A Conformationally Mobile Cysteine Residue (Cys-561) Modulates Na+ and H+ Activation of Human CNT3

Slugoski, Melissa D.; Smith, Kyla M.; Mulinta, Ras; Ng, Amy M. L.; Yao, Sylvia Y. M.; Morrison, Ellen; Lee, Queenie O.T.; Zhang, Jing; Karpinski, Edward; Cass, Carol E.; Baldwin, Stephen A.; Young, James D.

doi:10.1074/jbc.m801793200

Cited by 24 publications

(50 citation statements)

References 41 publications

(80 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Immunoblots were probed with hCNT3 polyclonal antibodies (37) directed against amino acid residues 45-69 of the intramembranous N-terminal region of the protein. Similar to previous findings (38) and consistent with the presence of multiple C-terminal sites of N-linked glycosylation, cell-surface hCNT3 exhibited discrete immunostained bands at 100 and 86 kDa (Fig. 3).…”

Section: Resultssupporting

confidence: 78%

“…Kinetic parameters derived from the curves are summarized alongside those for Na ϩ in Table 3. In good agreement with previous studies, H ϩ -coupled uridine uptake by wild-type hCNT3 occurred with an apparent K 50 value of 495 nM and a corresponding V max value of 9 pmol/oocyte⅐min Ϫ1 (11,19,38). Consistent with Fig.…”

Section: Hcnt3 Glutamate Residuessupporting

confidence: 81%

“…In good agreement with previous studies (11,19,38), Na ϩ activation of uridine uptake by wild-type hCNT3 occurred with an apparent K 50 value of 11 mM and a V max value of 10 pmol/ oocyte⅐min Ϫ1 . In marked contrast, E343Q exhibited very low affinity for Na ϩ , such that the curve appeared linear with concentration, although uridine uptake at 100 mM Na ϩ was marginally greater than that in the absence of Na ϩ (see also Fig.…”

Section: Hcnt3 Glutamate Residuessupporting

confidence: 80%

“…3). Both these immunostained bands correspond to glycosylated forms of the transporter, aglycohCNT3 having a lower molecular mass of 75 kDa (38). Antibody specificity was confirmed by lack of immunoreactivity in membranes from control, water-injected oocytes.…”

Section: Resultsmentioning

confidence: 85%

“…Na ϩ -containing medium at pH 8.5 was used to avoid the small, but significant, amount of hCNT3 H ϩ activation that occurs at pH 7.5 (11,19). Na ϩ -coupled uridine transport by hCNT3 at pH 8.5 is kinetically indistinguishable from that at pH 7.5 (19,38).…”

Section: Hcnt3 Glutamate Residuesmentioning

confidence: 99%

See 4 more Smart Citations

Conserved Glutamate Residues Glu-343 and Glu-519 Provide Mechanistic Insights into Cation/Nucleoside Cotransport by Human Concentrative Nucleoside Transporter hCNT3

Slugoski¹,

Smith²,

Ng³

et al. 2009

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Resultssupporting

confidence: 78%

Section: Hcnt3 Glutamate Residuessupporting

confidence: 81%

Section: Hcnt3 Glutamate Residuessupporting

confidence: 80%

Section: Resultsmentioning

confidence: 85%

Section: Hcnt3 Glutamate Residuesmentioning

confidence: 99%

See 3 more Smart Citations

Conserved Glutamate Residues Glu-343 and Glu-519 Provide Mechanistic Insights into Cation/Nucleoside Cotransport by Human Concentrative Nucleoside Transporter hCNT3

Slugoski¹,

Smith²,

Ng³

et al. 2009

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

Transporters that translocate nucleosides and structural similar drugs: structural requirements for substrate recognition

Cano-Soldado

Pastor‐Anglada

2011

Medicinal Research Reviews

View full text Add to dashboard Cite

Nucleoside transporters (NT) are integral membrane proteins implicated in the salvage of natural nucleobases and nucleosides for nucleic acid synthesis. These proteins also play a crucial role as carriers of nucleoside analogs used in anticancer and antiviral therapies. In fact, differential expression patterns of NT subtypes among tissues and individuals as well as the existence of genetic variants affect nucleoside-derived drug permeation, and consequently, their pharmacokinetic and cytotoxic properties. Thus, NT expression patterns may be effective predictive markers of therapeutic response. While the structures of NT proteins are yet to be solved, specific residues responsible for interaction with substrates and inhibitors have been identified, providing further insights into their structure-function relationship. In addition to transporter structural features, several experimental approaches have been used to identify the structural requirements of nucleosides for interaction with Concentrative Nucleoside Transporters and Equilibrative Nucleoside Transporters (SLC28 and SLC29 gene families, respectively). Pharmacophore models proposed for both protein families may prove suitable for optimizing drug design. Additional transporter proteins, including Organic Anion Transporters, Organic Cation Transporters (members of the SLC22 gene family), and Peptide Transporters (SLC15 gene family), have been implicated in the uptake of nucleoside-derived drugs, particularly those currently used in antiviral therapies. In this review, we focus on the pharmacological profiles of these transporter proteins, summarizing the documented studies covering structure-function and substrate structural requirement properties that determine drug-carrier interaction and efficient substrate translocation across the plasma membrane of target cells.

show abstract

SLC28 and SLC29

Yao¹,

Young²

2017

Encyclopedia of Signaling Molecules

View full text Add to dashboard Cite

A Conformationally Mobile Cysteine Residue (Cys-561) Modulates Na+ and H+ Activation of Human CNT3

Cited by 24 publications

References 41 publications

Conserved Glutamate Residues Glu-343 and Glu-519 Provide Mechanistic Insights into Cation/Nucleoside Cotransport by Human Concentrative Nucleoside Transporter hCNT3

Conserved Glutamate Residues Glu-343 and Glu-519 Provide Mechanistic Insights into Cation/Nucleoside Cotransport by Human Concentrative Nucleoside Transporter hCNT3

Transporters that translocate nucleosides and structural similar drugs: structural requirements for substrate recognition

SLC28 and SLC29

Contact Info

Product

Resources

About