Biomedical subjects

F Gambale

Publications and source records attributed to F Gambale.

At least 37 records · Page 2Linked to original sources

PubMed1993

Fast and slow activation of voltage-dependent ion channels in radish vacuoles.

The molecular processes associated with voltage-dependent opening and closing (gating) of ion channels were investigated using a new preparation from plant cells, i.e., voltage and calcium-activated ion channels in radish root vacuoles. These channels display a main single channel conductance of approximately 90 pS and are characterized by long activation times lasting several hundreds of milliseconds. Here, we demonstrate that these channels have a second kinetically distinct activation mode which is characterized by even longer activation times. Different membrane potential protocols allowed to switch between the fast and the slow mode in a controlled and reversible manner. At transmembrane potentials of -100 mV, the ratio between the fast and slow activation time constant was around 1:5. Correspondingly, activation times lasting several seconds were observed in the slow mode. The molecular process controlling fast and slow activation may represent an effective modulator of voltage-dependent gating of ion channels in other plant and animal systems.

Biophysical Phenomena↗

PubMed1992

Properties of Pseudomonas aeruginosa exotoxin A ionic channel incorporated in planar lipid bilayers.

Acidic conditions induce the incorporation of Pseudomonas aeruginosa exotoxin A into phospholipid planar bilayers and the formation of pores permeable to electrolytes. Channel openings occur as single events, although they may occasionally cluster in bursts. In 100 mM KCl, the elementary single channel current amplitude is 3.1 pA (at a transmembrane voltage of 100 mV), the mean open time is 1.3 ms, while bursts may last for several seconds. Noise analysis gave results identical to single channel analysis. Voltage pulse protocols and continuous cycling voltage ramps showed that the toxin channel is voltage dependent, having a higher probability of being open at positive voltages.

ADP Ribose Transferases↗

PubMed1991

Lipid interaction of Pseudomonas aeruginosa exotoxin A. Acid-triggered permeabilization and aggregation of lipid vesicles.

We have investigated the interaction of Pseudomonas exotoxin A with small unilamellar vesicles comprised of different phospholipids as a function of pH, toxin, and lipid concentration. We have found that this toxin induces vesicle permeabilization, as measured by the release of a fluorescent dye. Permeabilization is due to the formation of ion-conductive channels which we have directly observed in planar lipid bilayers. The toxin also produces vesicle aggregation, as indicated by an increase of the turbidity. Aggregation and permeabilization have completely different time course and extent upon toxin dose and lipid composition, thus suggesting that they are two independent events. Both time constants decrease by lowering the pH of the bulk phase or by introducing a negative lipid into the vesicles. Our results indicate that at least three steps are involved in the interaction of Pseudomonas exotoxin A with lipid vesicles. After protonation of one charged group the toxin becomes competent to bind to the surface of the vesicles. Binding is probably initiated by an electrostatic interaction because it is absolutely dependent on the presence of acidic phospholipids. Binding is a prerequisite for the subsequent insertion of the toxin into the lipid bilayer, with a special preference for phosphatidylglycerol-containing membranes, to form ionic channels. At high toxin and vesicle concentrations, bound toxin may also induce aggregation of the vesicles, particularly when phosphatidic acid is present in the lipid mixture. A quenching of the intrinsic tryptophan fluorescence of the protein, which is induced by lowering the pH of the solution, becomes more drastic in the presence of lipid vesicles. However, this further quenching takes so long that it cannot be a prerequisite to either vesicle permeabilization or aggregation. Pseudomonas exotoxin A shares many of these properties with other bacterial toxins like diphtheria and tetanus toxin.

ADP Ribose Transferases↗

PubMed1990

Tetanus toxin channel in phosphatidylserine planar bilayers: conductance states and pH dependence.

