Water transport by the sodium glucose cotransporter

After more than a hundred years of investigations, the debate whether epithelial water transport may occur according to a secondary active mechanism is still unresolved. We will now address this question by monitoring water transport through monolayers consisting of cells expressing the sodium-glucose cotransporter SGLT1. If SGLT1 transports 250 molecules of water with every molecule of glucose as previously reported, we will observe a water flux in the presence of glucose and sodium gradients, despite the fact that the osmotic gradient is absent or directed oppositely to the solute gradient. The specially designed combination of scanning electrochemical microscopy with fluorescence techniques allows simultaneous detection of water, sodium and glucose fluxes and thus determination of transport stoichiometry for SGLT1 expressed in its native environment. In addition to the above- mentioned approaches we are going to genetically label SGLT1 to count the number of transporters in the plasma membrane by fluorescence correlation spectroscopy (FCS) so that the number of water molecules passing the SGLT1 pore can be derived with thus far unparalleled precision. We will show that the water pore is different from the glucose pathway by mutational analysis and we will exploit photon counting histograms to test the hypothesis that water pore formation is due to SGLT1 oligomerization. As an alternative to stoichiometric coupling, solute solvent coupling was proposed to occur by local osmosis within the unstirred layers. To test this hypothesis we measure tiny changes of the solute concentration adjent to the epithelia by scanning ion sensitive electrodes. The technical innovations are made without discharging well established methods for monitoring tight junction permeability: like measurements of transepithelial resistance (TER) or paracellular macromolecular fluxes. Last but not least, the application of scanning confocal microscopy in combination with FCS will allow (i) visualization of transcellular water flux via monitoring spatial intracellular differences of solute mobility and solute concentration and (ii) testing the possibility that the cytoskeleton has a role in regulating flux velocity.

Humans reabsorb about 8 to 10 liters water from the intestine daily. This amount of water is not part of the diet, but rather originates from various glands and is thus part of the saliva or gastric juice. Until now it was unclear how the water passes from the intestine to the blood. The epithelium of the intestine lacks highly conductive water channel proteins (so-called aquaporins) like the ones that can be found in the kidney. We have now shown that another protein acts as a water-conducting channel. Up to now this protein, the so-called sodium glucose cotransporter, was only known to be responsible for the uptake of glucose from the intestine. Its ability to transport water as fast as the renal aquaporins was unknown. We have demonstrated its permeability to water by two different means: on the one hand we grew epithelial cells on filters to form confluent monolayers; on the other hand we purified the protein from cells and reconstituted it into lipid vesicles (liposomes). The epithelial cells were genetically modified to produce a fluorescent variant of the transporter in large amounts. This enabled us to count the number of protein molecules in the plasma membrane. At the same time, we measured the amount of water that passed both the cell monolayer and the filter beneath it. Taken together, these data enabled calculation of the unitary transporter water permeability. We calculated the same unitary water permeability from observing the rate at which the vesicles shrunk when exposed to osmotic pressure. We used changes in the intensity of scattered light to assess the time-dependent changes of vesicle volume and we exploited transporter fluorescence to count the number of transporters per vesicle. We observed that an increase in bath glucose concentration led to decreased unitary water permeability suggesting that water and glucose take the same route through the protein. Mutations of the glucose binding pocket greatly diminished the effect, indicating that due to weaker binding, fewer glucose molecules obstructed the already narrow water pathway. The sodium glucose cotransporters new role as a highly efficient facilitator of water transport qualifies it as a new drug target in therapeutical efforts to maintain water balance.