Transport of purines and purine salvage pathway inhibitors by the Plasmodium falciparum equilibrative nucleoside transporter PfENT1

Paul M. Riegelhaupt, María B. Cassera, Richard F G Fröhlich, Keith Z. Hazleton, Jonathan J. Hefter, Vern L. Schramm, Myles Akabas

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

Plasmodium falciparum is a purine auxotroph. The transport of purine nucleosides and nucleobases from the host erythrocyte to the parasite cytoplasm is essential to support parasite growth. P. falciparum equilibrative nucleoside transporter 1 (PfENT1) is a major route for purine transport across the parasite plasma membrane. Malarial parasites are sensitive to inhibitors of purine salvage pathway enzymes. The immucillin class of purine nucleoside phosphorylase inhibitors and the adenosine analog, tubercidin, block growth of P. falciparum under in vitro culture conditions. We sought to determine whether these inhibitors utilize PfENT1 to gain access to the parasite cytosol. There is considerable controversy in the literature regarding the Km and/or Ki for purine transport by PfENT1 in the Xenopus oocyte expression system. We show that oocytes metabolize adenosine but not hypoxanthine. For adenosine, metabolism is the rate limiting step in oocyte uptake assays, making hypoxanthine the preferred substrate for PfENT1 transport studies in oocytes. We demonstrate that the Ki for PfENT1 transport of hypoxanthine and adenosine is in the 300-700 μM range. Effects of substrate metabolism on uptake studies may explain conflicting results in the literature regarding the PfENT1 adenosine transport Km. PfENT1 transports the tubercidin class of compounds. None of the immucillin compounds tested inhibited PfENT1 transport of [3H]hypoxanthine or [3H]adenosine. Although nucleobases are transported, modifications of the ribose ring in corresponding nucleoside analogs affect substrate recognition by PfENT1. These results provide new insights into PfENT1 and the mechanism by which purine salvage pathway inhibitors are transported into the parasite cytoplasm.

Original languageEnglish (US)
Pages (from-to)40-49
Number of pages10
JournalMolecular and Biochemical Parasitology
Volume169
Issue number1
DOIs
StatePublished - Jan 2010

Fingerprint

Equilibrative Nucleoside Transporter 1
Nucleoside Transport Proteins
Purines
Plasmodium falciparum
Adenosine
Parasites
Hypoxanthine
Oocytes
Tubercidin
purine
Cytoplasm
Purine-Nucleoside Phosphorylase
Purine Nucleosides
Ribose
Growth
Xenopus

Keywords

  • Immucillin
  • Malaria
  • Nucleoside
  • Purine
  • Transport
  • Tubercidin

ASJC Scopus subject areas

  • Molecular Biology
  • Parasitology

Cite this

Transport of purines and purine salvage pathway inhibitors by the Plasmodium falciparum equilibrative nucleoside transporter PfENT1. / Riegelhaupt, Paul M.; Cassera, María B.; Fröhlich, Richard F G; Hazleton, Keith Z.; Hefter, Jonathan J.; Schramm, Vern L.; Akabas, Myles.

In: Molecular and Biochemical Parasitology, Vol. 169, No. 1, 01.2010, p. 40-49.

Research output: Contribution to journalArticle

Riegelhaupt, Paul M. ; Cassera, María B. ; Fröhlich, Richard F G ; Hazleton, Keith Z. ; Hefter, Jonathan J. ; Schramm, Vern L. ; Akabas, Myles. / Transport of purines and purine salvage pathway inhibitors by the Plasmodium falciparum equilibrative nucleoside transporter PfENT1. In: Molecular and Biochemical Parasitology. 2010 ; Vol. 169, No. 1. pp. 40-49.
@article{2b5ac959d733452f819e4b404a9197e9,
title = "Transport of purines and purine salvage pathway inhibitors by the Plasmodium falciparum equilibrative nucleoside transporter PfENT1",
abstract = "Plasmodium falciparum is a purine auxotroph. The transport of purine nucleosides and nucleobases from the host erythrocyte to the parasite cytoplasm is essential to support parasite growth. P. falciparum equilibrative nucleoside transporter 1 (PfENT1) is a major route for purine transport across the parasite plasma membrane. Malarial parasites are sensitive to inhibitors of purine salvage pathway enzymes. The immucillin class of purine nucleoside phosphorylase inhibitors and the adenosine analog, tubercidin, block growth of P. falciparum under in vitro culture conditions. We sought to determine whether these inhibitors utilize PfENT1 to gain access to the parasite cytosol. There is considerable controversy in the literature regarding the Km and/or Ki for purine transport by PfENT1 in the Xenopus oocyte expression system. We show that oocytes metabolize adenosine but not hypoxanthine. For adenosine, metabolism is the rate limiting step in oocyte uptake assays, making hypoxanthine the preferred substrate for PfENT1 transport studies in oocytes. We demonstrate that the Ki for PfENT1 transport of hypoxanthine and adenosine is in the 300-700 μM range. Effects of substrate metabolism on uptake studies may explain conflicting results in the literature regarding the PfENT1 adenosine transport Km. PfENT1 transports the tubercidin class of compounds. None of the immucillin compounds tested inhibited PfENT1 transport of [3H]hypoxanthine or [3H]adenosine. Although nucleobases are transported, modifications of the ribose ring in corresponding nucleoside analogs affect substrate recognition by PfENT1. These results provide new insights into PfENT1 and the mechanism by which purine salvage pathway inhibitors are transported into the parasite cytoplasm.",
keywords = "Immucillin, Malaria, Nucleoside, Purine, Transport, Tubercidin",
author = "Riegelhaupt, {Paul M.} and Cassera, {Mar{\'i}a B.} and Fr{\"o}hlich, {Richard F G} and Hazleton, {Keith Z.} and Hefter, {Jonathan J.} and Schramm, {Vern L.} and Myles Akabas",
year = "2010",
month = "1",
doi = "10.1016/j.molbiopara.2009.10.001",
language = "English (US)",
volume = "169",
pages = "40--49",
journal = "Molecular and Biochemical Parasitology",
issn = "0166-6851",
publisher = "Elsevier",
number = "1",

