E498Q, and E532Q) and thus could not be assessed functionally. The remaining two (E308Q and E322Q) were present in normal quantities at cell surfaces but exhibited low intrinsic transport activities. Charge replacement with the alternate acidic amino acid enabled correct processing of D482E and E498D, but not of E532D, to cell surfaces and also yielded partially functional E308D and E322D. Relative to wild-type hCNT1, only D482E exhibited normal transport kinetics, whereas E308D, E308Q, E322D, E322Q, and E498D displayed increased K50(Na+) and/or Km(uridine) values and diminished Vmax(Na+) and Vmax(uridine) values. E322Q additionally exhibited uridine-gated uncoupled Na+ transport. Together, these findings demonstrate roles for Glu-308, Glu-322, and Glu-498 in Na+/nucleoside cotransport and suggest locations within a common cation/nucleoside translocation pore. Glu-322, the residue having the greatest influence on hCNT1 transport function, exhibited uridine-protected inhibition by p-chloromercuriphenyl sulfonate and 2-aminoethyl methanethiosulfonate when converted to cysteine.

Cation coupling properties of human concentrative nucleoside transporters hCNT1, hCNT2 and hCNT3.

The SLC28 family of concentrative nucleoside transporter (CNT) proteins in mammalian cells contains members of two distinct phylogenic subfamilies. In humans, hCNT1 and hCNT2 belong to one subfamily, and hCNT3 to the other. All three CNTs mediate inwardly-directed Na(+)/nucleoside cotransport, and are either pyrimidine nucleoside-selective (hCNT1), purine nucleoside-selective (hCNT2), or broadly selective for both pyrimidine and purine nucleosides (hCNT3). While previous studies have characterized cation interactions with both hCNT1 and hCNT3, little is known about the corresponding properties of hCNT2. In the present study, heterologous expression in Xenopus oocytes in combination with radioisotope flux and electrophysiological techniques has allowed us to undertake a side-by-side comparison of hCNT2 with other hCNT family members. Apparent K (50) values for Na(+) activation were voltage-dependent, and similar in magnitude for all three transporters. Only hCNT3 was also able to couple transport of uridine to uptake of H(+). The Na(+)/nucleoside stoichiometry of hCNT2, as determined from both Hill coefficients and direct charge/flux measurements, was 1:1. This result was the same as for hCNT1, but different from that of hCNT3 (2:1). The charge-to-(22)Na(+) uptake stoichiometry was 1:1 for all three hCNTs. In parallel with their division into two separate CNT subfamilies, hCNT2 shares common cation specificity and coupling characteristics with hCNT1, which differ markedly from those of hCNT3.

Specific mutations in transmembrane helix 8 of human concentrative Na+/nucleoside cotransporter hCNT1 affect permeant selectivity and cation coupling.

The Na+/nucleoside cotransporters hCNT1 (650 residues) and hCNT2 (658 residues) are 72% identical in amino acid sequence and contain 13 putative transmembrane helices (TMs). Both transport uridine and adenosine but are otherwise selective for pyrimidine (system cit) and purine (system cif) nucleosides, respectively. Previously, we used site-directed mutagenesis and functional expression in Xenopus oocytes to identify two pairs of adjacent residues in TMs 7 and 8 of hCNT1 (Ser319-Gln320 and Ser353-Leu354) that, when converted to the corresponding residues in hCNT2 (Gly-Met and Thr-Val, respectively), changed the permeant selectivity of the transporter from cit to cif. We now report an investigation of the effects of corresponding mutations in TM 8 alone and demonstrate unique S353T- and L354V-induced changes in nucleoside specificity and cation coupling, respectively. hCNT1 mutation S353T produced a profound decrease in cytidine transport efficiency (Vmax/Km ratio) and, in combination with L354V (S353T/L354V), resulted in a novel uridine-preferring transport phenotype. In addition, the L354V mutation markedly increased the apparent affinity of hCNT1 for Na+ and Li+. Both hCNT1 TM 8 residues exhibited uridine-protectable inhibition by p-chloromercuribenzene sulfonate when converted to Cys, suggesting that they occupy positions within or closely adjacent to a common cation/nucleoside translocation pore.

Characterization of the transport mechanism and permeant binding profile of the uridine permease Fui1p of Saccharomyces cerevisiae.

The uptake of Urd into the yeast Saccharomyces cerevisiae is mediated by Fui1p, a Urd-specific nucleoside transporter encoded by the FUI1 gene and a member of the yeast Fur permease family, which also includes the uracil, allantoin, and thiamine permeases. When Fui1p was produced in a double-permease knock-out strain (fur4Deltafui1Delta) of yeast, Urd uptake was stimulated at acidic pH and sensitive to the protonophore carbonyl cyanide m-chlorophenylhydrazone. Electrophysiological analysis of recombinant Fui1p produced in Xenopus oocytes demonstrated that Fui1p-mediated Urd uptake was dependent on proton cotransport with a 1:1 stoichiometry. Mutagenesis analysis of three charged amino acids (Glu(259), Lys(288), and Asp(474) in putative transmembrane segments 3, 4, and 7, respectively) revealed that only Lys(288) was required for maintaining high Urd transport efficiency. Analysis of binding energies between Fui1p and different Urd analogs indicated that Fuip1 interacted with C(3')-OH, C(2')-OH, C(5)-H, and N(3)-H of Urd. Fui1p-mediated transport of Urd was inhibited by analogs with modifications at C-5', but was not inhibited significantly by analogs with modifications at C-3', C-5, and N-3 or inversions of configuration at C-2' and C-3'. This characterization of Fui1p contributes to the emerging knowledge of the structure and function of the Fur family of permeases, including the Fui1p orthologs of pathogenic fungi.

