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Lipid raft components cholesterol and sphingomyelin increase H+/OH- permeability of phosphatidylcholine membranes.

ABSTRACT: H+/OH- permeation through lipid bilayers occurs at anomalously high rates and the determinants of proton flux through membranes are poorly understood. Since all life depends on proton gradients, it is important to develop a greater understanding of proton leak phenomena. We have used stopped-flow fluorimetry to probe the influence of two lipid raft components, chol (cholesterol) and SM (sphingomyelin), on H+/OH- and water permeability. Increasing the concentrations of both lipids in POPC (palmitoyl-2-oleoyl phosphatidylcholine) liposomes decreased water permeability in a concentration-dependent manner, an effect that correlated with increased lipid order. Surprisingly, proton flux was increased by increasing the concentration of chol and SM. The chol effect was complex with molar concentrations of 17.9, 33 and 45.7% giving 2.8-fold (P<0.01), 2.2-fold (P<0.001) and 5.1-fold (P<0.001) increases in H+/OH- permeability from a baseline of 2.4x10(-2) cm/s. SM at 10 mole% effected a 2.8-fold increase (P<0.01), whereas 20 and 30 mole% enhanced permeability by 3.6-fold (P<0.05) and 4.1-fold respectively (P<0.05). Supplementing membranes containing chol with SM did not enhance H+/OH- permeability. Of interest was the finding that chol addition to soya-bean lipids decreased H+/OH- permeability, consistent with an earlier report [Ira and Krishnamoorthy (2001) J. Phys. Chem. B 105, 1484-1488]. We speculate that the presence of proton carriers in crude lipid extracts might contribute to this result. We conclude that (i) chol and SM specifically and independently increase rates of proton permeation in POPC bilayers, (ii) domains enriched in these lipids or domain interfaces may represent regions with high H+/OH- conductivity, (iii) H+/OH- fluxes are not governed by lipid order and (iv) chol can inhibit or promote H+/OH- permeability depending on the total lipid environment. Theories of proton permeation are discussed in the light of these results.

SUBMITTER: Gensure RH

PROVIDER: S-EPMC1559473 | biostudies-other | 2006 Sep

REPOSITORIES: biostudies-other

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Project description:The paradigm of biological membranes has recently gone through a major update. Instead of being fluid and homogeneous, recent studies suggest that membranes are characterized by transient domains with varying fluidity. In particular, a number of experimental studies have revealed the existence of highly ordered lateral domains rich in sphingomyelin and cholesterol (CHOL). These domains, called functional lipid rafts, have been suggested to take part in a variety of dynamic cellular processes such as membrane trafficking, signal transduction, and regulation of the activity of membrane proteins. However, despite the proposed importance of these domains, their properties, and even the precise nature of the lipid phases, have remained open issues mainly because the associated short time and length scales have posed a major challenge to experiments. In this work, we employ extensive atom-scale simulations to elucidate the properties of ternary raft mixtures with CHOL, palmitoylsphingomyelin (PSM), and palmitoyloleoylphosphatidylcholine. We simulate two bilayers of 1,024 lipids for 100 ns in the liquid-ordered phase and one system of the same size in the liquid-disordered phase. The studies provide evidence that the presence of PSM and CHOL in raft-like membranes leads to strongly packed and rigid bilayers. We also find that the simulated raft bilayers are characterized by nanoscale lateral heterogeneity, though the slow lateral diffusion renders the interpretation of the observed lateral heterogeneity more difficult. The findings reveal aspects of the role of favored (specific) lipid-lipid interactions within rafts and clarify the prominent role of CHOL in altering the properties of the membrane locally in its neighborhood. Also, we show that the presence of PSM and CHOL in rafts leads to intriguing lateral pressure profiles that are distinctly different from corresponding profiles in nonraft-like membranes. The results propose that the functioning of certain classes of membrane proteins is regulated by changes in the lateral pressure profile, which can be altered by a change in lipid content.

Project description:Mixtures of unsaturated lipids, sphingolipids, and cholesterol form coexisting liquid-disordered and sphingolipid and cholesterol-rich liquid-ordered (Lo) phases in water. The detergent Triton X-100 does not readily solubilize Lo domains, but does solubilize liquid-disordered domains, and is commonly used to prepare detergent-resistant membranes from cells and model membranes. However, it has been proposed that in membranes with mixtures of sphingomyelin (SM), 1-palmitoyl 2-oleoyl phosphatidylcholine (POPC), and cholesterol Triton X-100 may induce Lo domain formation, and therefore detergent-resistant membranes may not reflect the presence of preexisting domains. To examine this hypothesis, the effect of Triton on Lo domain formation was measured in SM/POPC/cholesterol vesicles. Nitroxide quenching methods that can detect ordered nanodomains with radii >12 Å showed that in the absence of Triton X-100 this mixture formed ordered state domains that melt with a midpoint (= T(mid)) at ?45°C. However, T(mid) was lower when detected using various fluorescence resonance energy transfer (FRET) pairs. Furthermore, the T(mid) value was Ro dependent, and decreased as Ro increased. Because FRET can only readily detect domains with radii >Ro, this result can be explained by domain radii that are close to Ro and decrease as temperature increases. An analysis of FRET and quenching data suggests that nanodomain radius gradually decreases from ?150 Å to <40 Å as temperature increases from 10 to 45°C. Interestingly, the presence of Triton X-100 or a transmembrane-type peptide did not stabilize ordered state formation when detected by nitroxide quenching, i.e., did not increase T(mid). However, FRET-detected T(mid) did increase in the presence of Triton X-100 or a transmembrane peptide, indicating that both increased domain size. Controls showed that the results could not be accounted for by probe-induced perturbations. Thus, SM/POPC/cholesterol, a mixture similar to that in the outer leaflet of plasma membranes, forms nanodomains at physiological temperatures, and TX-100 does not induce domain formation or increase the fraction of the bilayer in the ordered state, although it does increase domain size by coalescing preexisting domains.

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Lipid raft components cholesterol and sphingomyelin increase H+/OH- permeability of phosphatidylcholine membranes.

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