Silvia Micelli

Effect of Sterols on β-Amyloid Peptide (AβP 1–40) Channel Formation and their Properties in Planar Lipid Membranes

We investigate the role played by membrane composition on the interaction and self-assembly of b-amyloid peptide (AbP1-40) during pore formation in planar lipid membranes (PLMs). Incorporation studies showed that AbP does not interact with zwitterionic membranes made up of phosphatidylcholine, whereas the addition of cholesterol or ergosterol to the membranes leads to channel formation. Among the PLMs used, a higher propensity of AbP to form channels at low applied potential (620 mV) was observed in 7-dehydrocholesterol and in oxidized cholesterol PLMs. These channels present long lifetimes, high-occurrence frequencies, and are voltage dependent. In particular, the AbP channel in oxidized cholesterol showed anion selectivity. Thus cholesterol (and sterols in general) could be considered as targets for AbP, which prevents the fibrillation process by increasing incorporation into membranes. Furthermore, by switching the channel selectivity versus anions, cholesterol helps to reduce the imbalance of the cellular ions, calcium included, induced by membrane depolarization, which could be one of the factors responsible for cytotoxicity in Alzheimers disease.

Channel formation by salmon and human calcitonin in black lipid membranes.

In this study we investigated the interaction of salmon and human calcitonin (Ct) with artificial lipid bilayer membranes. Both peptides were able to form either transient or permanent channels in the model membranes. The channels formed by salmon Ct at concentration (125 nM) had, on average, a single-channel conductance of 0.58 +/- 0.04 nS in 1M KCl (+10 mV), which is voltage-dependent at lower voltages. Human Ct forms at the same concentration channels with a much lower probability, and high voltages of up to +150 mV were needed to initiate channel formation. The estimated single-channel conductance formed under these conditions was approximately 0.0119 +/- 0.0003 nS in 1 M KCl. Both salmon and human Ct channels were found to be permeable to calcium ions. The possibility is discussed that the superior therapeutic effect of salmon Ct as a tool to treat bone disorders, including Paget disease, osteoporosis, and hypercalcemia of malignancy, rather than human Ct is related to the lack of the fibrillating property of salmon Ct. Preliminary data indicate that also eel and porcine Ct and carbocalcitonin form channels in model membranes.

Kissper, a kiwi fruit peptide with channel-like activity: structural and functional features.

Kissper is a 39‐residue peptide isolated from kiwi fruit (Actinidia deliciosa). Its primary structure, elucidated by direct protein sequencing, is identical to the N‐terminal region of kiwellin, a recently reported kiwi fruit allergenic protein, suggesting that kissper derives from the in vivo processing of kiwellin. The peptide does not show high sequence identity with any other polypeptide of known function. However, it displays a pattern of cysteines similar, but not identical, to those observed in some plant and animal proteins, including toxins involved in defence mechanisms. A number of these proteins are also active on mammalian cells. Functional characterization of kissper showed pH‐dependent and voltage‐gated pore‐forming activity, together with anion selectivity and channeling in model synthetic PLMs, made up of POPC and of DOPS:DOPE:POPC. A 2DNMR analysis indicates that in aqueous solution kissper has only short regions of regular secondary structure, without any evident similarity with other bioactive peptides. Comparative analysis of the structural and functional features suggests that kissper is a member of a new class of pore‐forming peptides with potential effects on human health. Copyright

Magainin 2 channel formation in planar lipid membranes: the role of lipid polar groups and ergosterol

