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H Duclohier

Publications and source records attributed to H Duclohier.

At least 19 recordsLinked to original sources
PubMed1992

Prolines are not essential residues in the "barrel-stave" model for ion channels induced by alamethicin analogues.

In the "barrel-stave" model for voltage-gated alamethicin channels in planar lipid bilayers, proline residues, especially Pro14, are assumed to play a significant role. Taking advantage of a previous synthetic alamethicin analogue in which all eight alpha-aminoisobutyric acids were replaced by leucines, two new analogues were prepared in order to test the effects of Pro14 and Pro2 substitutions by alanines. The alpha-helical content of the three analogues in methanol solution remains predominant (between 63 and 80%). Macroscopic conductance experiments show that a high voltage dependence is conserved, although the apparent mean number of monomers forming the channels is significantly reduced when the substitution occurs at position 14. This is confirmed in single-channel experiments which further reveal faster fluctuations for the modified analogues. These results demonstrate that, although prolines, especially Pro14, are favorable residues for alamethicin-like events, they are not absolute prerequisites for the development of highly voltage-dependent multistate conductances.

Alamethicin↗
PubMed1992

Membrane-modifying properties of the pore-forming peptaibols saturnisporin SA IV and harzianin HA V.

Harzianin HA V and saturnisporin SA IV are alpha-amino isobutyric-containing peptides with 18- and 20-residue chain length, respectively. They were isolated from in vitro cultures of Trichoderma species and their sequences were determined by the combined use of positive ion FAB mass spectrometry and NMR. In organic solvent solution, both peptides exhibited the same predominant alpha-helical secondary structure including a hinge at the level of the central Pro residue, as deduced from NMR data. Their interaction with neutral phospholipid bilayers was shown to induce leakage of the material entrapped in small unilamellar vesicles composed of egg phosphatidylcholine/cholesterol (7/3). When incorporated into neutral planar lipid bilayers, they promoted voltage-gated channels. The concentration- and voltage-dependences of the ionic conductances induced by these peptides were studied in macroscopic current-voltage experiments. Single-channel measurements showed that whilst SA IV developed non-integral multi-open states similar to those induced by alamethicins, but with faster kinetics, the shorter analogue, HA V promoted much smaller-sized conducting aggregates in agreement with macroscopic conductance data.

Amino Acid Sequence↗
PubMed1991

Synthetic analogues of alamethicin: effect of C-terminal residue substitutions and chain length on the ion channel lifetimes.

In a previous study, a synthetic analogue of the peptaibol alamethicin, in the sequence of which all alpha-aminoisobutyric acid (Aib) were substituted by leucine residues and the C-terminal residue modified, was shown to display the same single-channel behaviour as alamethicin in planar lipid bilayer, except that the sublevel lifetimes were much reduced. New analogues differing in their C-terminal residue (Phe-NH2, Pheol, Trp-NH2) have now been tested for their single channel properties in neutral lipid bilayers. The conductance amplitudes and open channel lifetimes do not differ significantly from the previous analogue. Thus, the nature of the last residue, which may be located near the membrane interface, does not seem to play an important role in the destabilisation of the conducting aggregate observed after the Aib substitution by Leu. Since the deletion of one residue (Glu18) in the 14-20 moiety induces a slight decrease of the increment between the conductance levels, but has no effect upon the channel lifetimes, this residue and the length of this segment do not interfer much with the channel lifetime of peptaibols. In conclusion the factors influencing the aggregate stability may be sought in the helix-helix interactions.

Alamethicin↗
PubMed1991

[Purification of sodium channel from squid mantle and reincorporation in planar lipid bilayers].

The purification of voltage-dependent sodium channels from mantle muscles of the squid is monitored through increase in the specific binding of tritiated saxitoxin (3H-STX) of membrane extracts solubilized with Lubrol-PX, chromatographed over an ion-exchange resin then through an affinity column coupling lectins (WGA) and Sepharose. The achieved purification factor is 300 and the electrophoretic pattern of purified fractions shows a single band at 260 kD. Reincorporation of these fractions into planar lipid bilayers yields two conductance levels (in 0.5 M NaCl). The most frequently observed level, at 15 pS, is in agreement with in situ recordings. The other level, at 150 pS, equally blocked by tetrodotoxin, could be explained by some flexibility of the open state or alternatively by cooperative interactions between channels.

Animals↗
PubMed1989

The influence of the trichorzianin C-terminal residues on the ion channel conductance in lipid bilayers.

Four natural trichorzianin analogues, channel-forming peptaibols, differing in their C-terminal residues (Gln or Glu, Trpol or Pheol) were tested for their macroscopic and single-channel conductances in planar lipid bilayers. The results indicate that, as regards to the voltage threshold, the most efficient analogue is the charged Trpol-bearing one. In addition, Trpol brings about a drastic lengthening of the open channel life-times. This behaviour is attributed to the dipole moment of the end residues and to the bulkiness and hydrogen bonding ability of Trpol.

