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EFFECT OF DIVALENT CATIONS ON PORIN INCORPORATION IN PLANAR BLM
No full textBiological membranes contain as a basic structural unit a phospholipid bilayer.
Phospholipid membranes in the form of bilayer films (BLM) are currently used as experimental models for transport phenomena of the biological membranes. In particular, bilayers made of acidic phospholipids, present the advantage to investigate the bioelectrical phenomena across the biological membranes.
This class of phospholipids by having a fixed charge might bind ions that play an important role on many physiological processes. It is known that Ca++ and other alkaline cations change the electrostatic potential of the negatively charged bilayer membranes either absorbing to the phospholipids or accumulating in the aqueous diffuse double layer, and this potential change may induce conductance variation (1).
Cd++ another divalent cation has been shown to exert influence on the permeability of some anaelectrolytes across BLM made of negatively charged phospholipids, but not on the zwitterionic phospholipids (2).
We have studied the effect of divalent cations as Ca++ and Ba++ that exert a cross linking together with a screening effect respectively, on the kinetics of incorporation of the mithocondrial porin in bimolecular lipid membranes made with phosphatydilinositol.
In order to understand how lipids of the bilayer affect protein incorporation at the molecular level we must consider that protein crossing the bilayer has to surmount different regions characterized by different properties as interfacial region, head groups, lipid backbone and hydrocarbon core.
In this study we focused our attention to the role of the interface and head group regions of the BLM in the porin incorporation.
The artificial membranes were made of phosphatydilinositol cromatografically pure in n-decane (1% w/w). The bathing media were KC1 1M and KC1 1M plus CaC12 or BaCl2 10 mM. Porin was added at membrane "black" on the two aqueous bathing solutions. Bilayers conductivity and capacitance measurements were carried out at temperature of 25 ± 2 °C and studied by means of alternate current (f = 1 Hz) recording the voltage after a current to voltage converter in series with the membrane; simultaneously electrical capacitance was measured at 1 KHz before and during porin incorporation (3) .
We noticed that when the membranes are in the presence of Ca++ or Ba++ there was an exceptional stability.
With the different bathing solutions no significant differences were observed in membrane conductance values whereas small capacitance variations were recorded.
Once the membrane has became black porin, at a fixed concentration, was added to the aqueous solutions, and after a lag time due to the diffusion of the protein in the solutions and of the time to overcome the membrane barriers, an increase of the voltage output due to the channels formation was recoded. This phenomenon shows a "S-shaped" kinetics. In contrast to the kinetics of incorporation of the porin in oxidized cholesterol lipid membranes (4), showing an hyperbolic shape. These different kinetics may be explained by the different interface barrier; in fact when the experiment were conducted with porin already present in the bathing solutions before membrane formation, the different kinetics persisted in the phosphatydilinositol and in oxidized cholesterol BLM.
The S shaped kinetics persists also when porin is incorporated in phosphatydilinositol membranes in the presence of Ca++ or Ba++; but the curve in presence of the Ca++ is shifted to the right.
This shift is much more pronounced in the presence of Ba++.
It is known that Ca++ affects the surface potential of BLM containing negatively charged lipids. The protein incorporation into BLM can be regarded as a two steps process: I) the interaction at the surface barrier, and II) the penetration into hydrocarbon region.
In the first step the protein encounter an higher resistance in phosphatydilinositol membrane because it has to overcome the interface region consisting of a diffuse double layer, the membrane surface and the polar head groups.
Ca++, by exerting a cross-linking and screening effect on negatively charged membranes, retards the porin incorporation.
At the moment we are not able to distinguish which of the two effect is prevalent; but results in presence of Ba++, that exerts a screening effect only seem to indicate that the latter effect is prevalent. In order to establish the pure cross-linking effect experiments are in progress.
The influence of the divalent cations on the protein incorporation into BLM, that are sensitive to changes in environmental conditions, make this study appealing in order to provide a better knowledge in the molecular mechanism of the protein incorporation and channel formation.
Work supported by 40% grant from MURST 1991
1) S. G. A. McLaughlin, G. Szabo and G. Eisenman, 1971: J. Gen. physiol., 58, 667.
2) S. Micelli, E. Gallucci and C. Lippe, 1978: Arch. Intern. Physiol. Biochim., 86, 755-759.
3) G. Monticelli, E. Gallucci and S. Micelli, 1990: Proc. X School on Biophysics of Membrane Transport, 1: 328-343, Poland.
4) S. Micelli, G. Monticelli and E. Gallucci, 1990: IBST 1: 221
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 KINETICS IN PHOSPHATIDYLINOSITOL AND OXIDIZED CHOLESTEROL MEMBRANES
No full textConductivity of planar lipid bilayer membranes can be modulated by insertion of protein molecules (1).
