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.
Highlights
Most animal cells are able to regulate their volume in anisotonic media [1,2]
In agreement with previous studies on numerous mammalian cell lines [23,25], hypotonically swollen HEK293 cells are capable of regulatory volume decrease (RVD) in strongly hypotonic sucrose-substituted solutions (Fig. 1)
In the following experiments we address the solute carrier protein SLC5A3 as a possible candidate for a swelling-activated transporter of myo-inositol in cells exposed to severe hypotonic conditions
Summary
Most animal cells are able to regulate their volume in anisotonic media [1,2]. Efficient volume regulatory mechanisms are essential for cell survival because they protect cells against excessive osmotic shrinkage or swelling [3]. Shrunken cells undergo regulatory volume increase (RVI) via the uptake of extracellular electrolytes (e.g. NaCl) and osmotic water influx, whereas hypotonically swollen cells release KCl and shrink via regulatory volume decrease (RVD). A variety of channels and transporters are involved in the uptake or release of electrolytes (K+, Na+, Cl-, etc.) during cell volume regulation [4,5,6,7,8]. A key role of these molecules in cell volume regulation is supported by the fact that various cell types possess considerable pools of SOOs [12]. Myo-inositol, as one of the most abundant SOOs, is synthesized by cells or accumulated from extracellular medium by the sodium-dependent cotransporter SMIT [1,5,14]. SMIT belongs to the solute carrier (SLC) superfamily and it is referred hereafter as SLC5A3, according to the established nomenclature [15]
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