title: Hypotonic activation of the myo-inositol transporter SLC5A3 in HEK293 cells probed by cell volumetry, confocal and super-resolution microscopy.
abstract: 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 (dSTORM). dSTORM in hypotonic cells revealed a surface density of membrane-associated SLC5A3 proteins of 200-2000 localizations/μm2. 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 dSTORM 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.
authors.affiliation: Department of Biotechnology and Biophysics, University of Würzburg, Biozentrum, Am Hubland, Würzburg, Germany.
authors.forename: Joseph
authors.initials: J
authors.lastname: Andronic
authors.affiliation: Institute of Industrial Science, The University of Tokyo, Tokyo, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Shirakashi
authors.affiliation: Department of Biotechnology and Biophysics, University of Würzburg, Biozentrum, Am Hubland, Würzburg, Germany.
authors.forename: Simone U
authors.initials: SU
authors.lastname: Pickel
authors.affiliation: Department of Biotechnology and Biophysics, University of Würzburg, Biozentrum, Am Hubland, Würzburg, Germany.
authors.forename: Katherine M
authors.initials: KM
authors.lastname: Westerling
authors.affiliation: Department of Biotechnology and Biophysics, University of Würzburg, Biozentrum, Am Hubland, Würzburg, Germany.
authors.forename: Teresa
authors.initials: T
authors.lastname: Klein
authors.affiliation: Department of Biotechnology and Biophysics, University of Würzburg, Biozentrum, Am Hubland, Würzburg, Germany.
authors.forename: Thorge
authors.initials: T
authors.lastname: Holm
authors.affiliation: Department of Biotechnology and Biophysics, University of Würzburg, Biozentrum, Am Hubland, Würzburg, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Sauer
authors.affiliation: Department of Biotechnology and Biophysics, University of Würzburg, Biozentrum, Am Hubland, Würzburg, Germany.
authors.forename: Vladimir L
authors.initials: VL
authors.lastname: Sukhorukov
mesh_terms: D001692:Biological Transport; D048429:Cell Size; D015870:Gene Expression; D057809:HEK293 Cells; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007294:Inositol / Q000378:metabolism; D018613:Microscopy, Confocal; D009997:Osmotic Pressure; D021381:Protein Transport; D027981:Symporters / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D015854:Up-Regulation
chemical_list: D006360:Heat-Shock Proteins; D027981:Symporters; C073884:SLC5A3 protein, human; D007294:Inositol