The broadly selective human Na+/nucleoside cotransporter (hCNT3) exhibits novel cation-coupled nucleoside transport characteristics.

The concentrative nucleoside transporter (CNT) protein family in humans is represented by three members, hCNT1, hCNT2, and hCNT3. hCNT3, a Na+/nucleoside symporter, transports a broad range of physiological purine and pyrimidine nucleosides as well as anticancer and antiviral nucleoside drugs, and belongs to a different CNT subfamily than hCNT1/2. H+-dependent Escherichia coli NupC and Candida albicans CaCNT are also CNT family members. The present study utilized heterologous expression in Xenopus oocytes to investigate the specificity, mechanism, energetics, and structural basis of hCNT3 cation coupling. hCNT3 exhibited uniquely broad cation interactions with Na+, H+, and Li+ not shared by Na+-coupled hCNT1/2 or H+-coupled NupC/CaCNT. Na+ and H+ activated hCNT3 through mechanisms to increase nucleoside apparent binding affinity. Direct and indirect methods demonstrated cation/nucleoside coupling stoichiometries of 2:1 in the presence of Na+ and both Na+ plus H+, but only 1:1 in the presence of H+ alone, suggesting that hCNT3 possesses two Na+-binding sites, only one of which is shared by H+. The H+-coupled hCNT3 did not transport guanosine or 3'-azido-3'-deoxythymidine and 2',3'-dideoxycytidine, demonstrating that Na+- and H+-bound versions of hCNT3 have significantly different conformations of the nucleoside binding pocket and/or translocation channel. Chimeric studies between hCNT1 and hCNT3 located hCNT3-specific cation interactions to the C-terminal half of hCNT3, setting the stage for site-directed mutagenesis experiments to identify the residues involved.

Allelic isoforms of the H+/nucleoside co-transporter (CaCNT) from Candida albicans reveal separate high- and low-affinity transport systems for nucleosides.

Contigs 19-10196 and 19-20196 of the Stanford Candida albicans genome sequence databank encode two putative allelic isoforms of C. albicans CaCNT, a recently characterized 608 amino acid residue H+-coupled fungal member of the CNT family of concentrative nucleoside transport proteins. The single Ser/Gly difference between CaCNT/19-20196 and CaCNT occurs at position 328 in putative TM 7, and corresponds to a Ser/Gly substitution previously shown to contribute to the contrasting pyrimidine and purine nucleoside selectivities of human (h) and rat (r) Na+-dependent CNT1 and CNT2. CaCNT/19-10196 differs from CaCNT by four amino acids, but has Gly at position 328. These new proteins were recreated by site-directed mutagenesis of CaCNT and characterized functionally by heterologous expression in Xenopus laevis oocytes. In marked contrast to h/rCNT1/2, both CaCNT/19-10196 and CaCNT/19-20196 exhibited permeant selectivities for purine nucleosides (adenosine, guanosine and inosine) and uridine similar to that of CaCNT. However, although H+-coupled, CaCNT/19-20196 exhibited a approximately 10-fold higher apparent Km for uridine than either CaCNT or CaCNT/19-10196. CaCNT/19-20196 also exhibited a low apparent affinity for inosine. We conclude that the three proteins correspond to high-affinity (CaCNT, CaCNT/19-10196) and low-affinity (CaCNT/19-20196) allelic isoforms of the C. albicans CNT nucleoside transporter. This is the first example of a single amino acid residue substitution altering a CNT protein's overall apparent affinity for nucleosides.

Transport of physiological nucleosides and anti-viral and anti-neoplastic nucleoside drugs by recombinant Escherichia coli nucleoside-H(+) cotransporter (NupC) produced in Xenopus laevis oocytes.

The recently identified human and rodent plasma membrane proteins CNT1, CNT2 and CNT3 belong to a gene family (CNT) that also includes the bacterial nucleoside transport protein NupC. Heterologous expression in Xenopus oocytes has established that CNT1-3 correspond functionally to the three major concentrative nucleoside transport processes found in human and other mammalian cells (systems cit, cif and cib, respectively) and mediate Na(+) - linked uptake of both physiological nucleosides and anti-viral and anti-neoplastic nucleoside drugs. Here, one describes a complementary Xenopus oocyte transport study of Escherichia coli NupC using the plasmid vector pGEM-HE in which the coding region of NupC was flanked by 5'- and 3'-untranslated sequences from a Xenopus beta-globin gene. Recombinant NupC resembled human (h) and rat (r) CNT1 in nucleoside selectivity, including an ability to transport adenosine and the chemotherapeutic drugs 3'-azido-3'-deoxythymidine (AZT), 2',3'- dideoxycytidine (ddC) and 2'-deoxy-2',2'-difluorocytidine (gemcitabine), but also interacted with inosine and 2',3'- dideoxyinosine (ddl). Apparent affinities were higher than for hCNT1, with apparent K(m) values of 1.5-6.3 microM for adenosine, uridine and gemcitabine, and 112 and 130 microM, respectively, for AZT and ddC. Unlike the relatively low translocation capacity of hCNT1 and rCNT1 for adenosine, NupC exhibited broadly similar apparent V(max) values for adenosine, uridine and nucleoside drugs. NupC did not require Na(+) for activity and was H(+) - dependent. The kinetics of uridine transport measured as a function of external pH were consistent with an ordered transport model in which H(+) binds to the transporter first followed by the nucleoside. These experiments establish the NupC-pGEM-HE/oocyte system as a useful tool for characterization of NupC-mediated transport of physiological nucleosides and clinically relevant nucleoside therapeutic drugs.