}

TY - JOUR

T1 - Transport of purines and purine salvage pathway inhibitors by the Plasmodium falciparum equilibrative nucleoside transporter PfENT1

AU - Riegelhaupt, Paul M.

AU - Cassera, María B.

AU - Fröhlich, Richard F G

AU - Hazleton, Keith Z.

AU - Hefter, Jonathan J.

AU - Schramm, Vern L.

AU - Akabas, Myles

PY - 2010/1

Y1 - 2010/1

N2 - Plasmodium falciparum is a purine auxotroph. The transport of purine nucleosides and nucleobases from the host erythrocyte to the parasite cytoplasm is essential to support parasite growth. P. falciparum equilibrative nucleoside transporter 1 (PfENT1) is a major route for purine transport across the parasite plasma membrane. Malarial parasites are sensitive to inhibitors of purine salvage pathway enzymes. The immucillin class of purine nucleoside phosphorylase inhibitors and the adenosine analog, tubercidin, block growth of P. falciparum under in vitro culture conditions. We sought to determine whether these inhibitors utilize PfENT1 to gain access to the parasite cytosol. There is considerable controversy in the literature regarding the Km and/or Ki for purine transport by PfENT1 in the Xenopus oocyte expression system. We show that oocytes metabolize adenosine but not hypoxanthine. For adenosine, metabolism is the rate limiting step in oocyte uptake assays, making hypoxanthine the preferred substrate for PfENT1 transport studies in oocytes. We demonstrate that the Ki for PfENT1 transport of hypoxanthine and adenosine is in the 300-700 μM range. Effects of substrate metabolism on uptake studies may explain conflicting results in the literature regarding the PfENT1 adenosine transport Km. PfENT1 transports the tubercidin class of compounds. None of the immucillin compounds tested inhibited PfENT1 transport of [3H]hypoxanthine or [3H]adenosine. Although nucleobases are transported, modifications of the ribose ring in corresponding nucleoside analogs affect substrate recognition by PfENT1. These results provide new insights into PfENT1 and the mechanism by which purine salvage pathway inhibitors are transported into the parasite cytoplasm.

AB - Plasmodium falciparum is a purine auxotroph. The transport of purine nucleosides and nucleobases from the host erythrocyte to the parasite cytoplasm is essential to support parasite growth. P. falciparum equilibrative nucleoside transporter 1 (PfENT1) is a major route for purine transport across the parasite plasma membrane. Malarial parasites are sensitive to inhibitors of purine salvage pathway enzymes. The immucillin class of purine nucleoside phosphorylase inhibitors and the adenosine analog, tubercidin, block growth of P. falciparum under in vitro culture conditions. We sought to determine whether these inhibitors utilize PfENT1 to gain access to the parasite cytosol. There is considerable controversy in the literature regarding the Km and/or Ki for purine transport by PfENT1 in the Xenopus oocyte expression system. We show that oocytes metabolize adenosine but not hypoxanthine. For adenosine, metabolism is the rate limiting step in oocyte uptake assays, making hypoxanthine the preferred substrate for PfENT1 transport studies in oocytes. We demonstrate that the Ki for PfENT1 transport of hypoxanthine and adenosine is in the 300-700 μM range. Effects of substrate metabolism on uptake studies may explain conflicting results in the literature regarding the PfENT1 adenosine transport Km. PfENT1 transports the tubercidin class of compounds. None of the immucillin compounds tested inhibited PfENT1 transport of [3H]hypoxanthine or [3H]adenosine. Although nucleobases are transported, modifications of the ribose ring in corresponding nucleoside analogs affect substrate recognition by PfENT1. These results provide new insights into PfENT1 and the mechanism by which purine salvage pathway inhibitors are transported into the parasite cytoplasm.

KW - Immucillin

KW - Malaria

KW - Nucleoside

KW - Purine

KW - Transport

KW - Tubercidin

UR - http://www.scopus.com/inward/record.url?scp=71549117071&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=71549117071&partnerID=8YFLogxK

U2 - 10.1016/j.molbiopara.2009.10.001

DO - 10.1016/j.molbiopara.2009.10.001

M3 - Article

VL - 169

SP - 40

EP - 49

JO - Molecular and Biochemical Parasitology

JF - Molecular and Biochemical Parasitology

SN - 0166-6851

IS - 1

ER -