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Channel formation by b-amyloid peptide AbP(1-40) in black lipid membranes made of oxidized cholesterol
No full textBeta-amyloid peptide (AbP1-40) forms a pathway for ion flux across neuronal membranes. When incorporated into a bilayer, AbP forms cation selective channels (Arispe N. et al., 1992, Proc.Natl. Acad. Sci., 90:567-571.). Recent studies have suggested the importance of cholesterol in AbP aggregation and binding to membranes (Wang S. et al., 2001, J.Biol.Chem., 276:42027-42034). In this study, we used a voltage-clamp technique (Stipani V. et al., 2001, Biophys. J., 81:3332-3338) and obtained the same results of Arispe N. et al. in phosphatidylserine:phosphatidylethanolamine (50:50, w:w) membranes; then we extended the investigation to the interaction of AbP with artificial lipid membranes made of oxidized cholesterol. In these membranes, AbP (AbP concentration of 5*10-8 M; 50 mM KCl; ph=7; T=22 +/-2 °C) was able to form channels with different conductance levels, life times and occurrence frequenties. It is worth recalling that oxidized cholesterol membranes contain cholesterol and other products characteristic of aged cholestherol such as 7-dihydrocholesterol wich could enable them to simulate physiologically-aged membranes. These results indicate the importance of cholesterol domains in the incorporation and assemly of AbP in the membrane
Kinetic paremeters of motochondrial porin incorporation into phospholipid membranes with different surface charge
No full textThe kinetics of pore formation by mitochondrial porin in lipid bilayer membranes with different polar heads was investigated. Pore formation was estimated by the total current flowing through the membrane. The time course of the current revealed a "pore activation process" or "cooperative process" and was well described by a novel mathematical model in which two of the four parameters, K1 and K2, are directly related to the processes of pore insertion into and disappearance from the membrane, respectively.
At 1M of KCl, for fixed porin concentration, the K1 value decreases in the following order: phosphatidylserine, phosphatidylcholine membrane, indicating a different affinity of protein to membranes bearing a different polar head.
According to the Guy-Chapman theory, at 0.1 M of KCI, the surface charge of negatively-charged membranes is -137 mV, as compared to -80 mV at 1 M of KCI, and the corresponding K1 value decreased considerably under these experimental conditions; on the other hand, in zwitterionic phospholipid membranes of phosphatidylcholine, the value of K1 remains unmodified.
When diluting the negative charge of phosphatidylserine by the addition of phosphatidylethanolamine (1: 1 w: v), the K1 value increased strongly. The K2 parameter seems to be less influenced by the magnitude of surface potential present on negatively-charged membranes or by the nature of the lipid head.
These results indicate that the electrostatic coupling between porin and the polar head of lipid membranes could play an important role in porin incorporation
Magainin 2 channel formation in planar lipid membranes: the role of lipid polar groups and ergosterol
No full textMagainin 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 selfassociation
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 in different black lipid membranes and its gating mechanism studied by means of alternating current
No full textThe kinetics of pore formation by mitochondrial porin in black lipid membranes of phosphatidylinositol and oxidized cholesterol was investigated as a function of external applied voltage. During the insertion process, the total current passing through the membrane is easily monitored by means of an ac device, and is interpreted as a probe of the process involved during channel incorporation and assembly. The time course of the total current flowing through the membrane was well-described by means of a mathematical model, that gives kinetic parameters describing two concurrent processes which can be interpreted as positive/negative cooperativity.
In the growth rate of pore incorporation we observe a process similar to a phase transition taking place at a critical time, an aspect which has been neglected in most previous investigations of insertion into model membranes.
The behavior around the instant of transition phase is compatible with what is typically expected in other kinetics growth processes. By using data obtained under steady state conditions, with a well-defined number of pores inserted into the membranes, the conductance measurements provided indirect indications of two possible gating mechanisms.
Moreover, our measurement device makes it easy to acquire information on the capacitance characteristics of black membranes. While before porin incorporation we do not observe a significant capacitance dependence on the external applied voltage, afterwards we find evidence of a dual-exponential voltage dependence of capacitance, a result similar to that found for conductance
Different states of assembly of mitochondrial porin in various lipid bilayer membranes
No full textIn natural membranes the large variety of proteins and lipids is correlated with different membrane functions. Although there is evidence that specific non-bilayer-forming lipids are needed for the correct functioning of certain cell proteins, it is not known how lipid properties influence protein functions. Several recent experiments have shown that lipid variations induce changes in the configuration of intrinsic membrane proteins.
