The single-channel properties generated by alamethicin (Alm) F50/5 and its analog [L-Glu(OMe)7,18,19] were compared when inserted in a cell (the isolated rod outer segment of vertebrate photoreceptor, OS, recorded in whole-cell) and in excised patches from giant unilamellar vesicles (GUVs), with the long-term goal to understand how the peptide-lipid interactions produce membrane permeabilization. GUVs (diameter >10 μm) were prepared by electroformation, using different lipid compositions, which include fluorescent-labeled lipids, in order to optimize their mechanical properties, stability, and visualization. The best composition found was: 40% phosphatidylcholine, 20% phosphatidylglycerol, 30% cholesterol, and 10% nitrobenzoxadiazole-labeled phosphatidylethanolamine. The GUVs were viewed by exploiting a contrast-intensified camera, that was used to check also their unilamellarity in fluorescence. Peptides were applied to, and removed from, the membranes in ~50 ms. In the presence of symmetric KCl or NaCl, Alms F50/5 and [L-Glu(OMe)7,18,19] produced inward rectifying single-channel events of different sizes, at 2.5 µM concentration in GUVs and at 250 nM in OS, activating and deactivating in less than 10 s from the peptide application and withdrawal, respectively. The most probable amplitudes recorded in GUVs were 250 pS for Alm F50/5, and 350 and 500 pS for [L-Glu(OMe)7,18,19]Alm, that gave events up to 1 nS. For OS, Alms F50/5 and [L-Glu(OMe)7,18,19] afforded most probable events of 50 pS and 500 pS, respectively. These results show that the single-channel properties of Alms strongly vary from natural to artificial membranes.
Single-Channel Properties of Peptides Inserted in Natural and Artificial Membranes
BENEDUSI, Mascia;FASOLI, Anna;RISPOLI, Giorgio
2011
Abstract
The single-channel properties generated by alamethicin (Alm) F50/5 and its analog [L-Glu(OMe)7,18,19] were compared when inserted in a cell (the isolated rod outer segment of vertebrate photoreceptor, OS, recorded in whole-cell) and in excised patches from giant unilamellar vesicles (GUVs), with the long-term goal to understand how the peptide-lipid interactions produce membrane permeabilization. GUVs (diameter >10 μm) were prepared by electroformation, using different lipid compositions, which include fluorescent-labeled lipids, in order to optimize their mechanical properties, stability, and visualization. The best composition found was: 40% phosphatidylcholine, 20% phosphatidylglycerol, 30% cholesterol, and 10% nitrobenzoxadiazole-labeled phosphatidylethanolamine. The GUVs were viewed by exploiting a contrast-intensified camera, that was used to check also their unilamellarity in fluorescence. Peptides were applied to, and removed from, the membranes in ~50 ms. In the presence of symmetric KCl or NaCl, Alms F50/5 and [L-Glu(OMe)7,18,19] produced inward rectifying single-channel events of different sizes, at 2.5 µM concentration in GUVs and at 250 nM in OS, activating and deactivating in less than 10 s from the peptide application and withdrawal, respectively. The most probable amplitudes recorded in GUVs were 250 pS for Alm F50/5, and 350 and 500 pS for [L-Glu(OMe)7,18,19]Alm, that gave events up to 1 nS. For OS, Alms F50/5 and [L-Glu(OMe)7,18,19] afforded most probable events of 50 pS and 500 pS, respectively. These results show that the single-channel properties of Alms strongly vary from natural to artificial membranes.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
