Biochem. J. (2006) 398
(485–495) (Printed in Great Britain)
Lipid raft components cholesterol and sphingomyelin increase H+/OH- permeability of phosphatidylcholine membranes
Rebekah H. GENSURE, Mark L. ZEIDEL and Warren G. HILL1
Laboratory of Epithelial Cell Biology, Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, Pittsburgh, PA 15261, U.S.A.
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 2.4×10-2 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.
Key words: cholesterol, diffusion, leak, lipid bilayer, sphingomyelin, water and proton permeability.
Abbreviations used: AQP1, aquaporin 1; CF, 5,6-carboxyfluorescein; chol, cholesterol; DPH, diphenylhexatriene; PC, phosphatidylcholine; DPPC, dipalmitoyl PC; NEFA, non-esterified fatty acid; POPC, palmitoyl-2-oleoyl PC; SBPL, soya-bean polar lipids; SM, sphingomyelin.
1To whom correspondence should be addressed, at the present address: Beth Israel Deaconess Medical Center, 840 Memorial Drive, Cambridge, MA 02139, U.S.A. (email whill@bidmc.harvard.edu).
Received 3 October 2005/25 April 2006; accepted 17 May 2006
Published as BJ Immediate Publication 17 May 2006, doi:10.1042/BJ20051620
The Biochemical Society, London ©2006
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