Search PubMed⌕ Search

Biomedical subjects

R A Nogueira

Publications and source records attributed to R A Nogueira.

17 recordsLinked to original sources
PubMed2000

Hurst analysis applied to the study of single calcium-activated potassium channel kinetics.

The gating of ion channels has been modeled by assuming that the transitions between open and closed states is a memoryless process. Nevertheless, analysis of records of unitary current events suggests that the kinetic process presents short-term memory, i.e. the open- and closed-dwell times are short-term correlated. Here the rescaled range analysis (R/S Hurst analysis) is used as a method to test long-term correlation, in single calcium-activated potassium channels present in Leydig cells. The Hurst coefficients, calculated for four different voltages (V) are: 0.634+/-0.022 (n=3) for V=+20 mV; 0.635+/-0.012 (n=4) for V=+40 mV; 0.606+/-0.020 (n=4) for V=+60 mV and 0.608+/-0.026 (n=4) for V=+80 mV. This indicates that open- and closed-dwell times are long-term correlated and do not change with the voltage applied to the patch at a 5% significance level (F=2.2402;p=0.140715). Randomly shuffling the experimental data removes the correlation in all voltages. When the Hurst method was applied to the results from a simulated three-state Markovian model, it could not account for the long-term correlation found in the experimental data. In this case, H has the following values: 0. 5498+/-0.018 (n=100) for V=+20 mV; 0.5557+/-0.0202 (n=100) for V=+40 mV; 0.5565+/-0.0246 (n=100) for V=60 mV and 0.5595+/-0.0247 (n=100) for V=+80 mV. Even a four-state Markovian model was not adequate to correctly simulate the long-term memory found experimentally, with H values significantly different from those found for the experimental data, in the same voltage range (F=15.0355;p=0.00001). In conclusion, this paper shows that: (1) the open- and closed-dwell times of the single calcium-activated potassium channel of Leydig cells are long-term correlated; (2) three- and four-state Markovian models, which describe very well the dwell time distributions, are not adequate to describe the long-term correlation found between the open and closed states of this ion channel.

Animals↗
PubMed1999

Heparin influence on alpha-staphylotoxin formed channel.

The effects of heparin on ion channels formed by Staphylococcus aureus alpha-toxin (ST channel) in lipid bilayers were studied under voltage clamp conditions. Heparin concentrations as small as 100 pM induced a sharp dose-dependent increase in channel voltage sensitivity. This was only observed when heparin was added to the negative-potential side of lipid bilayers in the presence of divalent cations. Divalent cations differ in their efficiency: Zn2+>Ca2+>Mg2+. The apparent positive gating charge increased 2-3-fold with heparin addition as well as with acidification of the bathing solution. 'Free' carboxyl groups and carboxyl groups in ion pairs of the protein moiety are hypothesized to interact with sulfated groups of heparin through divalent cation bridges. The cis mouth of the channel (that protrudes beyond the membrane plane on the side of ST addition and to which voltage was applied) is less sensitive to heparin than the trans-mouth. It is suggested that charged residues which interact with heparin at the cis mouth of ST channels and which contribute to the effective gating charge at negative voltage may be physically different from those at the trans mouth and at positive voltage.

Bacterial Toxins↗
PubMed1998

A novel approach to study the geometry of the water lumen of ion channels: colicin Ia channels in planar lipid bilayers.

This paper describes a new approach to evaluate the inner structure (including a main constriction and its localization) of the water lumen of an ion channel. The method is based on the determination of channel filling by different nonelectrolyte molecules through each side of an ion channel. The method has two characteristic features that make its use attractive: (i) the possibility to ascertain the existence, localization and size of a narrow part inside an ion channel water lumen and (ii) the chances to determine the maximal size of both entrances of an ion channel and to obtain additional information about the geometry of its water lumen at the same time. Determinations were made on colicin Ia ion channels inserted into planar lipid bilayers. This channel was chosen because there is an apparent contradiction between its low single channel conductance and the large diameter of its water lumen. Our results show that the water lumen of the colicin Ia channel has a funnel-like structure with a small trans-entrance, with a diameter of about 1.0 nm, and a large cis-entrance, with a diameter of approximately 1.8 nm. A constriction with a diameter of approximately 0.7 nm is shown to be located close to the trans-entrance of the channel. The method can also be applied to patch clamp studies of single ion channels.

Colicins↗
PubMed1998

Channel-sizing experiments in multichannel bilayers.

The possibility of obtaining information about the radius of high and low conductance states of channels in multichannel membranes was tested experimentally. In spite of the interference of non-electrolytes on the numbers of channels that appeared in the membrane, the non-electrolyte-exclusion method was successfully adapted to multichannel bilayers to estimate the radius of the larger opening of the low conductance state of the channel induced by Staphylococcus aureus alpha-toxin. At the pH used, the channel transition to a low conductance state was accompanied by a decrease of the opening radius from 1.3 +/- 0.2 nm to 0.9 +/- 0.1 nm. The determination criteria for maximum size of a channel opening when using the non-electrolyte exclusion method is discussed.