Tetanus toxin (TeTx) forms ionic channel in phosphatidylserine bilayers. TeTx channels exhibit different modes of channel bursting activity, from a closed state to well defined open states of different amplitudes. At positive applied voltages, TeTx channels flicker continuously between a closed state and the various distinct open states. Furthermore, fast transitions into subconductance states are discernible within the bursts of channel activity. Elementary conductance steps submultiple of the open states were not identified in single channel records owing to rapid transitions between different states. However, statistical analysis shows that conductances cluster with amplitudes multiple of an elementary value: e.g. 25-30 pS at neutral pH. Single channel current amplitudes decrease with the pH of the bulk electrolyte solution. Conductance decrements can be accounted for by the relative decrease of permeant cation concentration at the membrane-water interface, by a relative enrichment of protons that block the channel or by the stabilization of a conformational state of the channel protein.

Hydrogen-Ion Concentration↗

PubMed1990

Voltage-gated sodium channels expressed in the human cerebellar medulloblastoma cell line TE671.

A characterization of the properties of voltage-gated sodium channels expressed in the human cerebellar medulloblastoma cell line TE671 is presented. Membrane currents were recorded under voltage clamp conditions using the patch clamp technique in both the whole-cell and the excised-patch configurations. Macroscopic sodium currents display a typical transient time course with a sigmoidal rise to a peak followed by an exponential decay. The rates of early activation and subsequent inactivation accelerate and approach a maximum in response to test potentials, V, of greater depolarization. The magnitude of peak sodium current increased from negligible values below V = -50 mV and reached a maximum at V = -3.6 mV +/- 2.7 mV (mean +/- S.E.M., n = 12). Sodium currents reversed at V = + 70 mV, near the predicted Nernst equilibrium potential for a Na+ selective channel. The peak sodium conductance, gpeak increased with depolarizing voltages to a maximum at V = approximately 0 mV, exhibiting half-activation voltage at V approximately equal to -36.8 mV and an e-fold change in gpeak/9.5 mV. The Hodgkin-Huxley inactivation parameter h infinity indicates that at V = -73.6 mV half of the sodium currents were inactivated. Single channel current recordings demonstrated the occurrence of discrete events: the latency for first opening was shorter as the depolarizing pulse became more positive. The single-channel current amplitude was ohmic with a slope conductance, gamma = 17.13 pS +/- 0.66 pS. Sodium channel currents were reversibly blocked by tetrodotoxin (TTX).(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗

PubMed1990

Phencyclidine blocks voltage-dependent potassium currents in murine thymocytes.

We investigated the effect of phencyclidine (PCP) on the voltage-dependent K+ current in whole cell recordings from murine thymocytes. PCP caused a dramatic, reversible and dose-dependent decrease of the current. An analysis of the dose-response relationship suggests a single site of action for PCP with an IC50 of 4.7 microM. Dextrorphan and naloxone also inhibited the current, although their effects were of a lower magnitude than those of PCP. Neither 10 microM dextrorphan nor 100 microM naloxone antagonized the inhibitory action of 10 microM PCP. The analysis of the dose-response curve for PCP in presence of 100 microM naloxone suggested that the two drugs act at the same site. We also investigated the effect of morphine on the K+ current. Morphine, in concentrations up to 100 microM, inhibited the K+ current less than dextrorphan or PCP. The kinetics and voltage dependence of the currents in the presence of morphine suggest that it interacts with a different site or different conformation of the channel than the other three compounds. Our findings show that certain opioids can act on thymocytes through a system completely different from the typical opioid receptors.

Animals↗

PubMed1989

Computerized analysis of ionic channel multi-state conductance in planar lipid bilayers.