Abstract. Magainin 2, a polycationic peptide, displays bactericidal and tumoricidal activity, presumably interacting with negatively charged phospholipids in the membrane hosts. In this work, we investigate the role played by the lipid head-group in the interactions and self-association of magainin 2 during pore formation in lipid bilayers. Two methods are used: single-channel and macroscopic incorporation into planar lipid membranes. Single-channel incorporation showed that magainin 2 did not interact with zwitterionic membranes, while the addition of negatively charged dioleoylphosphatidylglycerol to the membrane leads to channel formation. On the other hand, magainin 2 did not form channels in membranes made up of dioleoylphosphatidylserine (DOPS), although the addition of ergosterol to DOPS membranes leads to channel formation. This finding could indicate that ergosterol may be a possible target of magainin 2 in fungal membranes. Further support for this hypothesis comes from experiments in which the addition of ergosterol to palmitoyloleoylphosphatidylcholine membranes induced channel formation. Besides the role of negatively charged membranes, this study has shown that magainin 2 also forms channels in membranes lacking heads, such as monoolein and oxidized cholesterol, indicating an interaction of magainin 2 with acyl chains and cholesterol, respectively. This finding provides further evidence that peptide binding and assembly in lipid membranes is a complex process driven by electrostatic and/or hydrophobic interactions, depending on the structure of the peptide and the membrane composition.

Mitochondrial porin incorporation into black lipid membranes: ionic and gating contribution to the total current

We present a new ac device useful for simultaneous measurements of ionic charge movement (conductance) and gating charge displacement (capacitance) in mitochondrial porin channels incorporated in two kinds of black lipid membranes (BLMs), made up of phosphatidylinositol (charged surface) and oxidized cholesterol (neutral surface). In particular, we investigated the conductance/capacitance variations during the process of porin incorporation (VDAC) at different porin concentrations. While conductance variations are present throughout the porin concentration range investigated, a threshold value seems to be necessary in order to detect a significant capacitance variation. A clear steady state in both conductance and capacitance is reached for the phosphatidylinositol bilayer, while for the oxidized cholesterol membranes, the steady state is reached only for the conductance. The dependence of capacitance characteristics on the membrane applied voltage V(m) is investigated before porin incorporation and at the ionic current steady state. The results obtained confirm that before porin incorporation, there is a small dependence on V(m)(2), while afterwards we find evidence of a dual exponential voltage dependence (a result similar to that found for conductance). Finally, we investigated the capacitance dependence on the radius of the hole separating the two compartments of the cell used in the measurements. In this study, performed only with oxidized cholesterol, the radius was varied from 200 to 1050 microm. We observed a significant variation in the specific capacitance in particular for smaller radii. The results were interpreted by a simple geometrical model taking into account the influence of the torus.

Interaction of the aminoglycoside antibiotic dihydrostreptomycin with the H+-ATPase of mitochondria

In this paper a study is presented of the effect of dihydrostreptomycin on the H+-ATPase of the inner mitochondrial membrane. The antibiotic caused at concentrations of 1-5 X 10(-3)M a marked enhancement of the hydrolytic activity of the H+-ATPase complex in intact mitochondria and submitochondrial particles which was accompanied, in the latter, by enhancement of passive transmembrane proton conduction by the complex. The stimulation by dihydrostreptomycin of ATP hydrolysis resulted in a suppression of the sensitivity of this activity to inhibition by oligomycin. On the other hand the dihydrostreptomycin-promoted proton conduction in submitochondrial particles was suppressed by oligomycin. At concentrations above 10(-2)M dihydrostreptomycin caused inhibition of the activity of both membrane bound and isolated H+-ATPase. In submitochondrial particles devoid of the catalytic moiety (F1) of the H+-ATPase complex, dihydrostreptomycin caused partial inhibition of proton conductivity. It is concluded that the antibiotic uncouples the hydrolytic activity of the catalytic moiety (F1) from transmembrane proton conduction by the membrane sector (F0) of the ATPase complex. This effect can be followed at higher concentrations of dihydrostreptomycin by inhibition of the catalytic activity of F1 and, when F1 is removed from the membrane, by inhibition of transmembrane proton conduction by F0.

Serosal and mucosal facilitated transport of urea in urinary bladder of bufo bufo evidence for an alleged water uptake

In Bufo bufo urinary bladder an urea facilitated transport has been localised on the luminal membrane. The transport fulfils the criteria for such a mechanism, i.e. is saturable and is inhibited by phloretin, a specific inhibitor for urea transport. Similarly to that of Bufo marinus and Rana esculenta the luminal membrane of Bufo bufo urinary bladder shows an ADH stimulated facilitated transport. Experiments wtih Amphotericin B, serosal phloretin (with and without ADH), have demonstrated the presence of a facilitated urea transport localised on basolateral membrane. Urea uptake on the isolated epithelial cells of Bufo bufo urinary bladder shows a characteristic feature, different from molecules passively transported such as glycerol yet inhibited by phloretin. Allegedly with urea, water flows in to the cells by a dragging or osmotic effect.

