Transport of volatile solutes through AQP1

Abstract

For almost a century it was generally assumed that the lipid phases of all biological membranes are freely permeable to gases. However, recent observations challenge this dogma. The apical membranes of epithelial cells exposed to hostile environments, such as gastric glands, have no demonstrable permeability to the gases CO2 and NH3. Additionally, the water channel protein aquaporin 1 (AQP1), expressed at high levels in erythrocytes, can increase membrane CO2 permeability when expressed in Xenopus oocytes. Similarly, nodulin-26, which is closely related to AQP1, can act as a conduit for NH3. A key question is whether aquaporins, which are abundant in virtually every tissue that transports O2 and CO2 at high levels, ever play a physiologically significant role in the transport of small volatile molecules. Preliminary data are consistent with the hypothesis that AQP1 enhances the reabsorption of HCO3- by the renal proximal tubule by increasing the CO2 permeability of the apical membrane. Other preliminary data on Xenopus oocytes heterologously expressing the electrogenic Na+-HCO3- cotransporter (NBC), AQP1 and carbonic anhydrases are consistent with the hypothesis that the macroscopic cotransport of Na+ plus two HCO3- occurs as NBC transports Na+ plus CO3(2-) and AQP1 transports CO2 and H2O. Although data - obtained on AQP1 reconstituted into liposomes or on materials from AQP1 knockout mice - appear inconsistent with the model that AQP1 mediates substantial CO2 transport in certain preparations, the existence of unstirred layers or perfusion-limited conditions may have masked the contribution of AQP1 to CO2 permeability.

Figure 1. Model of aquaporin 1

A, schematic representation of the structure of AQP1. The molecule has intracellular amino and carboxy termini, six membrane-spanning segments, and five loops. Loops B and E fold back into the membrane, contain the tandem repeat of the amino-acid sequence NPA. B, the hourglass model predicts that loops B and E overlap in the membrane to allow the formation of a channel pore. (Data from Nielsen et al. 2002.)

Figure 2. Unique permeability properties of the apical membranes of the gastric gland

A, low H⁺ permeability. The experiments in all three panels were performed at 37 °C on isolated perfused gastric glands from the rabbit, and the records are from a single parietal cell. Intracellular pH (pH_i) was monitored using the fluorescent pH-sensitive dye BCECF in conjunction with a digital imaging system. Reducing the pH of the luminal fluid from 7.4 to 1.4 in a CO₂/HCO₃⁻-free system produced no detectable pH_i change, whereas reducing bath (i.e. basolateral) pH by only 1 pH unit caused a large and rapid fall in pH_i. B, low NH₃/NH₄⁺ permeability. Exposing the lumen to a Hepes-buffered solution containing 20 mm NH₄⁺/NH₃ at pH 8.0 ([NH₃]_o ∼ 1.46 mm) produced no detectable pH_i increase, whereas exposing the bath to 20 mm NH₄⁺/NH₃ at pH 7.4 ([NH₃]_o ∼ 0.4 mm) produced a rapid pH_i increase followed by the usual series of changes. C, low CO₂/HCO₃⁻ permeability. Exposing the lumen to a solution equilibrated with 100 % CO₂ and containing 22 mm HCO₃⁻ (pH 6.1, [CO₂]_o ∼ 24 mm) produced no discernable pH_i decrease, whereas exposing the bath to 1 % CO₂/4.4 mm HCO₃⁻ (pH 7.4, [CO₂]_o ∼ 0.24 mm) or 5 % CO₂/22 mm HCO₃⁻ (pH 7.4, [CO₂]_o ∼ 1.2 mm) caused the expected pH_i decreases. The gland was bathed in 200 μM DIDS to eliminate the pH_i recovery from the intracellular acid load that would otherwise have occurred. (In A, data from Waisbren et al. 1994a. In B, data from Waisbren et al. 1994b.)

Figure 3. Expression of aquaporin 1 increase the permeability of Xenopus oocytes to CO₂

A, experiments on oocytes with vitelline membrane intact. Switching the extracellular buffer from Hepes to 1.5 % CO₂/10 mm HCO₃⁻ (constant pH_o = 7.5) causes a sustained fall of intracellular pH, the initial rate of which is indicated by the filled bars. The initial rate of pH_i recovery (i.e. alkalinization) caused by removing the CO₂/HCO₃⁻ solution is summarized by the open bars. Injection of carbonic anhydrase (CA) caused a significant increase in the initial CO₂-induced rate in both water-injected oocytes and those expressing AQP1. Moreover, in CA-injected oocytes, expression of AQP1 significantly increased the CO₂-induced acidification rate. B, experiments on oocytes with vitelline membrane removed, and in absence of injected CA. Each symbol represents data from a separate experiment (i.e. oocyte). There was a linear relationship between the initial rate of CO₂-induced acidification and lysis time (an index of the AQP1 expression level), with acidification rate increasing as lysis time decreased. (In A, data from Nakhoul et al. 1998. In B, data from Cooper & Boron, 1998a.)

Figure 4. Effect of pCMBS on the CO₂-induced acidification

A, H₂O-injected control oocyte. Two records represent paired data from an experiment on a single oocyte exposed to 1.5 % CO₂/10 mm HCO₃⁻ before and after a 15-min exposure to 1 mm pCMBS. B, AQP1-expressing oocyte. C, C189S-expressing oocyte. D, change in CO₂-induced acidification rate produced by pCMBS. Each bar represents the mean of paired differences, before and after treating the oocyte with pCMBS. (Reproduced from Cooper & Boron, 1998a with permission of the American Physiological Society.)

Figure 5. Aquaporin 1 increases the CO₂ permeability of liposomes made from E. coli membrane lipids

A, effect of incorporating AQP1 into liposomes. In vesicles prepared from E. coli membranes, adding AQP1 produced a significant increase in the rate at which intravesicular pH decreased upon exposure to a CO₂-containing solution. B, effect of blocking AQP1 with HgCl₂. The AQP1-dependent increase in CO₂ permeability was reduced by addition of HgCl₂, and reversed by β-mercaptoethanol. (Reproduced from Prasad et al. 1998 with permission of the Journal of Biological Chemistry.)