No facilitator required for membrane transport of hydrogen sulfide

Abstract

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.

Fig. 1.

Scheme of H₂S transport across lipid membranes. δ denotes the size of the unstirred layer (USL) present on both sides. Transport of H₂S across a membrane includes four steps: (i) all participating molecules (protonated and deprotonated forms) diffuse from the cis bulk to the membrane; (ii) at the membrane surface, HS⁻ gets protonated; (iii) only the uncharged form of hydrogen sulfide (H₂S) permeates the membrane; (iv) after passing the membrane, most of the H₂S molecules release a proton; and (v) all molecules diffuse from the membrane to trans bulk. The presence of buffer molecules (B⁻, BH) is essential for providing stable bulk.

Fig. 2.

Near-membrane pH and H₂S concentration distributions for bulk pH 7.4. (A) Experimental pH profiles (bulk pH = 7.5) in the trans unstirred water layer. A H₂S gradient was induced by different NaHS concentrations (as indicated) in the cis compartment. Increasing H₂S gradients resulted in an increased proton accumulation in the trans unstirred layer. The analytical model was fitted to the first 50 μm of the experimental pH profiles. The best fit resulted in P_M,H2S = 0.05 cm/s (black lines). The bathing solution contained 100 mM NaCl and 5 mM Mops adjusted to pH = 7.5. (B) The analytical model (see Appendix) allowed visualization of the corresponding H₂S concentration distributions in the cis and trans USLs. As an example, the concentration profiles for the HS⁻ bulk concentration of 215 μM is shown. The lack of a transmembrane H₂S gradient indicates that membrane resistance to H₂S diffusion is negligible.

Fig. 3.

Near-membrane pH and H₂S concentration distributions for bulk pH 8.9. (A) Representative recordings of the acidification in the trans unstirred layer at different NaHS concentrations in the cis compartment. The bulk solutions contained 100 mM NaCl and 5 mM Tris adjusted to pH = 8.9. The black lines represent the best fit of the analytical model to the experimental profiles (P_M,H2S = 0.5 cm/s). (B) Corresponding theoretical H₂S profiles are shown in the cis and trans USLs for a membrane permeability P_M,H2S = 0.5 cm/s (bulk HS⁻ concentration 5 mM). Even at pH = 8.9, the USLs in the immediate membrane vicinity are rate-limiting to the H₂S transport process.

Fig. 4.

Calculated H₂S profiles for size-reduced USLs at 20 °C and 80 °C. The total bulk concentrations of the weak acid (H₂S + HS⁻) were set to 1 mM (cis) and 0 mM (trans) compartments. The differential equations were solved for δ = 0.3 and 1 μm (20 °C). For the calculation at 80 °C, we took into account the following: (i) the different H₂S diffusion coefficients of 2 and 4.62 × 10⁻⁵ cm²/s at 20 °C and 80 °C, respectively (46); (ii) the increase of the diffusion coefficients of all other substances by a factor of 3.4; (iii) the decrease of pK of H₂S from 6.89 to 6.33; (iv) the increase of δ from 300 to 396 nm [δ scales with the third root of D (28)]; and (v) a tenfold-increased H₂S permeability from P_f,H2S = 0.5 cm/s at 20 °C to P_f,H2S = 5 cm/s at 80 °C. Comparison with the increase in water permeability (Arrhenius plot) shows that this factor is most likely an underestimation. Nevertheless, more than 95% of the H₂S gradient is lost within the stagnant water layers next to the membrane. If at 80 °C, P_f,H2S > 5 cm/s, the USL would account for more than 95% of the resistance to H₂S flow. Thus, facilitated H₂S transport is very unlikely, especially at elevated temperatures.

Fig. 5.

Experimental pH profiles induced by H₂S flux from cis to trans side at a bulk pH equal to 7.5. The pH changes did not significantly differ from those measured for sphingomyelin and cholesterol (cholesterol:E.coli lipid:sphingomyelin = 3:2:1 by mass) containing membranes and E. coli lipid bilayers reconstituted with AfAQP (mass ratio of 1:75).

Fig. 6.

Representative concentration profiles showing that osmotic water flow is accompanied by sodium retention at the hypotonic side of the lipid bilayer. The hyperosmotic compartment contained 1 M urea. Water permeability was P_f = 23 ± 2 μm/s for the bare lipid bilayer and increased to P_f = 41 ± 3 μm/s after AfAQP reconstitution at a protein:lipid mass ratio of 1:75. Buffer solution contained 100 mM NaCl and 5 mM Mops, pH = 7.5.