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Swelling-activated pathways for  myo -inositol, one of the most abundant organic osmolytes in mammalian cells, have not yet been identified. The present study explores the SLC5A3 protein as a possible transporter of  myo -inositol in hyponically swollen HEK293 cells. To address this issue, we examined the relationship between the hypotonicity-induced changes in plasma membrane permeability to  myo -inositol  P   ino  [m/s] and expression/localization of SLC5A3.  P   ino  values were determined by cell volumetry over a wide tonicity range (100–275 mOsm) in  myo -inositol-substituted solutions. While being negligible under mild hypotonicity (200–275 mOsm),  P   ino  grew rapidly at osmolalities below 200 mOsm to reach a maximum of ∼3 nm/s at 100–125 mOsm, as indicated by fast cell swelling due to  myo -inositol influx. The increase in  P   ino  resulted most likely from the hypotonicity-mediated incorporation of cytosolic SLC5A3 into the plasma membrane, as revealed by confocal fluorescence microscopy of cells expressing EGFP-tagged SLC5A3 and super-resolution imaging of immunostained SLC5A3 by  direct  stochastic optical reconstruction microscopy ( d STORM).  d STORM in hypotonic cells revealed a surface density of membrane-associated SLC5A3 proteins of 200–2000 localizations/μm 2 . Assuming SLC5A3 to be the major path for  myo -inositol, a turnover rate of 80–800  myo -inositol molecules per second for a single transporter protein was estimated from combined volumetric and  d STORM data. Hypotonic stress also caused a significant upregulation of SLC5A3 gene expression as detected by semiquantitative RT-PCR and Western blot analysis. In summary, our data provide first evidence for swelling-mediated activation of SLC5A3 thus suggesting a functional role of this transporter in hypotonic volume regulation of mammalian cells.

Hypotonic Activation of the Myo-Inositol Transporter SLC5A3 in HEK293 Cells Probed by Cell Volumetry, Confocal and Super-Resolution Microscopy