Ionic transport through cell membranes controls many physiological processes in living organisms. Transport across the cell surface is mediated by membrane proteins (channels), which form hydrophilic pathways permeable to various ions. Also, many synthetic peptides and bacterial toxins form ionic channels when incorporated in lipid bilayer membranes. Changes in boundary physico-chemical conditions and modifications of protein conformation, or sequence, give rise to ionic current signals with different characteristics. A low-cost MS-DOS-based system interfaced to a 12 bit analog/digital/analog (A/D/A) board was used for data acquisition and analysis of square-shaped signals due to the open-closed transitions of single-channel molecules. A dedicated software was developed to investigate the conductance of channels that generate bursts of current, i.e. a sequence of current events due to the rapid flickering of the protein between the open and closed configurations. Up to three current levels present within the same burst can be identified from the best fit of current histogram distribution with the sum of three gaussians. Results obtained from the incorporation of a tetanus toxin channel into phosphatidylserine planar bilayers are reported.

Analog-Digital Conversion↗

PubMed1989

Interaction of tetanus toxin with lipid vesicles. Effects of pH, surface charge, and transmembrane potential on the kinetics of channel formation.

We investigated the interaction of tetanus toxin with small unilamellar vesicles composed of different phospholipids as a function of pH, toxin concentration, temperature, and ionic strength of the solution. Tetanus toxin increased the permeability of the vesicles to fluorescent markers of molecular weight up to 700. The time course of the permeabilization was described as the sum of two exponential components of which the faster accounts for more than 70% of the total effect. Both time constants decreased when the pH of the solution was lowered and when vesicles contained negative lipids. These results can be explained in terms of a phenomenological model based on reaction rate theory. The model assumes that tetanus toxin, after equilibrating with the local pH existing at the surface of the vesicles, inserts into the lipid bilayer forming an ionic channel through which solutes can diffuse. Trigger event for the insertion of the toxin is the protonation, and consequent neutralization of one charged group which makes the molecule more hydrophobic. The intrinsic pK of this group was found to be 3.4 +/- 0.2, suggesting that it may be a carboxyl group. Since the toxin equilibrates with the local pH, the enhancing effect of acidic phospholipids is merely explained by the creation of a negative surface potential which increases the local proton concentration. This was confirmed by the inhibitory effect of high Na+ concentration which reduced the surface charge by screening and specific binding. We found still small differences between the lipids tested and the following order of sensitivity to the action of the toxin: phosphatidylinositol greater than phosphatidylserine greater than phosphatidylcholine approximately cholesterol. The activation energy for the two time constants was found to be 19.8 and 14.8 kcal/mol, fast and slow component, respectively, i.e., slightly larger than that for pure diffusion through the bilayer. The permeabilization induced by tetanus toxin is a voltage-dependent process because vesicles bearing an inner negative potential were depolarized very quickly whereas those bearing an inner positive voltage were barely depolarized at all.

Cholesterol↗

PubMed1988

Membrane action of synthetic N-terminal peptides of influenza virus hemagglutinin and its mutants.

Synthetic peptides corresponding to the N-terminal of the cleaved hemagglutinin (HA2) of influenza virus induce an increase in conductance of planar phospholipid bilayers, and cause the release of encapsulated molecules from large unilamellar liposomes. Two mutant peptides, derived from hemagglutinins of mutant viruses with no or reduced fusion activity, do not alter the membrane conductance significantly. These observations support the hypothesis that influenza virus fuses with its target membrane by inserting the HA2 N-terminal into the membrane.

Amino Acid Sequence↗

PubMed1988

Characterization of the channel properties of tetanus toxin in planar lipid bilayers.