Studies on the Effect of Salts on the Channel Activity of Kissper, a Kiwi Fruit Peptide

Fruits and vegetables are known to possess health benefits: indeed, they contain a large number of bioactive molecules with a positive impact on human health. Dietary proteins possess specific biological properties which make these components potential ingredients of functional or health-promoting foods. Many of these properties are attributed to physiologically-active peptides encrypted in protein molecules. Kissper, a 39-residue peptide isolated from kiwi fruit (Actinidia deliciosa), derives from the processing of kiwellin, a well-represented novel allergenic protein present in the edible part of the fruit. Kissper has six cysteine residues forming three disulfide bridges. This cysteine pattern is similar, but not identical, to those observed in some plant and animal proteins, including toxins involved in defence mechanisms. We show that kissper forms ion channels in DOPS:DOPE:POPC PLMs in different media, such as KCl, potassium gluconate, potassium citrate and potassium phosphate monobasic. Kisspers channels show a strong voltage dependence in all media used, and an estimated pore diameter in the range from 1.9 to 7.6 A in KCl medium. Finally, kisspers pores shift selectivity from anions to neutral to cations, depending on the nature of the salt solutions. Therefore we propose the kissper as a novel nutraceutical component of kiwi fruit that may have a positive impact on human health due to its channel-like pathways which modulate membrane permeability. The kissper action might promote the adsorption of substances, in particular, within the gastrointestinal tract, helping digestion, preventing constipation and harmful accumulation of metabolites.

Effects of n-Octyl-β-D-Glucopyranoside on Human and Rat Erythrocyte Membrane Stability Against Hemolysis

The practical importance for the pharmaceutical and cosmetics industries of the interactions between biological membranes and surfactant molecules has led to intensive research within this area. The interactions of non-ionic surfactant n-octyl-β-D-glucopyranoside (OG) with the human and rat erythrocyte membranes were studied. The in vitro hemolytic and antihemolytic activities were determined by employing a method in which both erythrocytes were added to the hypotonic medium containing OG at different concentrations, and the amount of haemoglobin released was determined. n- octyl-β-D-glucopyranoside was found to have a biphasic effect on both types of erythrocyte membrane. We also investigated the interactions of OG with the erythrocyte membrane in isotonic medium; the dose-dependent curves show similar behaviour in both human and rat erythrocytes. Our results showed that OG has greater antihemolytic potency on rat than on human erythrocytes; furthermore, rat erythrocytes were more sensitive than human erythrocytes to hypotonic shock. How the different lipoprotein structure of these erythrocytes determines a difference in antihemolytic activity is discussed.

Choline Modulation of the Aβ P1-40 Channel Reconstituted into a Model Lipid Membrane

Nicotinic acetylcholine receptors (AChRs), implicated in memory and learning, in subjects affected by Alzheimers disease result altered. Stimulation of α7-nAChRs inhibits amyloid plaques and increases ACh release. β-amyloid peptide (AβP) forms ion channels in the cell and model phospholipid membranes that are retained responsible in Alzheimer disease. We tested if choline, precursor of ACh, could affect the AβP1-40 channels in oxidized cholesterol (OxCh) and in palmitoyl-oleoyl-phosphatidylcholine (POPC):Ch lipid bilayers. Choline concentrations of 5 × 10−11 M–1.5 × 10−8 M added to the cis- or trans-side of membrane quickly increased AβP1-40 ion channel frequency (events/min) and ion conductance in OxCh membranes, but not in POPC:Ch membranes. Circular Dichroism (CD) spectroscopy shows that after 24 and 48 hours of incubation with AβP1-40, choline stabilizes the random coil conformation of the peptide, making it less prone to fibrillate. These actions seem to be specific in that ACh is ineffective either in solution or on AβP1-40 channel incorporated into PLMs.