Abstract
Background/Aims: The permeability of the plasma membrane for some substances increases during osmotic swelling of the cell. Transport systems for cationic dyes were identified previously, but the hypotonic uptake of anionic substances remained elusive. We aimed to test for the hypotonic uptake of the anionic dye fluorescein and to identify the transport pathway. Methods: We quantified the uptake of fluorescein in hypoosmotic medium by fluorescence microscopy in several cell lines. To investigate the uptake mechanism, we used pharmacological inhibitors for the volume-regulated anion channel VRAC and a genome-edited cell line lacking the essential LRRC8A VRAC subunit. Results: Fluorescein was specifically taken up under hypotonic conditions in HeLa, MDCK, and 3T3 cells. Both pharmacological inhibition and genetic disruption of VRAC strongly diminished hypotonicity-driven dye uptake. Conclusion: Cellular uptake of fluorescein is mediated by the volume-regulated anion channel VRAC. The measurement of fluorescein accumulation can be used as a convenient method to detect and study VRAC activation.
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