Water, solute and gas transport across membranes and proteins:

Despite intense study over many years the mechanisms by which water and small nonelectrolytes cross lipid bilayers remain unclear. We are interested in studying the role of lipid structural parameters and its influence on membrane permeability. Our results along with mathematical modeling suggest that area occupied by the lipid is the major determinant and the hydrocarbon thickness is a secondary determinant for membrane permeability to water. The water permeability decreases with added cholesterol and it correlates in a different way from pure lipids with area per lipid, bilayer thickness, and also with area compressibility (2,3). 

Aquaporins are primarily known to transport water but some homologues also transport small molecules such as urea and glycerol. In addition aquaporins were also shown to transport gases. However, CO2 transport through aquaporins is debatable and H2S transport through archeal aquaporins has been speculated. Gas transport is difficult to quantify using kinetic methods as they underestimate the unstirred layer effects. In a collaborative study, we recently measured the CO2 and H2S permeability across planar lipid membranes using novel membrane scanning microelectrode methods which circumvent the unstirred layer effects. We have shown that the rate limiting step for gas permeability is the unstirred layer adjacent to the membrane and not the membrane itself. These results suggest that a protein for transport of gases such as CO2 and H2S may be redundant (1, 5). 

We are also interested in studying urea transporters as urea is a major metabolite of nitrogen metabolism and is involved in urine concentration mechanisms in the kidney. Although the genetics of urea transporters are well known, not much is known about structure and kinetics of urea transporter. We are studying the urea transporter from bacteria and humans in an effort to understand the structure function relationships. We have overexpressed, purified and reconstituted urea transporters into proteoliposomes for functional characterization and structural studies.

Urea Transporters

We are studying urea transporters from mice as a model for UT function in humans as the protein isoforms and gene structure are very similar. There are two separate UT genes; the SLC14a1 gene encodes a 384 amino acid protein known as UT-B, which is found in a wide variety of tissues, but in relation to kidney function is found in circulating red blood cells and the vasa recta. A second gene SLC14a2 encodes a more complex set of proteins - in total 6 different isoforms have been described from various mammals. Mice are known to have five of these isoforms with the major kidney specific ones being UT-A1 (930aa), UT-A2 (397aa) and UT-A3 (461aa). UT-A2 and UT-A3 each effectively represent one half of UT-A1 (see figure 1). 
Our methodology primarily involves the use of Xenopus laevis oocytes as an expression system where we have two approaches; the use of whole oocytes for isotopic urea flux measurements and a plasma membrane purification protocol, developed by Warren Hill (Hill et al, 2005), that allows more precise kinetic measurements using stopped-flow fluorimetry. This latter approach has allowed us to undertake initial characterization of two mouse urea transporters isoforms, UT-A2 and UT-A3 and to determine that they differ insignificantly in urea, urea analog and water permeabilities (MacIver et al, 2008).


It was originally thought that water homeostasis in a cell was solely by plasma membrane diffusion, but this thinking was overturned by studies that showed certain tissues could increase their water permeabilities after hormone treatment. Subsequently the aquaporin protein water channels were discovered. Aquaporins have now been found in a wide range of life, from bacteria to plants to mammals. Our group has a number of collaborations underway related to the study of aquaporins. 
The European eel (Anguilla anguilla) is a fish that can acclimate from salt to fresh water (an euryhaline) and in order to do so it must alter its osmoregulation. Molecular cloning studies have revealed at least ten aquaporin-like genes in this animal. In collaboration with C. P. Cutler (Georgia Southern University) we have so far characterized four aquaporins with particular attention on those that are highly expressed in the gill and the gut, tissues where considerable osmoregulation is required in fish. 
Additionally in collaboration with J Brodsky, N Kaufmann and M Burstein at the University of Pittsburgh an aquaporin and an aquaglyceroporin from the fruit fly Drosophila melanogaster have been characterized. The fly's kidney equivalent is the malpighian tubule, which is composed of two major cell types; principal cells and stellate cells. The aquaporin is the product of the drip gene and is expressed in stellate cells (figure 3). The aquaglyceroporin gene is expressed in the principal cells and we have demonstrated that it conducts glycerol and urea in addition to water. The fly is a superb model organism to elucidate the role of these genes during development.

Relevant Publications

1: Missner A, Kügler P, Saparov SM, Sommer K, Mathai JC, Zeidel ML, Pohl P. Carbon dioxide transport through membranes., J Biol Chem. 2008 Jul 9. [Epub ahead of print] 

2: Nagle JF, Mathai JC, Zeidel ML, Tristram-Nagle S. Theory of passive permeability through lipid bilayers. J Gen Physiol. 2008 Jan;131(1):77-85. 

3: Mathai JC, Tristram-Nagle S, Nagle JF, Zeidel ML. Structural determinants of water permeability through the lipid membrane. J Gen Physiol. 2008 Jan;131(1):69-76. 

4: Mathai JC, Zeidel ML. Measurement of water and solute permeability by stopped-flow fluorimetry. Methods Mol Biol. 2007;400:323-32. Review. 

5: Mamonov AB, Coalson RD, Zeidel ML, Mathai JC. Water and deuterium oxide permeability through aquaporin 1: MD predictions and experimental verification. 
J Gen Physiol. 2007 Jul;130(1):111-6. 

6: Tenchov B, Vescio EM, Sprott GD, Zeidel ML, Mathai JC. Salt tolerance of archaeal extremely halophilic lipid membranes. J Biol Chem. 2006 Apr 14;281(15):10016-23. 

7. Hill WG, Southern NM, MacIver B, Potter E, Apodaca G, Smith CP, and Zeidel ML. Isolation and characterization of the Xenopus oocyte plasma membrane: a new method for studying activity of water and solute transporters. Am J Physiol Renal Physiol 289: F217-224, 2005. 

8. Maciver B, Smith CP, Hill WG, and Zeidel ML. 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: F956-964, 2008.


Peter Pohl, JKU, Linz, Austria 
John Nagle and Stephanie Tristram Nagle, CMU, Pittsburgh. 
Dennis Sprott, Institute for Biological Sciences, NRC, Canada 
Boris Tenchov, Northwestern University, Evanston, Illinois