Florian Zocher

Local Partition Coefficients Govern Solute Permeability of Cholesterol-Containing Membranes

The permeability of lipid membranes for metabolic molecules or drugs is routinely estimated from the solutes oil/water partition coefficient. However, the molecular determinants that modulate the permeability in different lipid compositions have remained unclear. Here, we combine scanning electrochemical microscopy and molecular-dynamics simulations to study the effect of cholesterol on membrane permeability, because cholesterol is abundant in all animal membranes. The permeability of membranes from natural lipid mixtures to both hydrophilic and hydrophobic solutes monotonously decreases with cholesterol concentration [Chol]. The same is true for hydrophilic solutes and planar bilayers composed of dioleoyl-phosphatidylcholine or dioleoyl-phosphatidyl-ethanolamine. However, these synthetic lipids give rise to a bell-shaped dependence of membrane permeability on [Chol] for very hydrophobic solutes. The simulations indicate that cholesterol does not affect the diffusion constant inside the membrane. Instead, local partition coefficients at the lipid headgroups and at the lipid tails are modulated oppositely by cholesterol, explaining the experimental findings. Structurally, these modulations are induced by looser packing at the lipid headgroups and tighter packing at the tails upon the addition of cholesterol.

The mobility of single-file water molecules is governed by the number of H-bonds they may form with channel-lining residues

Mobility of single-file water molecules determined by H-bonds. Channel geometry governs the unitary osmotic water channel permeability, pf, according to classical hydrodynamics. Yet, pf varies by several orders of magnitude for membrane channels with a constriction zone that is one water molecule in width and four to eight molecules in length. We show that both the pf of those channels and the diffusion coefficient of the single-file waters within them are determined by the number NH of residues in the channel wall that may form a hydrogen bond with the single-file waters. The logarithmic dependence of water diffusivity on NH is in line with the multiplicity of binding options at higher NH densities. We obtained high-precision pf values by (i) having measured the abundance of the reconstituted aquaporins in the vesicular membrane via fluorescence correlation spectroscopy and via high-speed atomic force microscopy, and (ii) having acquired the vesicular water efflux from scattered light intensities via our new adaptation of the Rayleigh-Gans-Debye equation.

Routes of Epithelial Water Flow: Aquaporins versus Cotransporters

The routes water takes through membrane barriers is still a matter of debate. Although aquaporins only allow transmembrane water movement along an osmotic gradient, cotransporters are believed to be capable of water transport against the osmotic gradient. Here we show that the renal potassium-chloride-cotransporter (KCC1) does not pump a fixed amount of water molecules per movement of one K(+) and one Cl(-), as was reported for the analogous transporter in the choroid plexus. We monitored water and potassium fluxes through monolayers of primary cultured renal epithelial cells by detecting tiny solute concentration changes in the immediate vicinity of the monolayer. KCC1 extruded K(+) ions in the presence of a transepithelial K(+) gradient, but did not transport water. KCC1 inhibition reduced epithelial osmotic water permeability P(f) by roughly one-third, i.e., the effect of inhibitors was small in resting cells and substantial in hormonal stimulated cells that contained high concentrations of aquaporin-2 in their apical membranes. The furosemide or DIOA (dihydroindenyl-oxy-alkanoic acid)-sensitive water flux was much larger than expected when water passively followed the KCC1-mediated ion flow. The inhibitory effect of these drugs on water flux was reversed by the K(+)-H(+) exchanger nigericin, indicating that KCC1 affects water transport solely by K(+) extrusion. Intracellular K(+) retention conceivably leads to cell swelling, followed by an increased rate of endocytic AQP2 retrieval from the apical membrane.

