Development of a Novel HEK293 Cell Model Lacking SLC29A1 to Study the Pharmacology of Endogenous SLC29A2 -Encoded Equilibrative Nucleoside Transporter Subtype 2.

Autor: Shahid N; Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada., Cromwell C; Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada., Hubbard BP; Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada., Hammond JR; Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada james.hammond@ualberta.ca.
Jazyk: angličtina
Zdroj: Drug metabolism and disposition: the biological fate of chemicals [Drug Metab Dispos] 2024 Sep 16; Vol. 52 (10), pp. 1094-1103. Date of Electronic Publication: 2024 Sep 16.
DOI: 10.1124/dmd.124.001814
Abstrakt: Equilibrative nucleoside transporters (ENTs) mediate the transmembrane flux of endogenous nucleosides and nucleoside analogs used clinically. The predominant subtype, ENT1, has been well characterized. However, the other subtype, ENT2, has been less well characterized in its native milieu due to its relatively low expression and the confounding influence of coexpressed ENT1. We created a cell model where ENT1 was removed from human embryonic kidney (HEK293) cells using CRISPR/cas9 [ENT1 knockout (KO) cells]; this cell line has ENT2 as the only functional purine transporter. Transporter function was assessed through measurement of [ 3 H]2-chloroadenosine uptake. ENT1 protein was quantified based on the binding of [ 3 H]nitrobenzylthioinosine, and ENT1/ENT2 protein was detected by immunoblotting. Changes in expression of relevant transporters and enzymes involved in purine metabolism were examined by quantitative polymerase chain reaction. Wild-type HEK293 cells and ENT1KO cells had a similar expression of SLC29A2 /ENT2 transcript/protein and ENT2-mediated [ 3 H]2-chloroadenosine transport activity (V max values of 1.02 ± 0.06 and 1.50 ± 0.22 pmol/ μ l/s, respectively). Of the endogenous nucleosides/nucleobases tested, adenosine had the highest affinity (K i ) for ENT2 (2.6 μ M), while hypoxanthine was the only nucleobase with a submillimolar affinity (320 μ M). A range of nucleoside/nucleobase analogs were also tested for their affinity for ENT2 in this model, with affinities (K i ) ranging from 8.6 μ M for ticagrelor to 2,300 μ M for 6-mercaptopurine. Our data suggest that the removal of endogenous ENT1 from these cells does not change the expression or function of ENT2. This cell line should prove useful for the analysis of novel drugs acting via ENT2 and to study ENT2 regulation. SIGNIFICANCE STATEMENT: We have created a cell line whereby endogenous ENT2 can be studied in detail in the absence of the confounding influence of ENT1. Loss of ENT1 has no impact on the expression and function of ENT2. This novel cell line will provide an ideal model for studying drug interactions with ENT2 as well as the cellular regulation of ENT2 expression and function.
(Copyright © 2024 by The Author(s).)
Databáze: MEDLINE
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