In this work, we describe various different assemblies of mitochondrial porin into black lipid membranes. Bilayers, composed of a single type of lipid (phosphatidylinositol in n-decane or oxidized cholesterol in octane-n-decane), are formed in a medium of variable KCl concentration. In phosphatidylinositol membranes, the kinetics of porin incorporation up to the steady state showed sigmoidal curves, indicating cooperative phenomena. The steepness of the curves decreased with a reduction in the ionic strength of the membrane medium. From the best fit to data point with a four- parameter logistic equation, we obtained slope factors or Hill coefficients indicating the formation of different protein assemblies. In oxidized cholesterol membranes, mitochondrial porin seems to be present as a monomer. This configuration was found at both high and low ionic strengths. Although there is much evidence that the aggregation of protein is related to salt concentration (Lin S. et al., Anal Chim. Acta, 383:101-107,1999), given that solubility increases at low salt concentrations (>0.5M), and B-sheet proteins in particular predominantly increase their stability at salt concentrations of >1.5 M (Bandyopadhyay A.K. and Sonawat H.M., Biophys. J., 790.501- 510,2000), our results could indicate a coupling of the protein and lipid polar head
Evidence for a different assembly of Gramicidin A in oxidized cholesterol black lipid membrane
No full textGramicidin A (GA) is a hydrophobic linear pentadocapeptide that forms cation- selective channels in natural and black lipid membranes (BLM). The channels are believed to consist of single-stranded helices with the NH2 terminals linked together by hydrogen bonds. Linear gramicidins require a lipid bilayer to fold properly. If the phospholipid acyl chains are less than eight carbons long, channel folding is not observed; if the acyl chains are too long, the membrane- spanning channels are destabilized. Besides, embedded proteins might influence the molecular packing lipids (R. E. Koeppe II et al. Biol. Skr. Dan. Selsk, 49-93-98, 1998). In our experiments, we chose oxidized cholesterol black lipid membranes because their thickness (40±10 A) (Ti Tien and A. L. Diana, In Chemistry and Physics of Lipids, 2, 1968) is suited to both gramicidin length (25-30 A) and folding. The time course of gramicidin incorporation into BLM, studied by means of alternating current (E. Gallucci et al., Biophys. J. 71:824- 831, 1986), was sigmoidal independently of the KCI concentration used, indicating a cooperative process of incorporation into BLM. By means of a simple mathematical model (S. Micelli et al, submitted) we estimated the tm value, i.e. the parameter that describes a phase transition process (or similar phenomenon). This phenomenon has been observed by AA as a function of temperature and GA concentration (J. A. Killian and B. De Kruijff, Biophys. 1. 53: 111-117, 1988; Mingtao Ge and J. H. Freed, Biophys. J., 76: 266-280, 1999). On the other hand, the average values of the steady-state conductance against gramicidin concentration, for different KCI concentrations, shows a sigmoidal behavior too. These curves were fitted by means of a four-parameter equation which allowed us to obtain the Hill coefficient, D, or slope factor. The Hill coefficient gives the number of single-stranded helices which form the channel. Our results suggest that the number of channel sub-units depends on the KCI concentration. In our case, at a KCI concentration of 1M, the channel is characterized by two sub-units, in accordance with AA's finding (D. U. Urry, Proc. Natl. Acad. Sci. USA, 86:672, 1971, W. R. Veatch and R. Blond, Biochemisty, 13:5249, 1974), while for KCI concentrations of 0.1 and 0.5 M. the channel is formed by more than two sub-units
The role of external applied voltage on kinetic parameters of porin incorporation into black lipid membranes of oxidized cholesterol
No full textThe time course of porin incorporation into black lipid membranes of oxidized cholesterol, as a function of on external applied voltage, was studied by means of an ac method previously reported (E. Gallucci et al., Biophys. J. 71: 824-831, 1996). The kinetic parameters was derived by means of a simple mathematical model before described (D. Meleleo et al., SIF, 1998, Р 53).