A detailed characterization of the properties of the channel formed by tetanus toxin in planar lipid bilayers is presented. Channel formation proceeds at neutral pH. However, an acidic pH is required to detect the presence of channels in the membrane rapidly and effectively. Acid pH markedly lowers the single-channel conductance, for phosphatidylserine at 0.5 M KCl gamma = 89 pS at pH 7.0 while at pH 4.8, gamma = 30 pS. The toxin channel is cation selective without significant selectivity between potassium and sodium (gamma [K+]/gamma [Na+] greater than or equal to 1.35). In all the lipids studied gamma is larger at positive than at negative voltages. The toxin channel is voltage dependent both at neutral and acidic pH: for phosphatidylserine membranes, the probability of the channel being open is much greater at positive than at negative voltage. In different phospholipids the channel exhibits different voltage dependence. In phosphatidylserine membranes the channel is inactivated at negative voltages, whereas in diphytanoylphosphatidylcholine membranes channels are more active at negative voltages than at positive. The presence of acidic phospholipids in the bilayers increases both the single-channel conductance as well as the probability of the channel being open at positive voltage. A subconductance state is readily identifiable in the single-channel recordings. Accordingly, single-channel conductance histograms are best fitted with a sum of 3 Gaussian distributions corresponding to the closed state, the open subconductance state and the full open state. Channel activity occurs in bursts of openings separated by long closings. Probability density analysis of the open dwell times of the toxin channel indicate the existence of a single open state with a lifetime greater than or equal to 1 ms in all lipids studied. Analysis of intra-bursts closing lifetimes reveals the existence of two components; the slow component is of the order of 1 ms, the fast one is less than or equal to 0.5 ms. The channel activity induced by tetanus toxin in lipid bilayers suggests a mechanism for its neurotoxicity: a voltage dependent, cation selective channel inserted in the postsynaptic membrane would lead to continuous depolarization and, therefore, persistent activation of the postsynaptic cell.

Electric Conductivity↗

PubMed1987

Effects of calcium on the gramicidin A single channel in phosphatidylserine membranes. Screening and blocking.

In phosphatidylserine membranes the decrease in the conductance of the gramicidin A single channel caused by calcium is attributed to a reduction of surface potential and to a direct blocking of the pore (Apell et al. 1979). The aim of this paper is to make a quantitative evaluation of these two effects. We recorded the conductance of gramicidin single channels in 100 mM KCl in the presence of different amounts of CaCl2, MgCl2 or TEACl. The ionic activities at the channel mouth were calculated using the Gouy-Chapman-Stern theory. Our experiments showed that even when the K+ activity at the channel mouth was estimated to be the same, the single channel conductance was lower if divalent cations were present. This effect is attributed to a blocking action of these ions.

Calcium↗

PubMed1986

A microcomputer-based system for data acquisition and analysis of step-like current jumps due to the opening of single ionic channels in model membranes.

At the cell surface, passive transport is controlled by membrane proteins forming hydrophilic pores (or channels) which span the hydrophobic core of the lipid bilayer. Gating mechanisms determine the occurrence of very small and fast step-like current jumps. We developed an intelligent system to acquire and to analyze signals due to the opening and closing of ionic single channels. A specific algorithm allows us to recognize channel current transitions and to discard false events due to noise peaks or to unwelcome fluctuations of the signal. Statistics of the single channel amplitude and mean life-time can be performed. We report the results obtained from analyzing the characteristics of the gramicidin A single channel in phosphatidylserine model membranes. Mean current values of 2.08 +/- 0.01 pA and life-times of 101 +/- 3 ms. were measured at 100mV applied potentials in KC1 100mM solution.

Biological Transport, Active↗

PubMed1984

A thermodynamic interpretation to formation of clusters at the cell surface.

We develop further the original paper by Gershon, concerning the formation of receptor caps at the cell surface. The phenomenon is interpreted in terms of simple thermodynamic factors like boundary tension and supersaturated concentration of membrane components, within the Gibbs free energy change. The hypothesis that the total number of mol receptors is conserved is a sufficient condition to have a minimum of delta G as a function of the radius of the aggregates. We pay particular attention to effects induced by electrostatic interactions among charged receptor molecules. We discuss extensively the simultaneous formation of N aggregates and the role played by either the electrostatic repulsive forces or boundary tension in controlling the size and the number of stable aggregates. Depending on supersaturation of membrane receptors and in the absence of electric charge up to 50% of solute molecules may condense in a cap-like aggregate.

