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...

For H(+) transport between protein pumps, lateral diffusion along membrane surfaces represents the most efficient pathway. Along lipid bilayers, we measured a diffusion coefficient of 5.8 x 10(-5) cm(2) s(-1). It is too large to be accounted for by vehicle diffusion, considering proton transport by acid carriers. Such a speed of migration is accomplished only by the Grotthuss mechanism involving the chemical exchange of hydrogen nuclei between hydrogen-bonded water molecules on the membrane surface, and the subsequent reorganization of the hydrogen-bonded network. Reconstitution of H(+)-binding sites on the membrane surface decreased the velocity of H(+) diffusion. In the absence of immobile buffers, structural (Grotthuss) diffusion occurred over a distance of 100 micro m as shown by microelectrode aided measurements of the spatial proton distribution in the immediate membrane vicinity and spatially resolved fluorescence measurements of interfacial pH. The efficiency of the anomalously fast lateral diffusion decreased gradually with an increase in mobile buffer concentration suggesting that structural diffusion is physiologically important for distances of approximately 10 nm.

The transport of ammonia/ammonium is fundamental to nitrogen metabolism in all forms of life. So far, no clear picture has emerged as to whether a protein channel is capable of transporting exclusively neutral NH 3 while excluding H ؉ and NH 4 ؉ . Our research is the first stoichiometric study to show the selective transport of NH 3 by a membrane channel. The purified water channel protein aquaporin-8 was reconstituted into planar bilayers, and the exclusion of NH 4 ؉ or H ؉ was established by ensuring a lack of current under voltage clamp conditions. The single channel water permeability coefficient of 1.2 ؋ 10 ؊14 cm 3 /subunit/s was established by imposing an osmotic gradient across reconstituted planar bilayers, and resulting minute changes in ionic concentration close to the membrane surface were detected. It is more than 2-fold smaller than the single channel ammonia permeability (2.7 ؋ 10 ؊14 cm 3 /subunit/s) that was derived by establishing a transmembrane ammonium concentration gradient and measuring the resulting concentration increases adjacent to the membrane. This permeability ratio suggests that electrically silent ammonia transport may be the main function of AQP8.

The channel formed by the SecY complex must maintain the membrane barrier for ions and other small molecules during the translocation of membrane or secretory proteins. We have tested the permeability of the channel by using planar bilayers containing reconstituted purified E. coli SecY complex. Wild-type SecY complex did not show any conductance for ions or water. Deletion of the "plug," a short helix normally located in the center of the SecY complex, or modification of a cysteine introduced into the plug resulted in transient channel openings; a similar effect was seen with a mutation in the pore ring, a constriction in the center of the channel. Permanent channel opening occurred when the plug was moved out of the way by disulfide-bridge formation. These data show that the resting channel on its own forms a barrier for small molecules, with both the pore ring and the plug required for the seal; channel opening requires movement of the plug.