Hydrogen sulfide (H2S) has emerged as a new and important member in the group of gaseous signaling molecules. However, the molecular transport mechanism has not yet been identified. Because of structural similarities with H 2O, it was hypothesized that aquaporins may facilitate H 2S transport across cell membranes. We tested this hypothesis by reconstituting the archeal aquaporin AfAQP from sulfide reducing bacteria Archaeoglobus fulgidus into planar membranes and by monitoring the resulting facilitation of osmotic water flow and H2S flux. To measure H2O and H2S fluxes, respectively, sodium ion dilution and buffer acidification by proton release (H 2S % H ؉ ؉ HS ؊ ) were recorded in the immediate membrane vicinity. Both sodium ion concentration and pH were measured by scanning ion-selective microelectrodes. A lower limit of lipid bilayer permeability to H 2S, P M,H 2 S > 0.5 ؎ 0.4 cm/s was calculated by numerically solving the complete system of differential reaction diffusion equations and fitting the theoretical pH distribution to experimental pH profiles. Even though reconstitution of AfAQP significantly increased water permeability through planar lipid bilayers, PM,H 2 S remained unchanged. These results indicate that lipid membranes may well act as a barrier to water transport although they do not oppose a significant resistance to H 2S diffusion. The fact that cholesterol and sphingomyelin reconstitution did not turn these membranes into an H2S barrier indicates that H 2S transport through epithelial barriers, endothelial barriers, and membrane rafts also occurs by simple diffusion and does not require facilitation by membrane channels. aquaporins ͉ gas transport ͉ membrane permeability ͉ unstirred layer ͉ signaling

Several membrane channels, like aquaporin-1 (AQP1) and the RhAG protein of the rhesus complex, were hypothesized to be of physiological relevance for CO 2 transport. However, the underlying assumption that the lipid matrix imposes a significant barrier to CO 2 diffusion was never confirmed experimentally. Here we have monitored transmembrane CO 2 flux (J CO2 ) by imposing a CO 2 concentration gradient across planar lipid bilayers and detecting the resulting small pH shift in the immediate membrane vicinity. An analytical model, which accounts for the presence of both carbonic anhydrase and buffer molecules, was fitted to the experimental pH profiles using inverse problems techniques. At pH 7.4, the model revealed that J CO2 was entirely rate-limited by near-membrane unstirred layers (USL), which act as diffusional barriers in series with the membrane. Membrane tightening by sphingomyelin and cholesterol did not alter J CO2 confirming that membrane resistance was comparatively small. In contrast, a pH-induced shift of the CO 2 hydration-dehydration equilibrium resulted in a relative membrane contribution of about 15% to the total resistance (pH 9.6). Under these conditions, a membrane CO 2 permeability (3.2 ؎ 1.6 cm/s) was estimated. It indicates that cellular CO 2 uptake (pH 7.4) is always USL-limited, because the USL size always exceeds 1 m. Consequently, facilitation of CO 2 transport by AQP1, RhAG, or any other protein is highly unlikely. The conclusion was confirmed by the observation that CO 2 permeability of epithelial cell monolayers was always the same whether AQP1 was overexpressed in both the apical and basolateral membranes or not.The widely accepted model that gases like NH 3 , CO 2 , and O 2 pass biological membranes by diffusion through the lipid matrix has been recently called into question. For example, the membrane protein channels AmtB and aquaporin-8 have been identified to transport NH 3 (1, 2). Protein channels such as the human aquaporin-1, the plant aquaporin NtAQP1, and the RhAG protein of the rhesus complex were reported to provide a pathway for CO 2 transport (3-5). The similarity in the findings for NH 3 and CO 2 is very surprising because Overtone's rule predicts that their permeabilities, P M , across the lipid phase of biological membranes differ 750-fold. The number was calculated assuming that NH 3 and CO 2 have comparable membrane diffusivities and that neither one of them belongs to those extremely rare exceptions from Overtone's rule (6, 7) so that the proportionality between P M and the biphasic partition coefficient (water/organic solvent) applies as shown in Equation 1,where K CO2 ϳ 1.5 (8), K NH3 ϳ 0.002 (6), and P M,NH3 ϭ 0.016 cm/s (9).A P M , CO2 of 12 cm/s suggests that the lipid matrix of biological membranes cannot act as a barrier to CO 2 diffusion. In fact, a stagnant water layer adjacent to the membrane that has the same thickness (␦) as the membrane would generate the same resistance to CO 2 flow as is caused by the membrane itself. Because these so-called unstirr...