Cell Membrane↗

PubMed1984

GM1 micelles modify the transport properties of the ionophore gramicidin D in artificial planar bilayers.

We have analyzed the effects induced in different phospholipid planar bilayers by monosialoganglioside micelles containing the ionophore gramicidin D. The membrane conductance increases after the addition of GM1 micelles at various ionophore/ganglioside ratios. We believe this fact may be ascribed to gramicidin molecules that incorporate into the bilayer together with gangliosides. In the presence of micelles the mean lifetime and the amplitude of the gramicidin single channel did not present relevant modifications when dioleoylphosphatidylcholine or phosphatidylserine were used to form the bilayer. Calcium proved to trigger the interaction between phosphatidylethanolamine membranes and GM1 micelles containing gramicidin. In this case the ionic pore presents a longer lifetime and a lower amplitude with respect to pure gramicidin. We suggest that different properties developed by gramicidin may depend on structural organization of gangliosides when incorporated into the phospholipid bilayer.

Biophysical Phenomena↗

PubMed1984

Effect of gangliosides on phospholipid bilayers: a study with the lipophilic ions relaxation method.

The presence of monosialoganglioside GM1 in dioleoylphosphatidylcholine black lipid membranes modifies the transport properties of the hydrophobic ion tetraphenylborate and the kinetics of relaxation of this ion after the application of a voltage step. At zero applied voltage, the difference in the relaxation time constants between pure phospholipid and ganglioside-phospholipid mixed membranes is large. This difference may possibly rise from changes in the membranes fluidity since it has been found that the two types of membranes do not show appreciable difference in thickness. A uniform distribution of GM1 in the membrane seems to be more probable than the presence of lateral phase separation phenomena. The partition coefficient of tetraphenylborate between the bathing NaCl solution and the membrane appears to depend on the ionic strength, which controls the screening effect of the Na+ ions on the COO- charged groups of the sialic acid of the ganglioside polar heads. Effects of dipolar potentials on the partition coefficient can be excluded, being the absorption plane of tetraphenylborate probably located outside the dipolar layer of the membrane.

Biological Transport↗

PubMed1983

Capacitance--voltage relationship in phospholipid bilayers containing gangliosides.

Changes in the position of the minimum of the parabolic capacitance-voltage curve allow the measurement of the amount of ganglioside present in artificial bilayers made with phosphatidylcholine-ganglioside mixtures and asymmetrically shielded with Ca2+. The screening effect of the ionic solution must be considered. With ganglioside/phospholipid molar ratios of up to 15%, all glycolipids can be found at the membrane surfaces.

Gangliosides↗

PubMed1982

Properties of ionic transport through phospholipid-glycolipid artificial bilayers.

The ionic transport properties of dioleoylphosphatidylcholine (DOPC) membranes containing various sphingolipids were studied. Particular attention was paid to membranes formed from beta-D-glucosylceramide (GlcCer) and DOPC. They showed a marked increase in ionic permeability (up to a factor 30 with respect to pure DOPC membranes), slight cation selectivity and almost linear behaviour of the current-voltage characteristic. Bilayers containing GlcCer showed a typical conductance decrease upon increasing the temperature. We suggest the formation of clusters containing GlcCer molecules in a solid-crystalline phase. The conductance increase might be due to ionic pathways through disordered boundary regions. An increase in the mechanical breakdown potential was observed in all membranes which contain sphingolipids.

Electric Conductivity↗

PubMed1977

Incorporation into lipid bilayer membranes of a photo--sensitive pigment from the honeybee compound eye.

An increase of electrical conductance up to a factor 10(2)--5-10(2) was obtained by adding, in the dark, the honeybee photopigment to a positively charged lipid bilayer. The increase in conductance was made slower by illuminating the system during the incorporation of the protein into the membrane and it was negligible when the photopigment was bleached before the incorporation. The interaction of the photopigment with the membrane is tentatively interpreted in terms of formation of channels.

Animals↗