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The involvement of the clathrin-mediated endocytic internalization route in the uptake of cholera toxin (CT) was investigated using different cell lines, including the human intestinal Caco-2 and T84 cell lines, green monkey Vero cells, SH-SY5Y neuroblastoma cells and Madin-Darby canine kidney cells. Suppression of the clathrin-mediated endocytic pathway by classical biochemical procedures, like intracellular acidification and potassium depletion, inhibited cholera toxin up - take by up to about 50% as well as its ability to raise intracellular levels of cAMP. Also prior exposure of these cell types to the cationic amphiphilic drug chlorpromazine reduced the func - tional uptake of cholera toxin, even to a greater extent. These effects were dose- and cell type- dependent, suggesting an involvement of clathrin-mediated endocytosis in the functional uptake of cholera toxin. For a more straightforward approach to study the role of the clathrin-mediated uptake in the internalization of cholera toxin, a Caco-2 eps- cell line was exploited. These Caco- 2eps- cells constitutively suppress the expression of epsin, an essential accessory protein of clath - rin-mediated endocytosis, thereby selectively blocking this internalization route. CT uptake was found to be reduced by over 60% in Caco-2 eps- paralleled by a diminished ability of CT to raise the level of cAMP. The data presented suggest that the clathrin-mediated uptake route fulfils an important role in the functional internalization of cholera toxin in several cell types.

Distinct role of clathrin-mediated endocytosis in the functional uptake of cholera toxin.