Transmembrane domain 5 of the LdNT1.1 nucleoside transporter is an amphipathic helix that forms part of the nucleoside translocation pathway

Raquel Valdés, Gayatri Vasudevan, David Conklin, Scott Landfear

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

Transporters of the equilibrative nucleoside transporter (ENT) family promote the uptake of nucleosides, nucleobases, and a variety of therapeutic drugs in eukaryotes from protozoa to mammals. Despite its importance, the translocation pathway that mediates the internalization of these substrates has not been identified yet in any of the ENT carriers. Previous genetic studies on the LdNT1.1 nucleoside transporter from Leishmania donovani defined two amino acid residues in predicted transmembrane domains (TMD) 5 and 7 that may line this translocation pathway. The role of TMD5 in forming a portion of the aqueous channel was investigated using the substituted-cysteine accessibility method. A series of 22 cysteine substitution mutants spanning predicted TMD5 were created from a fully functional, cysteine-less, parental LdNT1.1. Cysteine replacement at six positions (M176C, T186C, S 187C, Q190C, V193C, and K194C) produced permeases that were inhibited by incubation with sulfhydryl-specific methanethiosulfonate reagents, denoting their solvent accessibility to the translocation pathway. Adenosine was able to block this thiol modification, implying that access to the domain becomes restricted as a consequence of the substrate binding. Strikingly, the Q190C substitution interacted differentially with the substrates adenosine and uridine, suggesting that binding of adenosine but not uridine might directly occlude this position. When superimposed on a helical model, all six mutants clustered along one face of the amphipathic α-helix predicted for TMD5, strongly suggesting its involvement in the translocation pathway through LdNT1.1.

Original languageEnglish (US)
Pages (from-to)6793-6802
Number of pages10
JournalBiochemistry
Volume43
Issue number21
DOIs
StatePublished - Jun 1 2004

Fingerprint

Nucleoside Transport Proteins
Nucleosides
Cysteine
Adenosine
Uridine
Substitution reactions
Substrates
Protozoa
Leishmania donovani
Mammals
Membrane Transport Proteins
Eukaryota
Sulfhydryl Compounds
Amino Acids
Pharmaceutical Preparations

ASJC Scopus subject areas

  • Biochemistry

Cite this

Transmembrane domain 5 of the LdNT1.1 nucleoside transporter is an amphipathic helix that forms part of the nucleoside translocation pathway. / Valdés, Raquel; Vasudevan, Gayatri; Conklin, David; Landfear, Scott.

In: Biochemistry, Vol. 43, No. 21, 01.06.2004, p. 6793-6802.

Research output: Contribution to journalArticle

@article{dea632aa6142480abfb744ff26cecd03,
title = "Transmembrane domain 5 of the LdNT1.1 nucleoside transporter is an amphipathic helix that forms part of the nucleoside translocation pathway",
abstract = "Transporters of the equilibrative nucleoside transporter (ENT) family promote the uptake of nucleosides, nucleobases, and a variety of therapeutic drugs in eukaryotes from protozoa to mammals. Despite its importance, the translocation pathway that mediates the internalization of these substrates has not been identified yet in any of the ENT carriers. Previous genetic studies on the LdNT1.1 nucleoside transporter from Leishmania donovani defined two amino acid residues in predicted transmembrane domains (TMD) 5 and 7 that may line this translocation pathway. The role of TMD5 in forming a portion of the aqueous channel was investigated using the substituted-cysteine accessibility method. A series of 22 cysteine substitution mutants spanning predicted TMD5 were created from a fully functional, cysteine-less, parental LdNT1.1. Cysteine replacement at six positions (M176C, T186C, S 187C, Q190C, V193C, and K194C) produced permeases that were inhibited by incubation with sulfhydryl-specific methanethiosulfonate reagents, denoting their solvent accessibility to the translocation pathway. Adenosine was able to block this thiol modification, implying that access to the domain becomes restricted as a consequence of the substrate binding. Strikingly, the Q190C substitution interacted differentially with the substrates adenosine and uridine, suggesting that binding of adenosine but not uridine might directly occlude this position. When superimposed on a helical model, all six mutants clustered along one face of the amphipathic α-helix predicted for TMD5, strongly suggesting its involvement in the translocation pathway through LdNT1.1.",
author = "Raquel Vald{\'e}s and Gayatri Vasudevan and David Conklin and Scott Landfear",
year = "2004",
month = "6",
day = "1",
doi = "10.1021/bi049873m",
language = "English (US)",
volume = "43",
pages = "6793--6802",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "21",

