Biological membranes regulates fluxes of water and other small molecules over a 1000-fold permeability range, from apical membranes of low permeability barrier epithelial to high permeability membranes that contain water channels (AQPs) and urea transporters (UTs). This range of permeabilities is essential for homeostasis. The original grant, its first competing renewal and this competing renewal proposal seek to answer two fundamental physiological questions, defined as its specific aims: Aim #1: How do the lipids and proteins of barrier apical membranes reduce the permeabilities of these membranes to water and small solutes? To determine the role of lipids in barrier function, the lipid composition of the urothelial umbrella cell apical membrane will be determined, and the role of specific lipids in barrier function will be examined. In addition, studies of archaebacterial lipids will define, at a molecular level, the interactions of water and small solutes with the lipid bilayer. The role of proteins will be examined by determining the effects on barrier function and other epithelial cell functions, of ablation of uroplakins and membrane spanning mucins, using knockout mice lacking these specific proteins. Aim #2: How does the structure of AQPs and UTS determine their permeabilities to water and small solutes? To define the structure/function relationships for AQPs, AQP3 was chosen for study, because it plays a critical role in collecting duct water transport, and is abundant in the basolateral membranes of barrier epithelial. Unlike AQP1 and AQP2, AQP3 has relatively low water permeability, and transports solutes effectively. AQP3 will be expressed in yeast sec6 vesicles which will be isolated and used for careful definition of function, purification and reconstitution for structural studies. Because UTS also transport small solutes and can transport water, and because they are abundant along the distal nephron, they will be studied using approaches already developed for AQPs. The different UTS expressed along the nephron are splice variants of the same gene. At present, little is known about the relationship between UT structure and function, and the role of phosphorylation in regulating function in vivo is unclear. UTS will be expressed in the yeast sec6 system and their function when phosphorylated and dephosphorylated will be defined. These proteins will be reconstituted into membranes for functional and structural studies. The combined aims of the proposal will enhance our understanding of how organisms control the water and solute compositions of their cells and body compartments.