Uroplakins Do Not Restrict CO2 Transport through Urothelium

Background: The tightness of various membrane barriers to CO2 is of unknown molecular origin. Results: The bladder tissue lacks carbonic anhydrase. The resulting low intra-epithelial CO2 concentration gives rise to the apparent CO2 impermeability. Conclusion: Uroplakins do not act to decrease transepithelial CO2 flux. Significance: Enzymatic regulation of CO2 abundance rules out that aquaporins significantly contribute to the maintenance of acid base homeostasis. Lipid bilayers and biological membranes are freely permeable to CO2, and yet partial CO2 pressure in the urine is 3–4-fold higher than in blood. We hypothesized that the responsible permeability barrier to CO2 resides in the umbrella cell apical membrane of the bladder with its dense array of uroplakin complexes. We found that disrupting the uroplakin layer of the urothelium resulted in water and urea permeabilities (P) that were 7- to 8-fold higher than in wild type mice with intact urothelium. However, these interventions had no impact on bladder PCO2 (∼1.6 × 10−4 cm/s). To test whether the observed permeability barrier to CO2 was due to an unstirred layer effect or due to kinetics of CO2 hydration, we first measured the carbonic anhydrase (CA) activity of the bladder epithelium. Finding none, we reduced the experimental system to an epithelial monolayer, Madin-Darby canine kidney cells. With CA present inside and outside the cells, we showed that PCO2 was unstirred layer limited (∼7 × 10−3 cm/s). However, in the total absence of CA activity PCO2 decreased 14-fold (∼ 5.1 × 10−4 cm/s), indicating that now CO2 transport is limited by the kinetics of CO2 hydration. Expression of aquaporin-1 did not alter PCO2 (and thus the limiting transport step), which confirmed the conclusion that in the urinary bladder, low PCO2 is due to the lack of CA. The observed dependence of PCO2 on CA activity suggests that the tightness of biological membranes to CO2 may uniquely be regulated via CA expression.

Design of Peptide-Membrane Interactions to Modulate Single-File Water Transport through Modified Gramicidin Channels

Water permeability through single-file channels is affected by intrinsic factors such as their size and polarity and by external determinants like their lipid environment in the membrane. Previous computational studies revealed that the obstruction of the channel by lipid headgroups can be long-lived, in the range of nanoseconds, and that pore-length-matching membrane mimetics could speed up water permeability. To test the hypothesis of lipid-channel interactions modulating channel permeability, we designed different gramicidin A derivatives with attached acyl chains. By combining extensive molecular-dynamics simulations and single-channel water permeation measurements, we show that by tuning lipid-channel interactions, these modifications reduce the presence of lipid headgroups in the pore, which leads to a clear and selective increase in their water permeability.

Membrane Transport of CO2 and H2S: No Facilitator Required

The observation that some membranes and epithelia have no demonstrable gas permeability suggested that membrane channels may be involved in CO2 transport. Both aquaporins and Rhesus proteins were reported to serve as pathways for CO2 and H2S. In contrast, we show here that membrane lipid has such high CO2 and H2S permeabilities that the presence of a protein channel does not enhance the flux. Therefore we reconstituted aquaporins into lipid bilayers and used scanning microelectrodes to monitor pH in the immediate vicinity of planar lipid bilayers. The lower limits of lipid bilayer permeabilities to CO2 and to H2S were equal to 3.2±1.6 cm/s[1] and 0.5±0.4 cm/s[2], respectively. We also observed that the CO2 flux through the lipid bilayer decreases several fold when the rate of CO2 formation from HCO3- was not augmented by carbonic anhydrase (CA). Experiments with epithelial cell monolayers grown on permeable support revealed the same result. Inhibition of CA transformed these otherwise highly CO2 permeable cell monolayers into CO2 barriers. Finally we tested the CO2 permeability of the epithelium of the mammalian bladder. It was impermeable to CO2 even after uroplakin knock-out. We found that the lack of intrinsic intracellular CA activity of these epithelial cells hampers the CO2 exchange between blood and urine.[1] A. Missner, P. Kugler, S. M. Saparov, K. Sommer, J. C. Matthai, M. L. Zeidel, P. Pohl, J.Biol.Chem. 2008, 283 25340-25347.[2.] J. C. Mathai, A. Missner, P. Kugler, S. M. Saparov, M. L. Zeidel, J. K. Lee, P. Pohl, Proc.Natl.Acad.Sci.U.S.A. 2009, 106 16633-16638.