}

TY - JOUR

T1 - Transmembrane domain 5 of the LdNT1.1 nucleoside transporter is an amphipathic helix that forms part of the nucleoside translocation pathway

AU - Valdés, Raquel

AU - Vasudevan, Gayatri

AU - Conklin, David

AU - Landfear, Scott

PY - 2004/6/1

Y1 - 2004/6/1

N2 - Transporters of the equilibrative nucleoside transporter (ENT) family promote the uptake of nucleosides, nucleobases, and a variety of therapeutic drugs in eukaryotes from protozoa to mammals. Despite its importance, the translocation pathway that mediates the internalization of these substrates has not been identified yet in any of the ENT carriers. Previous genetic studies on the LdNT1.1 nucleoside transporter from Leishmania donovani defined two amino acid residues in predicted transmembrane domains (TMD) 5 and 7 that may line this translocation pathway. The role of TMD5 in forming a portion of the aqueous channel was investigated using the substituted-cysteine accessibility method. A series of 22 cysteine substitution mutants spanning predicted TMD5 were created from a fully functional, cysteine-less, parental LdNT1.1. Cysteine replacement at six positions (M176C, T186C, S 187C, Q190C, V193C, and K194C) produced permeases that were inhibited by incubation with sulfhydryl-specific methanethiosulfonate reagents, denoting their solvent accessibility to the translocation pathway. Adenosine was able to block this thiol modification, implying that access to the domain becomes restricted as a consequence of the substrate binding. Strikingly, the Q190C substitution interacted differentially with the substrates adenosine and uridine, suggesting that binding of adenosine but not uridine might directly occlude this position. When superimposed on a helical model, all six mutants clustered along one face of the amphipathic α-helix predicted for TMD5, strongly suggesting its involvement in the translocation pathway through LdNT1.1.

AB - Transporters of the equilibrative nucleoside transporter (ENT) family promote the uptake of nucleosides, nucleobases, and a variety of therapeutic drugs in eukaryotes from protozoa to mammals. Despite its importance, the translocation pathway that mediates the internalization of these substrates has not been identified yet in any of the ENT carriers. Previous genetic studies on the LdNT1.1 nucleoside transporter from Leishmania donovani defined two amino acid residues in predicted transmembrane domains (TMD) 5 and 7 that may line this translocation pathway. The role of TMD5 in forming a portion of the aqueous channel was investigated using the substituted-cysteine accessibility method. A series of 22 cysteine substitution mutants spanning predicted TMD5 were created from a fully functional, cysteine-less, parental LdNT1.1. Cysteine replacement at six positions (M176C, T186C, S 187C, Q190C, V193C, and K194C) produced permeases that were inhibited by incubation with sulfhydryl-specific methanethiosulfonate reagents, denoting their solvent accessibility to the translocation pathway. Adenosine was able to block this thiol modification, implying that access to the domain becomes restricted as a consequence of the substrate binding. Strikingly, the Q190C substitution interacted differentially with the substrates adenosine and uridine, suggesting that binding of adenosine but not uridine might directly occlude this position. When superimposed on a helical model, all six mutants clustered along one face of the amphipathic α-helix predicted for TMD5, strongly suggesting its involvement in the translocation pathway through LdNT1.1.

UR - http://www.scopus.com/inward/record.url?scp=2542572681&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=2542572681&partnerID=8YFLogxK

U2 - 10.1021/bi049873m

DO - 10.1021/bi049873m

M3 - Article

VL - 43

SP - 6793

EP - 6802

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 21

ER -