At the different voltages Vs examined, the kinetics remained unmodified in its cooperative behavior. The K1 or growth rate parameter describing porin insertion or positive cooperativity seems to be independent of the applied voltage. The K2 or growth rate parameter related to porin deinsertion or negative cooperativity is strongly dependent on applied voltage. In particular K2 decreases with increasing applied voltages (Vs). The v(tm) or value of the maximum depolarization rate, and the Vll(tm) or value of the output voltage when the derivative v(tm) reaches the maximum, both increase with applied voltage (Vs). The kinetic values found indicated that mitochondrial porin is not driven by potential in the membrane, but its insertion depends (for fixed porin concentration, temperature and pH) on the lipid-protein affinity. This result supports previous authors' findings of porin insertion into mitochondrial membranes and asymmetric lipid bilayers (H. Freitag et al., Eur.J. Biochem. 74:483-492, 1993; S.M. Gasser et al.,J. Biol. Chem. 25: 3427- 3430,1983, A. Viese et al., Biophys. J., 70:321-329,1996). On the other hand, as K2 was found to be related with the conductance at the end of porin incorporation, its correlation with Vs reflects the voltage- dependence characteristics of the porin channel
Kinetic parameters of porin incorporation into black lipid membranes of oxidized cholesterol evaluated by means of alternating current
No full textIn this work we report the results of systematical investigation of the time course of porin incorporation into bilayer membranes made of oxidized cholesterol using an ac method (E. Gallucci et al., Biophys. J. 71: 824-831, 1996). This method has the advantages of continuously monitoring resistance and capacitance as a function of time during pore formation until a steady state of the phenomenon is reached. In our case the bilayer membranes are represented, as usual, by an equivalent electrical circuit consisting of one resistance and one capacitance connected in parallel. The total current passing through the membranes is easily monitored, during the insertion process and is interpreted as a probe of the process involved during channel incorporation and assembly. By means of a simple mathematical model, we provide evidence that two concurrent processes are present during the incorporation time course which can be interpreted either as insertion/deinsertion or as positive/negative cooperativity. We observe a phase transition (or similae phenomenon) which seems to take place during the insrtion process, an aspect which has been neglected in most previous investigation of insertion into bilayer membranes. Moreover, the continuous monitoring of capacitance may prove useful in tracking membrane parameters, such as thickness and dielectrical constant, that depend on the lipid and incorporated proteins
Porin incorporation in BLM controlled by surface potencial
No full textPorin incorporation and channel formation in bilayer membranes have been investigated under different incorporation conditions (Gallucci et al Biophys. J. 71 824-831, 1996; Micelli et al., Biophys J.,1998 submitted) We have identified a process of positive/negative cooperativity which can be described by simple mathematical model depending. among others, on two growth rate parameters: K1 describing the insertion or positive cooperativity and K2 describing the negative cooperativity. This model takes into account for the kinetics growth and steady state phase of the insertion, using appropriate data acquisition (dependence of steady state conductance on porin concentration) and analytical techniques, we have provided indirect evidence that the porin channel shows well-defined properties of assambly into the bilayers. In this note we pursue our study in order to answer the following question: are the above-mentioned characteristics dependent on the surface charge ie membrane potential) of the bilayers?
For this reason, using alternating current, we investigate the kinetics of porin incorporation into black lipid membranes (BLM) as a function of ionic strength, the BLM were made up of phosphatidylinositol (PI), oxidized cholesterol (Ox Ch).
Our results indicate that bilayers with higher negative charge density are associated with a slower insertion rate K, and an almost constant negative cooperativity rate K1. These results are corroborated, especially at lower ionic strength, by experiments in which Ba++ is added to the solution as a screening agent. Indeed, the K, parameter for Ox Ch, a neutral bilayer, is higher than that of the PI charged bilayer, and adding Ba++ to Ox Ch does not seem to affect either K1 or K2. Finally, the assembly properties of porin, as evaluated by fitting the data regarding the dependence of steady state conductance Gmss on porin concentration for three different ionic strength (0.1, 0.5 and 1 M), seem to be dependent on the ionic strength and membrane substrat
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