Mithocondrial porins form water filled pores in the outer membrane and these channels act selectively on the permeation of hydrophilic solutes.
Aims of this research were: 1) the reconstitution of porin channels in planar lipid bilayer membranes, 2) the analysis and the comparison of porin incorporation kinetics in different lipid membranes: phosphatidylinositol (PI) and oxidized cholesterol, 3) the study of protein concentration effect on the incorporation kinetics, 4) the analysis of the channel reconstitution dependence on the applied voltage.
The artificial membranes were formed, with lipid in n-decane (1% w/w), brushing the solution on a small circular hole between two compartments, 4 ml volume, of a teflon experimental chamber. The two compartments were filled with KCl 1M solution and continuously stirred.
Pt electrodes in bathing solutions were connected to a current to voltage converter and to a sinusoidal voltage source (Vs=VS sin2nt; n=1Hz) so that the voltage at the converter (Vl) resulted directly related to the electrical current crossing the membrane (2).
Experiments were performed at room temperature: 24 ± 1 °C.
Membrane electrical conductances and capacitances were higher in oxidized cholesterol than in PI membranes.
Addition of porin to the solutions bathing a completely black membrane induced an increase of membrane current with time indicating protein fusion and channel formation into the lipid matrix.
In PI membranes the time course of current was S-shaped, irrespective of porin addition before or after membrane formation, but in the first case a delay in channel formation and a slower kinetics were observed (3).
In oxidized cholesterol membranes the current time behaviours were S-shaped when porin was added after membrane formation but resulted of exponential type when porin was present before membrane formation. In this second case faster kinetics were observed and the incorporation started with an higher conductance value.
The time lag between porin addition and the first current variation was shorter in oxidized cholesterol than in PI membranes. For a same porin concentration the incorporation kinetics were faster and higher steady levels were reached in oxidized cholesterol than in PI membranes.
In both types of membranes porin incorporation rate decreased lowering porin concentration; five kinetics of porin incorporation (protein added after membrane formation) in oxidized cholesterol membranes are reported in figure.
Besides the porin concentration also the applied voltage affects the incorporation kinetics and the channel formation.
At a given porin concentration faster kinetics are recorded and higher steady levels are reached increasing the voltage to a critical value after which incorporation is less effective, the kinetics are slower and accompanied by lower steady levels.
The voltage dependence of functional pore reconstitution was more evident in PI than in oxidized cholesterol membranes.
In oxidized cholesterol membrane, at the steady state, nonlinear current-voltage characteristics were obtained. The conductance increases with the voltage to reach a critical value after which inactivation occurs.
At a given applied voltage higher membrane conductances were calculated increasing porin concentration.
Significative differences in the current-voltage relations were obtained depending on the absence or the presence of porin at the membrane formation. In this latter case the membranes had higher conductance values but, at the tested voltages, the range of variability was smaller.
The nature of the lipid matrix can be considered responsible of the different protein incorporation; in particular the surface charge of the lipid bilayer membrane and the interface potential, due to the local accumulation of ions at the membrane-solution interface, play a key role in the incorporation of porin in PI planar bilayer lipid membranes.
These observations suggest the possibility to insert protein channels in lipid layers built on a solid substrate finalized to realize organic semiconductors to be used as biosensor or transducers.
Work supported in part by CNR grant 92.02202.CT14.
1) G. Monticelli - "Pore formation in planar lipid bilayer membranes" Proc. X School on Biophysics of Membrane Transport, Szczyrk (Poland) 1990, 1: 315-327.
2) G. Monticelli, E. Gallucci and S. Micelli - "Experimental data on incorporation of porin molecules in lipid bilayers" Proc. X School on Biophysics of Membrane Transport, Szczyrk (Poland) 1990, 1: 328-343.
3) G. Monticelli, E. Gallucci and S. Micelli - "Pore formation in lipid membranes" Proc. Int. Cong. Memb. and Memb. Processes, Chicago 1990, 1: 175-177
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
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
Effect of calcium ions on kinetic of porin pore formation in BL membranes
No full textPorins are a major class of proteins found in the outer membrane of enteric bacteria and mitochondria. These proteins form water-filled transmembrane channels and the pore appears to be formed by a-sheet rather than b-helices. Porin molecules from mitochondria were reconstituted in bilayers of phosphatidylinositol dissolved (1 %) in n-decane and the kinetic of pore formation has been studied by means of alternate current (f = 1Hz). Ionic solutions bathing the membrane were KC1 1 M and the effect of calcium ions (1-10 mM) on pore formation has been tested. After addition of porins to the aqueous phases bathing a completely black membrane the transmembrane electrical current increases with time, the behaviour is S-shaped and the steady state value is reached after many minutes depending on porin concentration. Porin already present in aqueous solutions prior to the formation of the membrane resulted in a delay in channel formation and a slower kinetic was obtained. The rate of porin incorporation decreased in the presence of calcium ions and decreasing porin concentration. The kinetic of pore formation depends on the applied voltage. Experimental data were fitted with a four-parameter logistic equation. In control conditions transmembrane current was calculated considering the changes of membrane electrical resistance and capacitance during porin incorporation
COMPARISON OF PORIN INCORPORATION KINETICS IN PLANAR BLACK LIPID MEMBRANES OF OXIDIZED CHOLESTEROL AND PHOSPHATIDYLINOSITOL
No full textPorin molecules (the mitochondrial outer membrane proteins) when reconstituted into planar lipid bilayer membranes (BLMs) form voltage-dependent channels.
