Biological membranes regulate fluxes of water and solutes over a 10,000-fold permeability (P) range, from apical membranes of low P barrier epithelia to high P membranes which contain aquaporins (AQPs) or urea transporters UT's). This grant addresses 2 fundamental physiological questions:
Aim #1. What are the lipid and protein structural features of barrier epithelial apical membranes that reduce their P's to water and to other small solutes such as urea, H+, and NH3? Aim #2. How do the structures of AQP's and UT's determine their ability to mediate the fluxes of water and small solutes across membranes? Question #1 of Aim #1 will focus on the lipid determinants of apical membrane barrier function. We will obtain the best structural information (volume and thickness of the hydrocarbon ore, hydration radius of head groups, elastic and compression moduli) available and we will measure the water content as a function of depth within the bilayer for 6 phospholipids with and without 50% cholesterol. We will then measure P's to water, urea, glycerol, NH3 and H+. Comparing structural parameters and water content with P's will define the mechanisms by which lipids can limit permeation. Question #2 of Aim #1 will examine the role of uroplakins, which are integral membrane proteins, in the barrier function of urothelia. We will examine P's, ultrastructure, membrane trafficking in response to stretch and emptying, and the ability of these proteins to enhance the unstirred layer of the epithelium using background strain mice and mice in which uroplakins have been ablated. Question #1 of Aim #2 will compare molecular dynamics (MD) simulations of H2O, D2O, CO2 and H2S conductance across 2 AQP's for which the structure of the pore has been defined (AQP1 and the bacterial AQP, AQPZ) to actual measurements of these conductances, permitting us to enhance the true predictive ability of these simulations. Question #2 of Aim #2 will exploit a newly developed expression system, Xenopus oocyte plasma membrane vesicles, to examine in detail the function of renal UT's (UT-A's) and the red cell UT (UT-B). Because these UT's share a great deal of sequence homology, detailed functional studies will relate amino acid sequence to transport function. We will define the mechanism of cAMP regulation of UT-A function. We have also obtained 2 bacterial UT's, and will use these for functional studies and efforts at reconstitution and structural studies. Building on substantial prior progress, this competing renewal seeks to define at a molecular level the mechanisms by which water and other small molecules cross barrier membranes, AQP's, and UT's. ? ? ?
| Yu, Weiqun; Hill, Warren G; Apodaca, Gerard et al. (2011) Expression and distribution of transient receptor potential (TRP) channels in bladder epithelium. Am J Physiol Renal Physiol 300:F49-59 |
| Tristram-Nagle, Stephanie; Kim, Dong Joo; Akhunzada, Nadia et al. (2010) Structure and water permeability of fully hydrated diphytanoylPC. Chem Phys Lipids 163:630-7 |
| Godara, Geeta; Smith, Craig; Bosse, Janine et al. (2009) Functional characterization of Actinobacillus pleuropneumoniae urea transport protein, ApUT. Am J Physiol Regul Integr Comp Physiol 296:R1268-73 |
| Guler, S Deren; Ghosh, D Dipon; Pan, Jianjun et al. (2009) Effects of ether vs. ester linkage on lipid bilayer structure and water permeability. Chem Phys Lipids 160:33-44 |
| MacIver, Bryce; Cutler, Christopher P; Yin, Jia et al. (2009) Expression and functional characterization of four aquaporin water channels from the European eel (Anguilla anguilla). J Exp Biol 212:2856-63 |
| Raunser, Stefan; Mathai, John C; Abeyrathne, Priyanka D et al. (2009) Oligomeric structure and functional characterization of the urea transporter from Actinobacillus pleuropneumoniae. J Mol Biol 387:619-27 |
| Mathai, John C; Missner, Andreas; Kügler, Philipp et al. (2009) No facilitator required for membrane transport of hydrogen sulfide. Proc Natl Acad Sci U S A 106:16633-8 |
| Missner, Andreas; Kugler, Philipp; Saparov, Sapar M et al. (2008) Carbon dioxide transport through membranes. J Biol Chem 283:25340-7 |
| Mathai, John C; Tristram-Nagle, Stephanie; Nagle, John F et al. (2008) Structural determinants of water permeability through the lipid membrane. J Gen Physiol 131:69-76 |
| Maciver, Bryce; Smith, Craig P; Hill, Warren G et al. (2008) Functional characterization of mouse urea transporters UT-A2 and UT-A3 expressed in purified Xenopus laevis oocyte plasma membranes. Am J Physiol Renal Physiol 294:F956-64 |
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