Effects of the cationic antimicrobial peptide eumenitin from the venom of solitary wasp Eumenes rubronotatus in planar lipid bilayers: Surface charge and pore formation activity

Eumenitin, a novel cationic antimicrobial peptide from the venom of solitary wasp Eumenes rubronotatus, was characterized by its effects on black lipid membranes of negatively charged (azolectin) and zwitterionic (1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) or DPhPC-cholesterol) phospholipids: surface potential changes, single-channel activity, ion selectivity, and pore size were studied. We found that eumenitin binds preferentially to charged lipid membranes as compared with zwitterionic ones. Eumenitin is able to form pores in azolectin (G(1) = 118.00 +/- 3.67 pS or G(2) = 160.00 +/- 7.07 pS) and DPhPC membranes (G = 61.13 +/- 7.57 pS). Moreover, cholesterol addition to zwitterionic DPhPC membranes inhibits pore formation activity but does not interfere with the binding of peptide. Open pores presented higher cation (K (+)) over anion (Cl-) selectivity. The pore diameter was estimated at between 8.5and 9.8 angstrom in azolectin membranes and about 4.3 angstrom in DPhPC membranes. The results are discussed based on the toroidal pore model for membrane pore-forming activity and ion selectivity. (c) 2007 Elsevier Ltd. All rights reserved.

Cys mutants in functional regions of Sticholysin I clarify the participation of these residues in pore formation

Experimental evidence shows that the mechanism of pore formation by actinoporins is a multistep process, involving binding of the water-soluble monomer to the membrane and subsequent oligomerization on the membrane surface, leading to the formation of a functional pore. However, as for other eukaryotic pore-forming toxins, the molecular details of the mechanism of membrane insertion and oligomerization are not clear. In order to obtain further insight with regard to the structure-function relationship in sticholysins, we designed and produced three cysteine mutants of recombinant sticholysin I (rStI) in relevant functional regions for membrane interaction: StI E2C and StI F15C (in the N-terminal region) and StI R52C (in the membrane binding site). The conformational characterization derived from fluorescence and CD spectroscopic studies of StI E2C, StI F15C and StI R52C suggests that replacement of these residues by Cys in rStI did not noticeably change the conformation of the protein. The substitution by Cys of Arg(52) in the phosphocholine-binding site, provoked noticeable changes in rStI permeabilizing activity; however, the substitutions in the N-terminal region (Glu(2), Phe(15)) did not modify the toxin`s permeabilizing ability. The presence of a dimerized population stabilized by a disulfide bond in the StI E2C mutant showed higher pore-forming activity than when the protein is in the monomeric state, suggesting that sticholysins pre-ensembled at the N-terminal region could facilitate pore formation. (C) 2011 Elsevier Ltd. All rights reserved.

Selectivity in the mechanism of action of antimicrobial mastoparan peptide Polybia-MP1

Many potent antimicrobial peptides also present hemolytic activity, an undesired collateral effect for the therapeutic application. Unlike other mastoparan peptides, Polybia-MP1 (IDWKKLLDAAKQIL), obtained from the venom of the social wasp Polybia paulista, is highly selective of bacterial cells. The study of its mechanism of action demonstrated that it permeates vesicles at a greater rate of leakage on the anionic over the zwitterionic, impaired by the presence of cholesterol or cardiolipin; its lytic activity is characterized by a threshold peptide to lipid molar ratio that depends on the phospholipid composition of the vesicles. At these particular threshold concentrations, the apparent average pore number is distinctive between anionic and zwitterionic vesicles, suggesting that pores are similarly formed depending on the ionic character of the bilayer. To prospect the molecular reasons for the strengthened selectivity in Polybia-MP1 and its absence in Mastoparan-X, MD simulations were carried out. Both peptides presented amphipathic alpha-helical structures, as previously observed in Circular Dichroism spectra, with important differences in the extension and stability of the helix; their backbone solvation analysis also indicate a different profile, suggesting that the selectivity of Polybia-MP1 is a consequence of the distribution of the charged and polar residues along the peptide helix, and on how the solvent molecules orient themselves according to these electrostatic interactions. We suggest that the lack of hemolytic activity of Polybia-MP1 is due to the presence and position of Asp residues that enable the equilibrium of electrostatic interactions and favor the preference for the more hydrophilic environment.

Interactions of mast cell degranulating peptides with model membranes: A comparative biophysical study

In the last decade, there has been renewed interest in biologically active peptides in fields like allergy, autoimmume diseases and antibiotic therapy. Mast cell degranulating peptides mimic G-protein receptors, showing different activity levels even among homologous peptides. Another important feature is their ability to interact directly with membrane phospholipids, in a fast and concentration-dependent way. The mechanism of action of peptide HR1 on model membranes was investigated comparatively to other mast cell degranulating peptides (Mastoparan, Eumenitin and Anoplin) to evidence the features that modulate their selectivity. Using vesicle leakage, single-channel recordings and zeta-potential measurements, we demonstrated that HR1 preferentially binds to anionic bilayers, accumulates, folds, and at very low concentrations, is able to insert and create membrane spanning ion-selective pores. We discuss the ion selectivity character of the pores based on the neutralization or screening of the peptides charges by the bilayer head group charges or dipoles. (C) 2009 Elsevier Inc. All rights reserved.

Potassium Current in Mature Bovine Spermatozoa

Ion channels have been assigned a pivotal importance in various sperm functions and are therefore promising targets for contraceptive development. The lack of data on channel functionality and pharmacology has hampered this goal. This is a consequence of technical problems of applying electrophysiological techniques to spermatozoa due to their small size and form. By using a laminin coating to increase adherence of spermatozoa and nystatin in the patch pipette for pore formation, we have adapted the whole-cell recording technique to study currents in mature uncapacitated bovine spermatozoa. Employing these conditions, in the head region, patched spermatozoa could be transferred into the whole-cell configuration. For the first time we document an outward rectifying current in mature bovine spermatozoa was blocked by tetraethyl ammonium (TEA) chloride. The observation of a shift in the reversal potential as a response to changes in the extracellular concentration of K+ ions allowed us to identify this current as K+ selective. This result shows that K+ channels in the head region of mature uncapacitated bovine spermatozoa can be suitably investigated using the whole-cell recording patch-clamp technique.