Water transport by the sodium glucose cotransporter (FWF)
Project Leader: Univ.-Prof. Dr. Peter Pohl
FWF Project P23574
1 August 2011 - 31 March 2015
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.
The Sodium Glucose Cotransporter SGLT1 is an Extremely Efficient Facilitator of Passive Water Transport
Liudmila Erokhova, Andreas Horner, Nicole Ollinger, Christine Siligan and Peter Pohl
J Biol Chem. 2016 April 29; 291 (18), pp. 9712-20.