Despite its versatility and effectiveness in numerous studies, the vaccinia/HeLa cell expression model may not be optimal for the study of all transport proteins. To evaluate an alternative expression model for amino acid transport Systems ASC and X(AG)/-, the mRNA content and transport activity encoded by human hippocampal ASCT1 cDNA and rat hippocampal EAAC1 cDNA, respectively, were measured in pDR2-cDNA-transfected human embryonic kidney 293 cells made competent by stable transfection with the Epstein-Barr neutral antigen-1 (EBNA-1) cDNA (293c18 cells) to evaluate the EBNA-1/293c18 expression system. The results show that (i) the EBNA-1/293c18 expression system results in a larger increase over background of Systems ASCT1 (6.4X) and EAAC1 (39X) transport activity than does the vaccinia/HeLa expression system (2.6X and 22X, respectively); (ii) transfection and hygromycin B selection for the pDR2 vector do not affect the endogenous transport velocities of Systems ASC, X-(AG), or A; and (iii) the endogenous transport velocities of Systems ASC and X(AG)/- in 293c18 cells were not affected by the expression of exogenous EAAC1 or ASCT1. We conclude that the EBNA- 1/293c18 cell expression model represents a useful transient expression regimen to characterize mammalian amino acid transport proteins, especially for transporters that may exhibit relatively low activity in transient expression systems lacking a selection mechanism.
|Number of pages||7|
|State||Published - Dec 15 1997|
Bibliographical noteFunding Information:
This research was supported by the National Institutes of Health, the National Institute of Child Health and Human Resources (HD-29934 to D.A.N.), the Institute for Diabetes, Digestive, and Kidney Diseases (DK-28374 to M.S.K.), and an Institutional National Research Service Award (DK07667 to J.C.M.).
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology