Reconstituted Aquaporin 1 Water Channels Transport CO2 across Membranes

G V Ramesh Prasad,Larry A Coury,Frances Finn,Mark L Zeidel

doi:10.1074/jbc.273.50.33123

G V Ramesh Prasad, Larry A Coury + Show 2 more

Open Access

https://doi.org/10.1074/jbc.273.50.33123

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Abstract

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 CO2 flux across membranes, we developed a fluorometric assay to measure CO2 entry into vesicles. Maximal CO2 flux was approximately 1000-fold above control values with 0.5 mg/ml carbonic anhydrase. Unilamellar phospholipid vesicles of varying composition gave widely varying water permeabilities but similar CO2 permeabilities at 25 degreesC. When AQP1 purified from human red blood cells was reconstituted into proteoliposomes, however, it increased water and CO2 permeabilities markedly. Both increases were abolished with HgCl2, and the mercurial inhibition was reversible with beta-mercaptoethanol. We conclude that unlike water and small nonelectrolytes, CO2 permeation is not significantly altered by lipid bilayer composition or fluidity. AQP1 clearly serves to increase CO2 permeation, likely through the water pore; under certain circumstances, gas permeation through membranes is protein-mediated.

Highlights

Membrane permeability to gases has been less extensively studied, because of the difficulty of accurately measuring gas fluxes
In liposomes or proteoliposomes with entrapped 5,6-carboxyfluorescein (CF)1 and carbonic anhydrase, we measured the rate of pH drop engendered when abrupt exposure to external CO2/HCO3Ϫ leads to rapid entry of CO2 into the vesicle and generation of carbonic acid
Flux Measurements—For CO2 permeability (Pgas) measurements, vesicles were abruptly exposed to a CO2 gradient by rapidly mixing with an equal volume of freshly made 0.1 M NaHCO3 and 20 mM HEPES, pH 7.40, that was kept capped throughout the experiment

Summary

Membrane permeability to gases has been less extensively

Studied, because of the difficulty of accurately measuring gas fluxes. Gross movement of O2 and CO2 across the lung has been measured, as has NH3 permeability of some membranes [5,6,7]. We have developed a method to measure CO2 fluxes across the membranes of unilamellar vesicles. In liposomes or proteoliposomes with entrapped 5,6-carboxyfluorescein (CF) and carbonic anhydrase, we measured the rate of pH drop engendered when abrupt exposure to external CO2/HCO3Ϫ leads to rapid entry of CO2 into the vesicle and generation of carbonic acid. Using this method we compared water and CO2 permeability of a variety of membranes and demonstrated that aquaporin 1 (AQP1) reconstituted as the sole protein in proteoliposomes mediates CO2 flux

MATERIALS AND METHODS

RESULTS AND DISCUSSION

Water Pf