Electrolytes↗
PubMed1997

Is the mammalian porin channel, VDAC, a perfect cylinder in the high conductance state?

The mammalian porin channel (VDAC, porin-31BM) was reconstituted in planar lipid bilayers under voltage clamp conditions. The radii of both entrances of the channel were examined using a method that consisted in filling the channel with different non-electrolytes through its cis or trans entrances while recording single channel conductances. As a result it was found that the geometry of channels formed by porin-31BM could not be approximated by a perfectly cylindrical pore. In fact there is an asymmetry in the geometry of the channel: the diameters of the cis and trans entrances were estimated to be approximately 2 nm and approximately 4 nm respectively.

Animals↗
PubMed1997

The hinge portion of the S. aureus alpha-toxin crosses the lipid bilayer and is part of the trans-mouth of the channel.

This paper compares the functional properties of ion channels formed in planar lipid membranes by the wild and mutant Staphylococcus aureus alpha-toxin. It was shown that replacement of the amino acid Gly at position 130 by Cys in the primary structure of the toxin decreases the single-channel conductance with a concomitant decrease in the pH at which the channel becomes unable to discriminate between Cl- and K+ ions. The mutation also induced an increase in the asymmetry in the current-voltage relationship of the channel. The results of our experiments suggest that the trans-mouth of the channel is responsible for all the observed changes in channel properties. It was assumed that this entrance is built by the glycine-rich hinge portion of the toxin and is situated close to the surface of monolayer facing the trans-compartment.

Bacterial Toxins↗
PubMed1997

Influence of Cys-130 S. aureus alpha-toxin on planar lipid bilayer and erythrocyte membranes.

Replacement of an amino acid residue at position 130 -Gly by Cys- in the primary structure of Staphylococcus aureus alpha-toxin decreases the single-channel conductance induced by the toxin in planar lipid bilayers. Concomitantly, the pH value at which the channel becomes unable to discriminate between Cl- and K+ ions is also decreased. By contrast, the pH dependence of the efficiency of the mutant toxin to form ion channels in lipid bilayers was unchanged (maximum efficiency at pH 5.5-6.0). The asymmetry and nonlinearity of the current-voltage characteristics of the channel were increased by the point mutation but the diameter of the water pore induced by the mutant toxin, evaluated in lipid bilayers and in erythrocyte membranes, was found to be indistinguishable from that formed by wild-type toxin and equal to 2.4-2.6 nm. Alterations at the "trans mouth" were found to be responsible for all observed changes of the channel properties. This mouth is situated close to the surface of the second leaflet of a bilayer lipid membrane. The data obtained allows us to propose that the region around residue 130 in fact determines the main features of the ST-channel and takes part in the formation of the trans entrance of the channel.

Animals↗
PubMed1997

Pore-forming properties of proteolytically nicked staphylococcal alpha-toxin: the ion channel in planar lipid bilayer membranes.

Staphylococcal alpha-toxin is a single-chain protein with a molecular mass of 33.2 kDa, which can form large water-filled pores both in lipid bilayers and in erythrocyte membranes. Limited proteolysis of the purified toxin with proteinase K led to time-dependent changes of all the functional features of the channels formed by the toxin. Single-channel conductance in planar bilayers was decreased about threefold. The anion selectivity of the channel was replaced with cation selectivity and the asymmetry in the current-voltage relationship of the channel became more pronounced. At the same time the nicked toxin kept its full ability to form ion channels in lipid bilayers, although it lost a considerable part of its hemolytic activity. In planar bilayers and in erythrocyte membranes, the proteolytically nicked toxin actually formed channels with a slightly smaller diameter (approximately 1.2 times) than that formed by the native toxin. This decrease was not marked enough to explain changes in the biological effects of the nicked toxin. The change in channel selectivity induced by the cleavage is considered to be the major determinant of the changes in the biological effects of the nicked toxin.

Animals↗
PubMed1996

Diameter of the mammalian porin channel in open and "closed" states: direct measurement at the single channel level in planar lipid bilayer.

Porin isolated from bovine skeletal muscle was reconstitute in planar lipid bilayers under voltage clamp conditions. A set of non-electrolytes were used as molecular probes for determining the pore diameter. The maximal diameter of the open channel was estimated to be 3.02 +/- 0.26 nm. As observed for other porin channels, a large transmembrane potential drove the channel into a "closed" state. The channel transition to the low conductance (closed) state was followed by a decrease in the maximal diameter of the channel to 2.4 +/- 0.08 nm.

Animals↗
PubMed1995

Non stochastic distribution of single channels in planar lipid bilayers.

The selectivity of the planar lipid bilayers modified by two channel-forming proteins (alpha-toxin S. aureus and colicin Ia) was examined. It was established that in all cases the value of zero current potential depended on the amount of open ion channels and increased with the number of channels (from one to about 5-7). These facts point out both the interactions among ion channels and their non stochastic distribution on the membrane surface.

Bacterial Toxins↗
PubMed1995

Hurst analysis in the study of ion channel kinetics.

