Measuring the Release of Fluorescein from MscL-Loaded Liposomes with Stressed Lipid Bilayers

Abstract

The bacterial mechanosensitive ion channel of large conductance (MscL) acts as an emergency release valve to protect against osmotic stress1,2; its pore diameter is estimated to increase by >25A on opening3,4. To investigate mechanisms for controlling the gating of this channel for use in targeted drug delivery systems, we have encapsulated the fluorescent dye 5,6-carboxyfluorescein (CF) into liposomes (diam. 100 nm) via sonication and extrusion using the LiposoFast system, followed by incorporation of MscL protein. Functional assays of MscL by patch-clamping confirmed that the dye did not affect the protein incorporation, and unencapsulated dye was removed by column purification. Liposomes were incubated with different concentrations of the amphipath L-α-Lysophosphatidylcholine (LPC) dissolved in ∼3% MeOH for 30 min to induce stress on liposomal membranes5. Dye release from the liposomes via MscL was monitored as increased fluorescence in the external medium. The percentage fluorescence change quantified using a BMG Omega Polarstar Reader (excitation at 485nm and emission at 535nm). Based on these results, we demonstrate cargo release by MscL by means of manipulating the curvature of liposome membranes4.1. Martinac, B., J. Cell Sci., 117, 2449-2460 (2004).2. Perozo, E., Nat. Rev. Mol. Biol. , 7, 109-119 (2006).3. Cruickshank, C.C., Minchin, R., Le Dain, A., and Martinac, B. Biophys J 73, 1925-1931 (1997)4. Perozo, E., Cortes, D.M., Sompornpisut, P. Kloda, A. and Martinac, B. Nature 418: 942-948 (2002)5 Perozo, E., Kloda, A., Cortes, D.M., Martinac, B., Nat. Str. Biol. 9, 696-703(2002)Supported by grants from the Australian Research Council (ARC) and National Health and Medical Research Council (NHMRC). A.Foo is a UQ Scholarship recipient.