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Updated: May 18, 2026

Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D
Published on: March 16, 2017
Modulation of function of sodium-dependent vitamin C transporter 1 (SVCT1) by Rab8a in intestinal epithelial cells:
Veedamali S Subramanian1, Sandeep B Subramanya, Abhisek Ghosal
1Departments of Medicine, Physiology and Biophysics, University of California, Irvine, CA 92697, USA. vsubrama@uci.edu
Background:
Ascorbic acid (AA) is required for normal human health and development. Human intestine expresses two sodium-dependent vitamin C transporters (hSVCT-1 and -2) that mediate cellular AA transport, with hSVCT1 targeting to the apical membrane of polarized epithelia. Studies have shown a role for the Rab8a in the apical membrane targeting of transporters in intestinal cells.
Aims:
The purpose of this study was to determine whether Rab8a impacts the function and/or targeting of hSVCT1, and intestinal AA uptake.
Methods:
We used human intestinal cells and cells from a Rab8a knockout mouse. (14)C-AA uptake was performed to determine functionality. PCR and western blotting were performed to determine RNA and protein expression, respectively. Confocal imaging was performed to determine co-localization.
Results:
We show that hSVCT1 co-localized with Rab8a in intestinal cells. Knockdown of Rab8a lead to a significant inhibition in AA uptake and cell surface biotinylation studies revealed a lower cell surface expression of hSVCT1 in Rab8a siRNA-treated cells. Similarly, in the small intestine of a Rab8a knockout mouse, AA uptake was significantly inhibited. This effect again resulted from a decreased expression level of mSVCT1 protein, even though mRNA expression of SVCT1 was similar in intestinal cells from Rab8a knockout and wild-type litter-mates. The latter data are suggestive of enhanced lysosomal degradation of hSVCT1 protein in Rab8a-deficient cells; indeed, confocal imaging of Rab8a siRNA-treated intestinal cells revealed a strong overlap between hSVCT1-YFP and LAMP1-RFP.
Conclusions:
These findings show a role for Rab8a in the physiological function of hSVCT1 in intestinal epithelia.
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