Biomedical subjects

H Duclohier

Publications and source records attributed to H Duclohier.

At least 37 records · Page 2Linked to original sources

PubMed1997

Investigating synthetic P-regions from voltage-gated sodium channel at the conformational and functional levels.

Four peptides mimicking the four P-regions of the electric eel sodium channel were chemically synthesized to characterize their secondary structure and their contribution to the channel selectivity. Circular dichroism spectra of these peptides in trifluoroethanol demonstrate an important beta-sheet conformational component. This beta-sheet content is much enhanced upon interaction with phosphatidylcholine small unilamellar vesicles. As expected (and except for P of domain III), no significant voltage-dependence is revealed in either macroscopic or single-channel conductance experiments. The concentrations-dependences of macroscopic conductances suggest that tetramers are the membrane conducting aggregates. In asymmetric ionic conditions, these channels made up of P-peptides were mostly specific for sodium over chloride whilst caesium was largely excluded. Single-channel conductance analysis discloses a moderate selectivity for sodium over potassium for PI and PII. This selectivity is larger with PIII but inverted for PIV. Finally, a control random peptide of the same length and with a comparable mean hydrophibicity was also tested. Its conformation in TFE is mainly unordered and no activity was detected in planar lipid bilayers. The data suggest that the presumed selectivity filter may not assume a circular symmetry and that molecular recognition between the different P-regions has to be taken into account.

Amino Acid Sequence↗

PubMed1997

Influence of proline position upon the ion channel activity of alamethicin.

Alamethicin, a 20-residue peptaibol, induces voltage-dependent ion channels in lipid bilayers according to the barrel-stave model. To study relationships between the proline-14-induced kink region and the channel-forming behavior of the peptide, a set of alamethicin analogs with proline incorporated at positions 11, 12, 13, 14, 15, 16, and 17, respectively, as well as an analog with alanine instead of proline at position 14 were synthesized. Macroscopic conductance experiments show that the voltage dependence of the peptides is conserved although slightly influenced, but the apparent mean number of monomers forming the channels is significantly reduced when proline is not located at position 14. This is confirmed in single-channel experiments. The analogs with proline next to position 14 (i.e., 13, 15, 16) show stable conductance levels, but of reduced number, which follows the order Alam-P14 > Alam-P15 > Alam-P16 > Alam-P13. This reduction in the number of levels is connected with changes in the lifetime of the channels. Analogs with proline at position 11, 12, or 17 produce erratic, extremely short-lived current events that could not be resolved. The changes in functional properties are related to structural properties as probed by circular dichroism. The results indicate that proline at position 14 results in optimal channel activity, whereas channels formed by the analogs bearing proline at different positions are considerably less stable.

Alamethicin↗

PubMed1996

The inositol 1,4,5-trisphosphate-gated Ca2+ channel: effect of the protein thiol reagent thimerosal on channel activity.

The solubilized partially purified Ins(1,4,5)P3-sensitive Ca2+ channel from rat cerebellum has been reconstituted into planar lipid bilayer membranes by the 'tip-dip' method [Ehrlich (1992) Methods Enzymol. 207, 463-471] allowing low noise current records. Single-channel events have been recorded. In the presence of 10 microM Ins(1,4,5)P3, 50 microM ATP, and 0.2 microM Ca2+ the Ins(1,4,5)P3 receptor channel opens to a conductance level of 53 pS. In the presence of 100 microM thimerosal (TMS), a sulphydryl-oxidizing agent, three subconductance levels (60 pS, 80 pS and 120 pS) were observed. More than one population of mean open times was found, both in the absence and presence of TMS, although TMS affected the length of the open time by decreasing the short opening significantly from 4.05 ms to 2.78 ms and increasing the longer open time from 27.8 ms to 94.8 ms. The results indicate that TMS enhances Ins(1,4,5)P3-induced Ca2+ release by both altering the open times of the channel significantly and causing a shift to higher subconductance levels.

Adenosine Triphosphate↗

PubMed1996

Polarity-dependent conformational switching of a peptide mimicking the S4-S5 linker of the voltage-sensitive sodium channel.

The S4-S5 linker (or S45) in voltage-sensitive sodium channels was previously shown to be involved in the permeation pathway. The secondary structure, investigated by circular dichroism, of a S4-S45 peptide from domain IV and its fragments (including S45) is reported here and compared with that of the homologous peptide from domain II as a function of the solvent dielectric constant. The reduction in helicity seen for S4-S45 (II) in polar media is cancelled in membrane-like environment. The most striking result-- a sharp alpha-helix --> beta-sheet transition upon exposure of the S45 moiety to aqueous solvents-- is discussed as regards channel activation and selectivity.

Amino Acid Sequence↗

PubMed1996

Lateral diffusion in planar lipid bilayers: a fluorescence recovery after photobleaching investigation of its modulation by lipid composition, cholesterol, or alamethicin content and divalent cations.

In spite of the fact that planar lipid bilayers are still the best-suited artificial membrane system for the study of reconstituted ion channels and receptors, data dealing with their physical characterization, especially as regards dynamics, are scanty. A combined electrical and optical chamber was designed and allowed fluorescence recovery after photobleaching recovery curves to be recorded from stable virtually solvent-free bilayers. D, the lateral diffusion coefficient of N-(7-nitrobenzoyl-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn- glycero-3-phosphoethanolamine, was found to be relatively insensitive to the phospholipid composition (headgroup, chain unsaturation, etc.), whereas inclusion of 33-50% cholesterol in the membrane reduced D by a factor of 2. Divalent cations significantly reduced D of negatively charged bilayers. These results compare well with data gathered on other model and natural systems. In addition, the incorporation of the voltage-dependent pore-former alamethicin did slightly reduce lipid lateral mobility. This study demonstrates the feasibility of such experiments with planar bilayers, which are amenable to physical constraints, and thus offers new opportunities for systematic studies of structure-function relationships in membrane-associating molecules.

