We investigate the role played by membrane composition on the interaction and self-assembly of beta-amyloid peptide (AbetaP1-40) during pore formation in planar lipid membranes (PLMs). Incorporation studies showed that AbetaP 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 AbetaP to form channels at low applied potential (+/-20 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 AbetaP channel in oxidized cholesterol showed anion selectivity. Thus cholesterol (and sterols in general) could be considered as targets for AbetaP, 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 Alzheimer's disease.
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.
Human Calcitonin (hCt) is a peptide hormone which has a regulatory action in calcium-phosphorus metabolism. It is currently used as a therapeutic tool in bone pathologies such as osteoporosis and Paget's disease. However, due to its amphiphilic property tends to form a gelatinous solution in water which consists of fibrils that limits its therapeutic use. Here we show that sodium dodecyl sulfate (SDS), an anionic detergent able to induce and stabilize alpha-helices in polypeptides, at a monomeric concentration ranging between 0.26 mM-5 pM (all concentrations are below the CMC), increases the rate and number of hCt channel formation in planar lipid membranes, at both high and low hCt concentrations, with a maximum increase at a molecular hCt/SDS ratio of 1000:1. This effect could be interpreted as a counteraction to the fibrillation process of hCt molecules by removing molecules available for aggregation from the fluid; furthermore, this action, independently of channel formation in the cell membrane, could improve the peptide-receptor interaction. The action of SDS could be attributable to the strength of the sulfate negative charge and the hydrophobic chain; in fact, a similar effect was obtained with lauryl sarcosine and not with a neutral detergent such as n-dodecyl-beta-D-maltoside. The very low molecular ratio between SDS and peptide is suggestive of a possible catalytic action of SDS that could induce alpha-helices, the appropriate structures for interacting with the membrane. Moreover, in the experimental conditions investigated, the addition of SDS does not modify the membrane's electrical properties and most of the channel properties. This finding may contribute to the knowledge of environment-folding diseases due to protein and peptides.
Calcium ions (Ca(2+)) are indispensable for life and are involved in important physiological actions, which makes maintaining a constant level of blood Ca(2+) essential. Ca(2+) is mainly stored in bones which serve as a reservoir and its homeostasis is modulated by various hormones. Human calcitonin (hCt) is a small peptide hormone that exerts its physiological effect on Ca(2+) metabolism by means of osteoclast-mediated bone resorption inhibition. Most of these actions are mediated through peptide/receptor interaction that acts via a second messenger. However, in vitro studies have shown that hCt can interact with membrane lipids to form ion channels in membrane models. This ability is due to the peptide's secondary structure and aggregation state, that can be modulated by different molecules. In our study, we evaluated the effect of Ca(2+), at different concentrations, both on the hCt ion channel incorporated into a planar lipid membrane made up of phosphatidylcholine containing 15% phosphatidylglycerol and on the secondary structure of hCt in an aqueous environment. Ca(2+) is able to interact with the hCt peptide by acting on the channel incorporated into the membrane as well as on the peptide in solution, both by increasing hCt channel frequency and in solution promoting α-helix formation, that counteracts the fibrillating process. These experimental observations, suggesting that hCt senses Ca(2+) concentration variations, strengthen the hypothesis that channel formation represents an extra source of Ca(2+) entry into osteoclasts in addition to the well-known interaction of the monomer with the specific receptor.
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