Human Na+-dependent vitamin C transporter 1 (hSVCT1): primary structure, functional characteristics and evidence for a non-functional splice variant

Abstract

We report here on the cloning and functional characterization of human Na(+)-dependent vitamin C transporter 1 (SVCT1). The human SVCT1 cDNA, obtained from a Caco2 cell cDNA library, encodes a protein of 598 amino acids with 12 putative transmembrane domains. The SVCT1-specific transcript, 2.4 kb in size, is expressed in kidney, liver, small intestine, thymus and prostate. When expressed heterologously in HRPE cells, SVCT1 mediates the transport of ascorbate, the reduced form of vitamin C, in a Na(+)-dependent manner. The transporter is specific for ascorbate with a K(t) of approximately 75 microM. The relationship between the cDNA-specific uptake rate of ascorbate and Na(+) concentration is sigmoidal with a Na(+):ascorbate stoichiometry of 2:1, indicating that the transport process is electrogenic. In Caco2 cells and in normal human intestine, SVCT1 also exists as a non-functional splice variant with a four amino acid sequence inserted between E-155 and V-156. The splice variant results from the use of a donor site 12 bp downstream of the normal donor site.

Typical and atypical stem cells in the brain, vitamin C effect and neuropathology

“…Scale bars: 20μm. Faaland et al, 1998;Tsukaguchi et al, 1999;Wang et al, 1999). Each transporter has a distinct distribution.…”

Morphology and DNA degeneration during autoschizic cell death in bladder carcinoma T24 cells induced by ascorbate and menadione treatment

“…Once ingested, ascorbate circulates and is eventually oxidized in the extracellular milieu to dehydroxyascorbic acid. The specificity of the antitumor activity of VC is related to its ability to be bioconcentrated through facilitated uptake and its intracellular transport by the hS-VCT1 transporter (Daruwala et al, 1999;Wang et al, 1999Wang et al, , 2000Liang et al, 2001) or via glucose transporter isoform GLUT1, GLUT3, and GLUT12 channels (Agus et al, 1999;Chandler et al, 2003). Once dehydroxyascorbic acid is in the cell, it is again reduced and retained as ascorbic acid that is not transportable through the bidirectional channels (Guaiquil et al, 1997;Agus et al, 1999).…”

Primary Structure, Genomic Organization, and Functional and Electrogenic Characteristics of Human System N 1, a Na+- and H+-coupled Glutamine Transporter

“…[ 3 H]Asparagine (specific radioactivity, 650 mCi/mmol) was purchased from Moravek (Brea, CA). Human retinal pigment epithelial (HRPE) cells were originally provided by Dr. M. A. Del Monte (University of Michigan, Ann Arbor, MI) and have been in use in our laboratory for several years for heterologous expression of a variety of cloned transporters (22)(23)(24). The human liver cell line HepG2 was obtained from the American Tissue Culture Collection (Manassas, VA).…”

“…Hybridization was carried out for 20 h at 60°C in a solution containing 5 ϫ SSPE (1 ϫ SSPE ϭ 0.15 M NaC1, 10 mM NaH 2 PO 4 , and 1 mM EDTA), 5 ϫ Denhardt's solution, 0.5% SDS, and 100 g/ml denatured salmon sperm DNA. Posthybridization washing was done as described earlier (22)(23)(24), which involved extensive washes with 3 ϫ SSPE, 0.5% SDS at room temperature. Positive clones were identified, and the colonies were purified by secondary screening.…”

“…Functional Expression in HRPE Cells-This was done using the vaccinia virus expression system as described before (22)(23)(24). Subconfluent HRPE cells grown on 24-well plates were first infected with a recombinant vaccinia virus (VTF 7-3 ) encoding T7 RNA polymerase and then transfected with the plasmid carrying the full-length cDNA.…”