Amino Acid Sequence↗
PubMed1989

Design and conformation of non-Aib synthetic peptides enjoying alamethicin-like ionophore activity.

Analogues of alamethicin, a 20-mer amphipathic helical peptide with ionophore activity, in the sequence of which all Aib residues were substituted by Ala (A1) or Leu (L1), were synthesized by the solid phase method, purified by high performance liquid chromatography and characterized by fast atomic bombardment mass spectrometry. Infrared and CD studies showed that A1 easily underwent a transconformation to beta-structure whereas L1 displayed a predominant alpha-helical character, thus being a potential ionophore model. Its voltage-dependent multistate activity in model membranes showed that Aib is not a requisite residue to observe an alamethicin-like behavior. However, as the lifetime of the single channels was much shorter than for alamethicin, the peptide chain was lengthened by a Leu (LL1) or a Ser (SL1) residue. The last peptide gave an increased channel lifetime, but the design of other non-Aib peptides, taking into account the hydroxyl C-terminus and side-chain interactions between helices in a barrel-stave bundle, is desirable to approach more closely the alamethicin activity.

Alamethicin↗
PubMed1989

Structure and supramolecular architecture of membrane channel-forming peptides.

Peptides gathering together to induce channels in lipid bilayers may be classified in several categories according to the spatial structures involved. For example, gramicidin A forms intramolecular tubes, alamethicin, bundles of helical rods with intermolecular pores, porins (being proteins, properly speaking) are rich in beta-sheets that may form barrels, whereas cyclic peptides might stack together resulting in the formation of pores. The chemical structure of these compounds is now well characterized. The transmembrane electrical signals that they transmit are also typical of the particular supramolecular configurations (or architecture). Investigations in this field are thus relevant to structure-function relationship studies due to the availability of natural or synthetic analogues allowing the measurement of the influence of physico-chemical parameters upon the energy profiles of the pores. Consequently, questions such as the existence and probabilities of conductance substrates, their voltage-dependence and their ion or molecular selectivity can be tackled. Today, the loosest aspect of these studies lies in the actual molecular conformations and architecture in the membranes of the peptide aggregates, the knowledge of which remains imprecise, even 'at rest' in the best-studied cases. This review attempts to point out still unresolved questions and to propose some plausible approaches concerning, for example: 1) the configurations of the molecular aggregates responsible for ion transfer; 2) the mechanisms for channel-opening and closing (gating); 3) the eventual cooperative phenomena between channels, via the bilayer or interfacial components. Possible applications of these structures will be tentatively outlined.

Alamethicin↗
PubMed1989

Antimicrobial peptide magainin I from Xenopus skin forms anion-permeable channels in planar lipid bilayers.

The ionophore properties of magainin I, an antimicrobial and amphipathic peptide from the skin of Xenopus, were investigated in planar lipid bilayers. Circular dichroism studies, performed comparatively with alamethicin, in small or large unilamellar phospholipidic vesicles, point to a smaller proportion of alpha-helical conformation in membranes. A weakly voltage-dependent macroscopic conductance which is anion-selective is developed when using large aqueous peptide concentration with lipid bilayer under high voltages. Single-channel experiments revealed two main conductance levels occurring independently in separate trials. Pre-aggregates lying on the membrane surface at rest and drawn into the bilayer upon voltage application are assumed to account for this behaviour contrasting with the classical multistates displayed by alamethicin.

Amino Acid Sequence↗
PubMed1988

Intracellular pH change does not appear as a prerequisite for triggering activation of Barnea candida (Mollusca, Pelecypoda) oocytes.

Barnea candida oocytes, exposed to excess KCl, ammonia, or digitonin, exhibit germinal vesicle breakdown (GVBD) and reinitiate meiosis, at least up to first polar body extrusion. While we confirm that KCl- but not ammonia-induced activation required external calcium, our findings that digitonin is effective at any pH from 6 to 8, in the presence of calcium, while the phorbol ester TPA and diacylglycerol fail to reinitiate meiosis, strongly suggests calcium as the main trigger for this process. Preliminary experiments using the fluorescent probes fluorescein diacetate and Quin 2/AM show, moreover, that KCl and ammonia produce both an intracellular calcium surge (30 nM) and a slight alkalinization of the intracellular cytoplasm from 7.84 to 8.05.

Animals↗
PubMed1988

Orientation of the tryptophans responsible for the photoinactivation of nerve sodium channels.

UV irradiation of squid giant axons at wavelengths of 280 or 290 nm produces nearly the same rate of irreversible decrease of sodium currents. The rate of photodeactivation is unaffected by extensive removal of axoplasm with pronase, and it is independent of temperature in the range 5 degrees to 20 degrees C. The photochemical effect appears to be all or nothing. It does not alter the time course and the voltage dependence for activation and inactivation of the residual currents. Similar deactivation rates were produced by irradiations of the same intensity, but linearly polarized either parallel or perpendicular to the axon. The efficiency of the deactivation process is close to that expected if it was caused by the photooxidation of a single tryptophan residue per sodium channel. Owing to the geometry of the preparation the lack of polarization asymmetry suggests that this residue assumes nearly random (or pseudo-random) orientation in the three-dimensional structure of the sodium channel corresponding to the closed state.

