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M Robello

Publications and source records attributed to M Robello.

54 records · Page 3Linked to original sources
PubMed1989

Conductance transition induced by an electric field in lipid bilayers.

A cooperative phenomenon showing a structural change in the organization of bilayer lipid membranes at a critical value of the applied electric field is presented. The transition is characterized by a sharp increase in conductance. The phenomenon can be observed under current-clamp conditions (rather than the usual voltage-clamp conditions) to avoid rupturing the membrane. At a critical potential value the conductance increases and therefore the potential decreases to keep the current constant. Results refer to membranes made of egg phosphatidylcholine (PC), diphytanoylphosphatidylcholine and cholesterol/egg PC. It is found that the critical potential at which the transition occurs depends dramatically on pH and ionic concentration, indicating that the electrical properties of the external surface determine the major characteristics of such a transition.

Cholesterol↗
PubMed1989

Dependence of the conductance of the alpha-latrotoxin channel on applied potential and potassium concentration.

alpha-Latrotoxin, with a molecular weight 130,000, is the main component of black widow spider venom, and acts at the presynaptical level, inducing a notable release of neurotransmitters in the synapses of all vertebrates. In artificial lipid membranes, this neurotoxin induces the formation of cation-selective ionic channels, whose conductance depends on the intensity and direction of the applied potential. In fact, also in the presence of symmetrical solutions of potassium chloride, the voltage-current characteristics of the single channel strongly rectify. Such rectification, which depends on the concentration of the ions in solution, can be described by a one-site, one-ion model for a channel. The data fit provides the values of the three parameters describing the model. Moreover, a statistical analysis of the amplitude of the single channel, as a function of the concentration of potassium chloride, has made it possible to verify the consistency of the model used.

Action Potentials↗
PubMed1989

A transient voltage-dependent outward current in cultured cerebellar granules.

Granule cells were dissociated from rat cerebella with a procedure that yields a 98% pure cell population. Potassium currents in these cells were studied using the patch-clamp technique. Depolarizing pulses of 10 mV step and 100 ms duration from a holding potential of -80 mV elicited two different potassium outward currents: a transient, low-voltage activated component and a long lasting, high-voltage activated component. At +30 mV, the total current reached an amplitude of 2 nA (mean value of 15 experiments). The reversal potential of the transient current, estimated by measuring tail currents, was -77 mV, close to that predicted by the Nernst equation. The transient current was half inactivated with a holding potential of -78 mV and completely inactivated with -50 mV or more positive holding potentials. Finally, the current decay could be fitted by the sum of two exponentials with time constants of about 20 and 250 ms.

Animals↗
PubMed1989

Guanosine-5'-O-(3-thiotriphosphate) modifies kinetics of voltage-dependent calcium current in chick sensory neurons.

Internal perfusion with the G-protein activator guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma S) mimics the effect of noradrenaline and dopamine on the voltage-dependent calcium current in chick dorsal root ganglion (DRG) cells. With 100 microM GTP-gamma S in the pipette, the current at +10 mV was depressed by approximately 50%, with a 10-fold increase of its time to peak. The activation time course of the control calcium current could be approximated with a single exponential curve, whereas with GTP-gamma S the activation time course was double exponential, with time constants tau 1 and tau 2. 2 mM Mg-ATP in the pipette prevented the GTP-gamma S-induced current decrease in 70% of the cells, but the time course of the current was always double exponential. From -50 mV, the current at +10 mV was best fitted with tau 1 = 1.7 +/- 0.5 and tau 2 = 25.6 +/- 5.5 in seven cells. Both time constants decreased with increasing depolarizations. In the first 2 min of recording, the current changed with time. However, both tau 1 and tau 2 were constant, whereas the relative contribution of the slow component increased from 10 to 70%. In addition, the effect was independent of the holding potential in the range from -100 to -30 mV. These results suggest that the activation of a G-protein causes a fraction of the high-threshold calcium channels to switch to a new closed state, with slower opening kinetics.

