Simultaneous Measurements of K+ and Calcein Release from Liposomes and the Determination of Pore Size Formed in a Membrane

Katsu, Takashi; Imamura, Tomonori; Komagoe, Keiko; Masuda, Kazufumi; Mizushima, Tohru

doi:10.2116/analsci.23.517

Cited by 45 publications

(36 citation statements)

References 30 publications

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“…This makes pure membrane disruption a very unlikely mechanism of action for eurocin. This was further substantiated by the data obtained on real phospholipids by vesicle disruption experiments; although the eurocin concentrations tested were above a protein-to-lipid ratio of 5 and between 30 and 240 ϫ MIC, we could not observe complete release of vesicle contents in contrast to the high efficiency reported for antimicrobial peptides such as Ac-RRWWRF-NH 2 (57), Hnp-2 (58), gramicidin A, gramicidin S, amphotericin (59), and melittin (60) that are proposed to work by either barrel-stave, torroidal pore, or carpet mechanism. The synthetic antimicrobial peptide novispirin (61,62) effects full release of calcein from 5 M lipid at 0.2 M peptide, which is around 2 orders of magnitude more efficient than that of eurocin (63).…”

Section: Discussionsupporting

confidence: 77%

Eurocin, a New Fungal Defensin

Oeemig

Lynggaard

Knudsen

et al. 2012

Journal of Biological Chemistry

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Background: Antimicrobial peptides are new antibiotics avoiding resistance problems. Results: Eurocin is a new antimicrobial peptide featuring a cysteine-stabilized ␣␤-fold. Eurocin binds the cell wall precursor lipid II but does not disrupt cell membranes. Conclusion: Eurocin acts by inhibiting cell wall synthesis. Its structure is typical for invertebrate defensins. Significance: Knowing the mode of action and structure is a prerequisite for pharmaceutical application of an antibiotic.

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Section: Discussionsupporting

confidence: 77%

Eurocin, a New Fungal Defensin

Oeemig

Lynggaard

Knudsen

et al. 2012

Journal of Biological Chemistry

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“…The data probably reflect the importance of the specific amino acid sequence in maintaining the ability of S6 peptide to permeabilize the phospholipid membrane. The efficacy of this S6 segment to permeabilize the lipid vesicles is comparable with the pore-forming peptides or pore- forming segment of a toxin or ion channel protein (35,(62)(63)(64)(65). Furthermore, fluorescently labeled S6 peptide also induced appreciable calcein release (ϳ90% as compared with its unlabeled version) from PC/PG lipid vesicles, suggesting that labeling of the peptide did not alter its activity.…”

Section: Detection Of Localization Of S6 Peptide and Its Analog Into mentioning

confidence: 87%

A Synthetic S6 Segment Derived from KvAP Channel Self-assembles, Permeabilizes Lipid Vesicles, and Exhibits Ion Channel Activity in Bilayer Lipid Membrane

Verma

Malik

Azmi

et al. 2011

Journal of Biological Chemistry

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KvAP is a voltage-gated tetrameric K؉ channel with six transmembrane (S1-S6) segments in each monomer from the archaeon Aeropyrum pernix. The objective of the present investigation was to understand the plausible role of the S6 segment, which has been proposed to form the inner lining of the pore, in the membrane assembly and functional properties of KvAP channel. For this purpose, a 22-residue peptide, corresponding to the S6 transmembrane segment of KvAP (amino acids 218 -239), and a scrambled peptide (S6-SCR) with rearrangement of only hydrophobic amino acids but without changing its composition were synthesized and characterized structurally and functionally. Although both peptides bound to the negatively charged phosphatidylcholine/phosphatidylglycerol model membrane with comparable affinity, significant differences were observed between these peptides in their localization, selfassembly, and aggregation properties onto this membrane. S6-SCR also exhibited reduced helical structures in SDS micelles and phosphatidylcholine/phosphatidylglycerol lipid vesicles as compared with the S6 peptide. Furthermore, the S6 peptide showed significant membrane-permeabilizing capability as evidenced by the release of calcein from the calcein-entrapped lipid vesicles, whereas S6-SCR showed much weaker efficacy. Interestingly, although the S6 peptide showed ion channel activity in the bilayer lipid membrane, despite having the same amino acid composition, S6-SCR was significantly inactive. The results demonstrated sequence-specific structural and functional properties of the S6 wild type peptide. The selected S6 segment is probably an important structural element that could play an important role in the membrane interaction, membrane assembly, and functional property of the KvAP channel.Ion channels belong to a large family of proteins that catalyze the diffusion of inorganic ions down their electrochemical gradients across the cell membranes (1-3). It is known that electric fields exist or arise in living cells and tissues affecting conductance of ion channels (1-3). These ion channel proteins are involved in versatile physiological activities and are responsible for all electrical signaling in living creatures (4 -6). Ion channels have been named according to the name of ions that they allow to diffuse through the pores they form. For example, K ϩ channels are responsible for the diffusion of K ϩ ions and constitute an important class of ion channels (7-9). K ϩ channels modulate the resting potential and action potential duration of neurons, myocytes, and endocrine cells and stabilize the membrane potential of excitable and non-excitable cells (10 -13). K ϩ channels that are activated by membrane depolarization are termed voltage-gated K ϩ (Kv) 4 channels. These Kv channels are tetramers containing six transmembrane segments and one pore region in each monomer. The first four transmembrane segments, S1-S4, form a module that somehow controls the opening and closing of the pore. The fifth (S5) and sixth (S6) transmembrane se...

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“…Liposomes were prepared using the reversed-phase evaporation method, 8 as follows. Aliquots of lipid stock solutions containing egg PC (10 mmol, 7.7 mg) and cholesterol (7.5 mmol, 2.9 mg) or ergosterol (7.5 mmol, 3.0 mg) dissolved in chloroform/methanol (1:2, v/v) were placed in a centrifuge test tube (10 ml; Nichiden-Rika, Kobe, Japan).…”

Section: Liposomesmentioning

confidence: 99%

“…The fluorescence intensity corresponding to 100% release was determined by adding melittin (final concentration, 20 mM). 8 …”

Section: Erythrocyte Suspensionmentioning

confidence: 99%

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Precise Size Determination of Amphotericin B and Nystatin Channels Formed in Erythrocyte and Liposomal Membranes Based on Osmotic Protection Experiments

et al. 2008

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Simultaneous Measurements of K+ and Calcein Release from Liposomes and the Determination of Pore Size Formed in a Membrane

Cited by 45 publications

References 30 publications

Eurocin, a New Fungal Defensin

Eurocin, a New Fungal Defensin

A Synthetic S6 Segment Derived from KvAP Channel Self-assembles, Permeabilizes Lipid Vesicles, and Exhibits Ion Channel Activity in Bilayer Lipid Membrane

Precise Size Determination of Amphotericin B and Nystatin Channels Formed in Erythrocyte and Liposomal Membranes Based on Osmotic Protection Experiments

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