No facilitator required for membrane transport of hydrogen sulfide.

Hydrogen sulfide (H(2)S) 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(2)O, it was hypothesized that aquaporins may facilitate H(2)S 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 H(2)S flux. To measure H(2)O and H(2)S fluxes, respectively, sodium ion dilution and buffer acidification by proton release (H(2)S left arrow over right arrow 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(2)S, P(M,H(2)S) >or = 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, P(M,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(2)S diffusion. The fact that cholesterol and sphingomyelin reconstitution did not turn these membranes into an H(2)S barrier indicates that H(2)S transport through epithelial barriers, endothelial barriers, and membrane rafts also occurs by simple diffusion and does not require facilitation by membrane channels.

Carbon dioxide transport through membranes.

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 mum. 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.

Fast and selective ammonia transport by aquaporin-8.

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 x 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 x 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.

Determining the conductance of the SecY protein translocation channel for small molecules.

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.

A new model of weak acid permeation through membranes revisited: does Overton still rule?

According to a recent publication by Thomae, A. V., H. Wunderli-Allenspach, and S. D. Krämer (2005. Biophys. J. 89:1802-1811), membrane bilayers are well-permeable to the charged species of aromatic carboxylic acids. At physiological pH, the anions were claimed to be the major diffusing species. In contrast, calculation of the Born energy barrier predicts a 10(5)-fold higher permeability for the uncharged (protonated) form. To test the new model, we now have measured both the current carried by the salicylate anion through solvent-free planar membranes and the amount of protons transported by the neutral species. The corresponding membrane permeabilities of the charged and protonated forms were 4 x 10(-7) cm/s and 1.2 cm/s. These data are in perfect agreement with literature data gathered in the last three decades (compare, e.g., Gutknecht, J., and D. C. Tosteson. 1973. Science. 182:1258-1261). They indicate that the report by Thomae at al. represents an experimental artifact. The well-documented role of neutral species in the permeation process of weak acids and bases across artificial and natural membranes is not in question. Overton still rules.

Mobility of a one-dimensional confined file of water molecules as a function of file length.

Confinement of water by pore geometry to a one-dimensional file of molecules interacting with the pore alters the diffusion coefficient D(W). Here we report an exponential dependence of D(W) on the number of water positions in the pore. The result is based on measurements of single channel water permeabilities of structurally similar peptidic nanopores of different length. The inconsistency with predictions from continuum or kinetic models indicates that pore occupancy is reduced in single file transport. In longer pores (e.g., in aquaporins) the presence of charged residues increases D(W).

Proton exclusion by an aquaglyceroprotein: a voltage clamp study.

BACKGROUND INFORMATION: In silico both orthodox aquaporins and aquaglyceroporins are shown to exclude protons. Supporting experimental evidence is available only for orthodox aquaporins. In contrast, the subset of the aquaporin water channel family that is permeable to glycerol and certain small, uncharged solutes has not yet been shown to exclude protons. Moreover, different aquaglyceroporins have been reported to conduct ions when reconstituted in planar bilayers. RESULTS: To clarify these discrepancies, we have measured proton permeability through the purified Escherichia coli glycerol facilitator (GlpF). Functional reconstitution into planar lipid bilayers was demonstrated by imposing an osmotic gradient across the membrane and detecting the resulting small changes in ionic concentration close to the membrane surface. The osmotic water flow corresponds to a GlpF single channel water permeability of 0.7x10(-14) cm(3).subunit(-1).s(-1). Proton conductivity measurements carried out in the presence of a pH gradient (1 unit) revealed an upper limit of the H(+) (OH(-)) to H(2)O molecules transport stoichiometry of 2x10(-9). A significant GlpF-mediated ion conductivity was also not detectable. CONCLUSIONS: The lack of a physiologically relevant GlpF-mediated proton conductivity agrees well with predictions made by molecular dynamics simulations.

Beyond the diffusion limit: Water flow through the empty bacterial potassium channel.

Water molecules are constrained to move with K+ ions through the narrow part of the Streptomyces lividans K+ channel because of the single-file nature of transport. In the presence of an osmotic gradient, a water molecule requires <10 ps to cross the purified protein reconstituted into planar bilayers. Rinsing K+ out of the channel, water may be 1,000 times faster than the fastest experimentally observed K+ ion and 20 times faster than the one-dimensional bulk diffusion of water. Both the anomalously high water mobility and its inhibition observed at high K+ concentrations are consistent with the view that liquid-vapor oscillations occur because of geometrical confinements of water in the selectivity filter. These oscillations, where the chain of molecules imbedded in the channel (the "liquid") cooperatively exits the channel, leaving behind a near vacuum (the "vapor"), thus far have only been discovered in hydrophobic nanopores by molecular dynamics simulations [Hummer, G., Rasaiah, J. C. & Noworyta, J. P. (2001) Nature 414, 188-190; and Beckstein, O. & Sansom, M. S. P. (2003) Proc. Natl. Acad. Sci. USA 100, 7063-7068].

Structural proton diffusion along lipid bilayers.

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.