Action Potentials↗
PubMed1987

Permeation of divalent cations through alpha-latrotoxin channels in lipid bilayers: steady-state current-voltage relationships.

alpha-Latrotoxin, a polypeptide neurotoxin known to cause massive release of transmitter from vertebrate nerve terminals, is thought to act by forming cation-selective channels in plasma membranes. This paper describes the steady-state current carried by Ca2+, Sr2+ and Ba2+ through pores of alpha-LaTx molecules incorporated in artificial bilayer membranes made of neutral lipids. Even when the solutions separated by the membrane are identical, the I-V relations rectify strongly, the current being higher when the side to which the toxin is added is positive. The polarity of the rectification is consistent with the hypothesis that the mechanism of action of the toxin is, at least in part, that of promoting inwardly directed flow of cations, and thus, accumulation of Ca2+ and other ions in the intracellular spaces. The dependence of the I-V characteristics on voltage and Ca2+ concentration is well described by a one-site, one-ion model for a channel. Three parameters of the model are deduced: the binding constant of the site for Ca2+, K = 1.5 M-1 (or K = 7 M-1 when activities are used instead of concentrations); the "electrical" distance of the site from the toxin-containing solution, alpha = 0.3; the free energy difference between the two barrier peaks, delta F = 0.26 kT. The values of the parameters deduced by studying the channel in the presence of Ca2+ give theoretical curves that also fit the data with Sr2+ and Ba2+, indicating a low level of discrimination among these three cations.

Arthropod Venoms↗
PubMed1987

Characterization and some properties of the venom gland extract of a theridiid spider (Steatoda paykulliana) frequently mistaken for black widow spider (Latrodectus tredecimguttatus).

The simplified purification protocol established for the isolation of alpha-latrotoxin from the venom of the spider Latrodectus tredecimguttatus, has been employed for the purification of toxic components present in the venom of the spider Steatoda paykulliana. The venom of this spider, frequently mistaken for L. tredecimguttatus, is by tradition considered to cause an envenomation potentially dangerous to man. The venom of S. paykulliana has little toxic effect on guinea-pigs but is extremely toxic to houseflies (Musca domestica). No proteolytic activity was detectable. Interaction of microgram/ml amounts of the venom extract with artificial lipid membranes produces an increase of membrane conductance through the formation of stable ion-permeable channels modulated by the direction and size of the electric potential differences across the membrane. Higher concentrations of this venom are able to stimulate the release of transmitters from neurosecretory cells in a fashion reminiscent of black widow spider venom. Antibodies against the whole L. tredecimguttatus venom gave a few positive cross-reactions in the immunodiffusion test with S. paykulliana venom gland extract indicating the presence of common molecular sequences in the two venoms. Polyclonal antibodies against alpha-latrotoxin did not cross-react in the immunodiffusion test with S. paykulliana venom extracts, nor in the immunofluorescence assay with its cephalothorax sections, thus suggesting that the venom glands do not contain alpha-latrotoxin. A partial characterization of S. paykulliana venom has been performed and a high molecular weight protein toxic to houseflies has been partially purified.

Animals↗
PubMed1985

Leptinotoxin-h action in synaptosomes, neurosecretory cells, and artificial membranes: stimulation of ion fluxes.

