Biological membranes provide selective barriers to a number of molecules and gases. However, the factors that affect permeability to gases remain unclear because of the difficulty of accurately measuring gas movements. To determine the roles of lipid composition and the aquaporin 1 (AQP1) water channel in altering CO 2 flux across membranes, we developed a fluorometric assay to measure CO 2 entry into vesicles. Maximal CO 2 flux was ϳ1000-fold above control values with 0.5 mg/ml carbonic anhydrase. Unilamellar phospholipid vesicles of varying composition gave widely varying water permeabilities but similar CO 2 permeabilities at 25°C. When AQP1 purified from human red blood cells was reconstituted into proteoliposomes, however, it increased water and CO 2 permeabilities markedly. Both increases were abolished with HgCl 2 , and the mercurial inhibition was reversible with ␤-mercaptoethanol. We conclude that unlike water and small nonelectrolytes, CO 2 permeation is not significantly altered by lipid bilayer composition or fluidity. AQP1 clearly serves to increase CO 2 permeation, likely through the water pore; under certain circumstances, gas permeation through membranes is protein-mediated.

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Aquaporins 1 (AQP1) and 2 (AQP2) were expressed in the yeast secretory mutant sec6-4. The mutant accumulates post-Golgi, plasma membrane-targeted vesicles and may be used to produce large quantities of membrane proteins. AQP1 or AQP2 were inducibly expressed in yeast and were localized within isolated sec6-4 vesicles by immunoblot analysis. Secretory vesicles containing AQP1 and AQP2 exhibited high water permeabilities and low activation energies for water flow, indicating expression of functional AQP1 and AQP2. AQP1 solubilized from secretory vesicles was successfully reconstituted into proteoliposomes, demonstrating the ability to use the yeast system to express aquaporins for reconstitution studies. The AQP2-containing secretory vesicles showed no increased permeability toward formamide, urea, glycerol, or protons compared with control vesicles, demonstrating that AQP2 is highly selective for water over these other substances. We conclude that the expression of aquaporins in yeast sec6 vesicles is a valid system to further study mammalian water channel function.

Chloride uptake into yeast was measured as a function of pH. A small amount of uptake was seen at pH values of 3.0 and 4.0; at pH 6.0 chloride uptake was substantially less than the uptake of phosphate and rubidium. Because chloride uptake is inefficient, we expressed the putative mammalian chloride channel, pI Cln , in yeast and observed a chloride-selective current when total membrane protein was reconstituted into lipid bilayers. The current was inhibited by a specific chloride channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoic acid. These results suggest that yeast may serve as a means to characterize chloride channels from other organisms. ß