Ion channels are protein molecules which can assume distinct open and closed conformational states. The transitions between these states can be controlled by the electrical field, ions and/or drugs. Records of unitary current events show that short open-time intervals are frequently adjacent to much longer closed-time intervals, and vice-versa, suggesting that the kinetic process has memory, i.e., the intervals are correlated in time. Here the rescaled range analysis (R/S Hurst analysis) is proposed as a method to test for correlation. Simulations were performed with a two-state Markovian model, which has no memory. The calculated Hurst coefficients (H) presented a mean +/- SD value of 0.493 +/- 0.025 (N = 100). For the Ca(2+)-activated K+ channels of Leydig cells, H was equal to 0.75, statistically different (1% level) from that calculated for the memoryless process. Randomly shuffling the experimental data resulted in an H = 0.55, not significantly different (1% level) from that found for the two-state Markovian model. For a linear three-state Markovian model, H was equal to 0.548 +/- 0.017 (N = 15), again not significantly different (1% level) from that of the memoryless process. Although the three-state Markovian model adequately describes the open- and closed-time distributions, it does not account for the correlation found in this Ca(2+)-activated K+ channel. Our results illustrate the efficacy of the R/S analysis in determining whether successive opening and closing events are correlated in time and can be of help in deciding which model should be used to describe the kinetics of ion channels.

Animals↗
PubMed1995

The diameter of water pores formed by colicin Ia in planar lipid bilayers.

The effective size of colicin Ia channel was tested by a recently described method (FEMS, Microbiology and Immunology (1992). 105: 93-100) in which the nonelectrolyte molecules with different hydrodynamic diameters (0.52 to 5.0 nm) were used as molecular tools. We have shown that despite low conductance (55-105 pS at 1.5 M KCl, pH 7.0) the ion channels formed by colicin Ia have a fairly large water pore diameter equal to 1.66-1.88 nm. The results are discussed in terms of an energetic barrier for ions passing into the channel lumen.

Colicins↗
PubMed1992

Antigen F1 from Yersinia pestis forms aqueous channels in lipid bilayer membranes.

Antigen F1 is a protein of 17 kDa produced by Yersinia pestis when it is cultured at 37 degrees C. When incorporated into planar lipid bilayer membranes this protein induces fluctuations on membrane conductance typical of the formation of ionic channels. These fluctuations reveal two distinct unitary conductance sizes, one in the range of 800 to 1400 pS and the other in the range of 140 to 600 pS. Zero current potential measurements in the presence of a salt gradient show that the channel is not significantly ion selective. The reversal potential measured in the presence of 0.5 M KCl on the cis side and 0.1 M KCl on the trans side was 3.58 +/- 3.98 mV (N = 7). The non-selectivity of the channel, in addition to its large conductance, suggests that it forms large aqueous pores. The present results, taken together with other data showing that antigen F1 inhibits the activity of phagocytic cells, suggest that antigen F1 acts by forming aqueous pores in the membrane of these target cells.

Antigens, Bacterial↗
PubMed1988

Gating properties of channels formed by Colicin Ia in planar lipid bilayer membranes.

Colicin Ia forms voltage-dependent channels when incorporated into planar lipid bilayers. A membrane containing many Colicin Ia channels shows a conductance which is turned on when high positive voltages (greater than +10 mV) are applied to the cis side (side to which the protein is added). The ionic current flowing through the membrane in response to a voltage step shows at first an exponential and then a linear rise with time. The relationship between the steady-state conductance, achieved immediately after the exponential portion, and voltage is S-shaped and is adequately fit by a Boltzmann distribution. The time constant (tau) of the exponential is also dependent on voltage, and the relation between these two parameters is asymmetric around Vo (voltage at which half of the channels are open). In both cases the steepness of the voltage dependence, a consequence of the number of effective gating particles (n) present in the channel, is greatly influenced by the pH of the bathing solutions. Thus, increasing the pH leads to a reduction in n, while acidic pH's have the opposite effects. This result is obtained either by changing the pH on both sides of the membrane or on only one side, be it cis or trans. On the other hand, changing pH on only one side by addition of an impermeant buffer fails to induce any change in n. At the single-channel level, pH had an effect both on the unitary conductance, doubling it in going from pH 4.5 to 8.2, as well as on the fraction of time the channels stay open, F(v). For a given voltage, F(v) is clearly diminished by increasing the pH. This titration of the voltage sensitivity leads to the conclusion that gating in the Colicin Ia molecule is accomplished by charged amino- acid residues present in the protein molecule. Our results also support the notion that these charged groups are inside the aqueous portion of the channel.

Colicins↗
PubMedDate not supplied

[How can we measure a diameter of each entrance of a ion channel recording only channel conductance?].

The channel formed by colicin Ia in planar lipid bilayer was used to develop a method determining a diameter of each channel entrance. The method is based on measuring of a channel filling with nonelectrolytes through cis- and trans-entrance separately. To do this, one need to establish the two things only: the conductance of an ion channel and the conductivity of used solutions. Using the method to predict the internal geometry of a channel lumen is discussed.

Colicins↗