Animals↗
PubMed1988

Ionophore properties of a synthetic alpha-helical transmembrane fragment of the mitochondrial H+ ATP synthetase of Saccharomyces cerevisiae. Comparison with alamethicin.

A 22-amino acid polypeptide was synthesized to model the central transmembrane segment of subunit 8 of the H+ ATP synthetase of Saccharomyces cerevisiae and to test ionophore properties. Solid-phase synthesis was conducted on benzhydrilamino resin, and purification followed by high pressure liquid chromatography allowed the isolation of the pure product whose NH2 terminal was acetylated and whose molecular weight determined by Fast Atomic Bombardment was the expected 2,666. The infrared spectrum of this peptide in the solid state reveals a fully alpha-helical conformation, whereas in low dielectric constant solvents the alpha-helical content is 60%, as determined by circular dichroism studies. Macroscopic current-voltage curves displayed by different planar lipid bilayers (monomyristoleoyl-glycerol and phosphatidylethanolamine) doped with this peptide suggest a weakly voltage-dependent conductance. Only one conductance level is observed in any given single-channel conductance experiment. However, a series of experiments shows a distribution of conductance states, most often 440 or 3,000 pS, and occasionally 80, 1,200, or 6,500 pS. This behavior contrasts with the usual behavior of alamethicin, chosen as a model of "aggregating-helices" ionophore and whose conductance fluctuates continually between substates, through uptake and release of monomers. Nevertheless, alamethicin too can display, under certain conditions, long-lived and mono-level conductance states similar to those reported here for the newly synthesized peptide. These properties could possibly be explained by the formation of large domains of helical rods with a set of allowed and independent ionic pathways.

Alamethicin↗
PubMed1987

Voltage-dependent and multi-state ionic channels induced by trichorzianines, anti-fungal peptides related to alamethicin.

The ionophore properties of two peptaibols of the trichorzianine family have been investigated in planar lipid bilayers and compared to those of alamethicin. Macroscopic conductance experiments reveal voltage-dependent channels only in the thinnest membranes and a greater efficiency of the neutral analog. In single-channel experiments, a multi-state behaviour, consistent with the usual barrel-stave model, is disclosed but the discrete current fluctuations are much more rapid than for alamethicin. The results indicate a stringent requirement for the helix length/bilayer thickness match in agreement with a previous model and suggest the design of new synthetic peptides.

Alamethicin↗
PubMed1987

Single chloride-permeable channels of large conductance in cultured cardiac cells of new-born rats.

Large conductance channels were observed in the membrane of cultured cardiac cells of newborn rats studied with the patch-clamp technique in cell-attached and inside-out configurations. These channels were observed in approximately equal to 4% of the patches. In the cell-attached configuration they exhibited outward rectification and partial inactivation. In the inside-out configuration no rectification occurred but inactivation was present, mainly during hyperpolarizations. Two channels with large single unit conductances (400-450 pS) and one with a smaller conductance (200-250 pS) were frequently observed in the same patch. The two large channels generally had different kinetics. Under steady-state conditions the opening probability of the faster channel appeared to be voltage-independent. The slower channel was activated by depolarization. In asymmetrical solutions the permeability ratios PNa/PCl were 0.03 and 0.24 for the larger and smaller channels, respectively; corresponding values for PBa/PCl were 0.04 and 0.09. It is proposed that in cardiac membranes the chloride permeability system is composed of widely dispersed microclusters forming grouped channels of different types and sizes.

Animals↗
PubMed1983

[Relation between the axonal membrane microfluidity and excitability].

The olfactory nerve of the garfish, the rabbit vagus nerve and the sciatic nerve of the frog labelled with pyrene or triethylammonium-butyl-pyrene show during the action potential a transient decrease in the Ie/Im ratio which suggest a small transient decrease in nerve membrane fluidity.

Animals↗
PubMed1979

Temperature dependence of the fluorescence of pyrene labeled crab nerve membranes.

A method, using albumin-pyrene complexes, has been developed for labeling, in a controlled manner, crab leg nerves whose excitability was preserved. The excimer-to-monomer fluorescence intensity ratio of pyrene, embedded in nerve membrane lipids and in their crude lipid extracts, is a fluidity parameter which displayed the following features with temperatures. a--a temperature-dependent increase of fluidity b--three breaks (6 degrees, 19 degrees and 37 degrees C) in the physiological medium c--In Ca++-depleted sea water, the 37 degrees characteristic temperature vanished. These breaks may reflect some lateral phase separations of the lipid components of nerve membranes. The calcium dependent temperature break may involve a segregation of acidic phospholipids while the other two breaks (6 degrees and 19 degrees C) may be due to neutral lipids phase separation. The relationship of these findings to nerve function is discussed.

Animals↗