In this work we studied the roles of protein concentration and of the applied voltage on the porin incorporation kinetics in: 1) negatively charged BLMs of phosphatidylinositol (PI), 2) BLMs of oxidized cholesterol (OxCh).
The porin, purified from heart beef mitochondria, was kindly given by Prof. F. Palmieri.
BLMs were obtained as previously reported (Proc. X School Biophys. Memb. Transp. 1:328-343, Poland 1990). Bathing media were KCI 1 M; at membrane black, the porin was added to the stirred aqueous solutions (24±2 °C) and the channel formation studied by means of alternate current.
The conductances and capacitances of BLMs were: PI) 2.27±0.09 μS/cm2, 251.0±9.3 nF/cm2 (n=61); OxCh) 4.77±0.53 μS/cm2, 422.0±23.5 nF/cm2 (n=30).
With PI BLMs the addition of porin yields an S-shaped increase of the current under a fixed set of experimental parameters (applied voltage, salt concentration, temperature).
Besides the porin concentration also the applied voltage affects the incorporation kinetics. We observed that increasing the voltage, between 20-40 mV, the magnitude of the conductance increases too; at lower and higher voltages the kinetics of incorporation are lower. The S-shaped kinetics is preserved at all the porin concentrations and voltages tested.
Porin incorporation kinetics in OxCh membranes follow an hyperbolic function and, as for PI, the incorporation is a function of the porin concentration and of the applied voltage.
The increase and the behaviour were different, in that PI BLMs needed as much as ten times the porin concentration to reach the maximum value of the membrane current as compared to OxCh BLMs.
The differences between the two types of kinetics persist when the protein is already present in bathing solutions before the membrane formation either in PI or in OxCh BLMs.
However the incorporation kinetics in OxCh start with an elevated conductance and continue to increase through hyperbolic curves whereas in PI BLMs the S-shaped kinetics are shifted on the right. It could be assumed that the protein incorporation into the two different BLMs is governed by the different lipid matrix. In particular, the surface charge of the BLM and the interface potential, due to the local accumulation of ions at the membrane-solution interface, play a key role in the incorporation of porin in PI BLMs
Channel reconstitution in planar lipid bilayer membranes
No full textPorins are a major class of proteins found in the outer membrane of gram-negative bacteria and mitochondria. These molecules form water-filled channels and act selectively on the permeation of hydrophilic solutes. Pore forming properties have been studied in reconstitution experiments with planar lipid bilayer membrane. The artificial membranes were formed with phosphatidylinositol (PI) in n-decane brushing the solution on a small circular hole of a teflon cell. PI was extracted from ox brain and porins were purified by mitochondria of different tissues of mammalia. Ionic soluctions were KCl 0.5-1 M and in some experiments CaCl2 1-10 mM was added to both compartments. Porin incorporation has been studied by means of alternate current (f = 1 Hz) recording the voltage after a current to voltage converter in series with the membrane. Electrical capacitance of the system was measured at 1 kHz at some times during porin incorporation. The membrane capacitance resulted 307.5 ± 24.1 (n = 5) and 225.3 ± 3.8 (n = 7) nF/cm2 in KC1 0.5 and 1 M respectively. The electrical resistance was calculated by using data from measurements in alternate current and from direct current measurements too. The addition of porins to the aqueous phases separated by a completely black membrane resulted in an increase of transmembrane current. As time passed the current amplitude increased, indicating protein fusion and pore formation into the lipid matrix. The current time behaviour was S-shaped. A time lag was observed between membrane formation and the first appreciable membrane current variation when porin was already present in aqueous solution prior to the formation of the membrane. This time varied in different experiments and decreased the higher were porin concentration values. Faster incorporation kinetics were observed increasing porin concentration or the applied voltage. The presence of calcium in bathing solution reduced the dependence on the voltage. Experimental data were fitted with a four-parameter logistic function and the different curves compared using the obtained constants (maximum dVl/dt, steady state value, etc.). Analytical time derivative of electrical potential was calculated from the obtained function and the transmembrane current calculated
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