Leptinotoxin-h (LPTx), a neurotoxin (otherwise designated beta-leptinotarsin-h) known to stimulate the release of neurotransmitters from synapses, was purified from the hemolymph of the potato beetle, Leptinotarsa haldemani, by a simplification of the procedure originally developed by Crosland et al. [Biochemistry 23, 734-741, (1984)]. Highly and partially purified preparations of the toxin were applied to guinea pig synaptosomes and neurosecretory (PC12) cells. When applied in a Ca2+-containing Ringer medium, at concentrations in the 10(-11) - 10(-10) M range, the toxin induced: (a) rapid depolarization of the plasma membrane, which was not inhibited by organic blockers of voltage-dependent Na+ and Ca2+ channels (tetrodotoxin or verapamil); (b) large 45Ca influx; and (c) increased free cytosolic Ca2+ concentration. These latter two effects were unaffected by verapamil. In Ca2+-free media the effects of the toxin were different in the two systems investigated. In synaptosomes, depolarization was still observed, even if the toxin concentrations needed were higher (approximately 10X) than those effective in the complete medium. In contrast, in PC12 cells no effect of the toxin on membrane potential was observed. Binding of LPTx to its cellular targets could not be investigated directly because the toxin was inactivated by the procedures used for its labeling. Indirect evidence suggested however that Ca2+ is necessary for toxin binding to PC12 cells. Interaction of LPTx with air/water interfaces, as well as with cholesterol/phospholipid mono- and bilayer membranes was investigated. The results indicate that the toxin has affinity for hydrophobic surfaces, but lacks the capacity to insert across membranes unless transpositive voltage is applied. Our results are inconsistent with the previous conclusion of Crosland et al. (1984), who suggested opening of the Ca2+ channel as the mechanism of action of LPTx. The effects of the toxin resemble those of alpha-latrotoxin (alpha-LTx) of the black widow spider venom, and therefore the two toxins might act by similar mechanisms. However, the sites recognized by the two toxins might be different, because LPTx does not inhibit alpha-LTx binding.

Adrenal Gland Neoplasms↗
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↗
PubMedDate not supplied

Adsorption of gamma-aminobutyric acid to phosphatidylserine membranes.

The interaction of the negatively-charged phosphatidylserine (PS) and gamma-Aminobutyric acid (GABA) is examined in black lipid membranes (BLM) and inverse micelles. GABA does not permeate through PS membranes and, in concentrations of 10(-5)-10(-4) M, it reduces the negative potential at the membrane-aqueous solution interface. The effect is owing to the adsorption of the GABA cationic species and the consequent decrease of the negative surface charge density of the membrane. When the intrinsic pH of the membrane-solution interface is considered, the Gouy-Chapman-Stern theory describes the GABA screening effect and makes it possible to calculate the GABA-PS binding constant. This value is compared with that obtained measuring the partition of 14C-GABA between an organic phase containing PS and the aqueous solution. The results presented strongly suggest that the electrostatic force plays a major role in GABA-PS interaction.

Adsorption↗
PubMedDate not supplied

Modulation by extracellular pH of GABAA receptors expressed in Xenopus oocytes injected with rat brain mRNA.

Rat brain poly(A)(+) mRNA was injected into Xenopus oocytes. After 72-96 hr, GABA(A) receptors expressed in this heterologous system were studied by perfusion of GABA and recording of GABA evoked chloride current under voltage-clamp conditions. The GABA activated currents were blocked by bicuculline and enhanced by flunitrazepam. Acidic (6.4) extracellular pH (pH(e) ) augmented, whereas basic pH (8.4) decreased the current evoked by 100 microM GABA in the respect of the current evoked at pH 7.4. Concentration-response curves for GABA evoked chloride currents were built at the three pHs. These data showed that acidic pH does not change the EC50 for GABA but it increases significantly I(max) in comparison to pH 7.4. At pH 8.4 there was a significant decrease of EC50 for GABA. However, there was also a very strong decrease of I(max), so that the overall effect at 100 microM GABA was a decrease of GABA activated chloride current in the respect of the one activated at neutral pH. These data may indicate that on average brain GABA(A) receptors are positively modulated by extracellular acidosis. The opposite may occur in extracellular alcalosis.

Animals↗
PubMedDate not supplied

Effect of protein kinase C activators on the uptake of GABA by rat brain synaptosomes.

Synaptosomes were prepared from rat brain by a discontinuous Ficoll gradient method and used for studying the uptake of labelled GABA. Two GABA uptake components were evidenced, a high (Km = 3.13 microM) and a low (Km = 92.4 microM) affinity one. Preincubation of synaptosomes with two different activators of protein kinase C, phorbol 12, 13-diacetate (PDAc) and oleyl-acetyl glycerol (OAG), resulted in a change of GABA uptake. In particular, the low affinity component increased its Vmax by 58-74%, with no change in the Km. No statistically significant modification was detected for the high affinity component.

Animals↗