Although water movement across plasma membranes is a universal property of cells, the precise route of such movement is unresolved. Against this background, we have recently found evidence of water movement across facilitative and sodium-dependent glucose transporters. In cells expressing them, specific inhibitors decrease cellular osmotic (Pf) and diffusional (Pd) permeabilities. Moreover, injecting mRNAs encoding these proteins results in an increase in oocyte Pf; that increase in turn is inhibited by the appropriate glucose transport inhibitors. These results are not consistent with prevailing views that sugar transporters behave as normally closed molecular gates, and instead suggest that they may have a constitutively open hydrophilic pore across their structure. The research proposed here will therefore examine several issues that arise from our results. We propose to: (1) Substantiate our observations by: a) correlating level of cellular transporter expression with Pf; b) examining the route of transmembrane water diffusional permeation; c) examining the temperature dependence (energy of activation) of Pf and Pd. (2) To determine whether water and glucose traverse the same respective pores of GLUTs and SGLTs by examining the effects of various transported and non- transported sugars on the kinetic parameters of glucose and water movements through these transporters. (3) To examine the unitary molecular Pf and Pd of these transporters by determining the numbers of transporters expressed by appropriate cells and measuring Pf by light scattering techniques and Pd by tritiated water diffusion in packed cells. (4) To investigate which elements in the three-dimensional structures of these transporters may account for their facilitating movements of both glucose and water. For this, we will perform site directed mutagenesis of the transporters, followed by expression in mammalian cells and measurement of their unitary molecular Pf. The results will be interpreted using theoretical kinetic analysis, and molecular modeling. these studies may lead to a better understanding of those health disorders which are characterized by altered fluid movements.
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