Intracellular Hypertonicity Is Responsible for Water Flux Associated with Na+/Glucose Cotransport

Abstract

Detection of a significant transmembrane water flux immediately after cotransporter stimulation is the experimental basis for the controversial hypothesis of secondary active water transport involving a proposed stoichiometry for the human Na(+)/glucose cotransporter (SGLT1) of two Na(+), one glucose, and 264 water molecules. Volumetric measurements of Xenopus laevis oocytes coexpressing human SGLT1 and aquaporin can be used to detect osmotic gradients with high sensitivity. Adding 2 mM of the substrate alpha-methyl-glucose (alphaMG) created mild extracellular hypertonicity and generated a large cotransport current with minimal cell volume changes. After 20, 40, and 60 s of cotransport, the return to sugar-free, isotonic conditions was accompanied by measurable cell swelling averaging 0.051, 0.061, and 0.077 nl/s, respectively. These water fluxes are consistent with internal hypertonicities of 1.5, 1.7, and 2.2 mOsm for these cotransport periods. In the absence of aquaporin, the measured hypertonicites were 4.6, 5.0, and 5.3 mOsm for the same cotransport periods Cotransport-dependent water fluxes, previously assumed to be water cotransport, could be largely explained by hypertonicities of such amplitudes. Using intracellular Na(+) injection and Na(+)-selective electrode, the intracellular diffusion coefficient for Na(+) was estimated at 0.29 +/- 0.03 x 10(-5) cm(2) s(-1). Using the effect of intracellular alphaMG injection on the SGLT1-mediated outward current, the intracellular diffusion coefficient of alphaMG was estimated at 0.15 +/- 0.01 x 10(-5) cm(2) s(-1). Although these intracellular diffusion coefficients are much lower than in free aqueous solution, a diffusion model for a single solute in an oocyte would require a diffusion coefficient three times lower than estimated to explain the local osmolyte accumulation that was experimentally detected. This suggests that either the diffusion coefficients were overestimated, possibly due to the presence of convection, or the diffusion in cytosol of an oocyte is more complex than depicted by a simple model.

α- and β-Monosaccharide transport in human erythrocytes

“…However, the coefficient for sugar self-diffusion must be reduced by at least 1,000-fold to explain limited intracellular diffusion over distances of 3.5 m or less (62). This seems untenable given estimates of intracellular tortuosity in neurons, brain, and red blood cells of 1-3 (7,25,29) and estimates of intracellular viscosity in red blood cells and Xenopus oocytes of 6 -15 cP (16,28). These increases in tortuosity and viscosity relative to aqueous solution could reduce intracellular glucose diffusion by as much as 20-fold but not by the necessary 1,000-fold.…”

Elaboration of a novel technique for purification of plasma membranes from Xenopus laevis oocytes

“…The variations in volume were used to determine water permeability values (P f) which are given in micrometers per second. For a more detailed description of this setup and procedure, see Duquette et al (9) and Charron et al (4).…”

GABA reverse transport by the neuronal cotransporter GAT1: influence of internal chloride depletion

“…The diffusion coefficients of ions and small organic molecules in the oocyte cytoplasm are reported to be in the order of D ϭ 0.2-0.6 ϫ 10 -5 cm 2 /s (35). The expected equilibration time constant in the oocyte cytoplasm is then in the order of a few hundred seconds, and this is confirmed by experimental observations as well (8,18).…”