Alamethicin↗

PubMed1996

Ion channel stabilization of synthetic alamethicin analogs by rings of inter-helix H-bonds.

Rings of inter-helix H-bonds due to Gln at position 7, a highly conserved residue in all pore-forming peptaibols, have been suggested to play an important role in the stabilization of alamethicin channels. In an attempt to test this hypothesis, experimental studies have been undertaken on four synthetic alamethicin non-Aib analogs (Alm-dUL) in which the Gln at position 7 (Q7) is substituted by Ala, Asn, or Ser (Q7A, Q7N, or Q7S). Voltage-dependent pore formation by these analogs in planar lipid bilayers is compared at the macroscopic and single-channel conductance levels. As anticipated, the Q7A substitution abolished all channel-forming activity. The voltage dependence of macroscopic current-voltage curves was conserved with the Q7N substitution but reduced in the Q7S analog. Normalized single-channel conductance ratios between substates follow the same pattern, with the Q7S analog yielding the highest unit conductances. Channel lifetimes were the most significantly modulated parameter with markedly faster kinetics when Gln or Asn was replaced by Ser. The effect of the Q7S substitution on channel lifetimes may be explained through a reduced stabilization of bundles by inter-helix H-bonds.

Alamethicin↗

PubMed1995

Conformation, pore-forming activity, and antigenicity of synthetic peptide analogues of a spiralin putative amphipathic alpha helix.

Current models depict spiralin as a bitopic transmembrane protein with the transbilayer domain being an amphipathic alpha helix. However, though secondary structure prediction methods suggest a helical conformation for the hypothetical transmembrane segment of spiralin, no potential transmembrane helices could be detected in this protein using the method of Von Heijne (Von Heijne, G. (1992) J. Mol. Biol. 225, 487-494). Therefore, we have reconsidered the spiralin topological model by investigating the properties of the chemically synthesized peptides SM-BC3 (LNAVNTYATLAKAVLDAIQN-NH2) and SC-R8A2 (LNAVNTYATLASAVLEAIKN-NH2), corresponding to the hypothetical transmembrane segments of spiralins of two distinct spiroplasma species. The hydrophobic moment plot method suggests that these spiralin amino acid stretches are class G amphipathic alpha helices (i.e., helices localized on the surface of a globular protein domain). Circular dichroism spectra showed that both peptides have little ordered structure in aqueous solutions but adopt a mainly helical conformation in the presence of 25% trifluoroethanol or in detergent micelles (up to 74% alpha helix). Both peptides formed concentration- and voltage-dependent pores in planar lipid bilayers with a unitary conductance of 130 pS in 1 M KCl and with mean numbers of monomers per conducting aggregates of 6 for SC-R8A2 and 9 for SM-BC3. However, the two peptides displayed a haemolytic activity only at high concentrations (> 250 microM) and reacted with antibodies raised against membrane-bound spiralin. Together with previously published results, these data suggest that spiralin is a monotopic membrane protein anchored at the surface of the spiroplasma cell and that the 20-residue amphipathic segment is most probably a class G helix containing a B-cell epitope.

Amino Acid Sequence↗

PubMed1995

Influence of proline-14 substitution on the secondary structure in a synthetic analogue of alamethicin.

Due to the bend introduced by proline 14 in the conformation of alamethicin (AcUPUAUAQUVUGLUPV UUEQFol), the role of this residue was assumed essential in the barrel-stave model for voltage-gated ion channels. Taking advantage of a previous synthetic alamethicin analogue (L2), in which all eight alpha-aminoisobutyric (U) were replaced by leucines (AcLPLALAQLV LGLLPV LLEQFol), another analogue (L5) was synthesized in order to test the effects of proline-14 substitution by an alanine (AcLPLALAQLVLGLLPVLLEQFol). Previous conductance experiments showed that both high voltage dependence and multistate behavior were conserved. In order to complement these functional results, a conformational study of L5 has been undertaken and compared to L2 using CD, high field nmr, and molecular dynamics. Results show that L5 presents a better ordered structure than L2 particularly in the region of the substitution and in the C-terminal part. These results are discussed as regards the previous hypothesis of the nonessential character of helix bending for the gating of voltage-dependent ion channels.

Alamethicin↗

PubMed1994

Anion pores from magainins and related defensive peptides.

The conformations and properties of magainins, powerful antimicrobial peptides isolated from Xenopus laevis skin, are reviewed. Although some emphasis will be placed upon membrane interactions both in living cells and liposomes, we will especially focus on pore-forming properties as studied with conductance measurements on doped planar lipid bilayers. The relevance to channel modelling and engineering will be stressed and finally the relations, analogies and differences, of magainins with other insect and mammalian defensive peptides, such as cecropins and defensins, will be presented.

Amino Acid Sequence↗

PubMed1994

Implication of segment S45 in the permeation pathway of voltage-dependent sodium channels.

A 34-mer peptide, encompassing the S4 and S45 segments of domain IV of the electric eel voltage-dependent sodium channel, was synthesized in order to test the potential implication of S45 in the gating or permeation pathway. The secondary structure of peptide S4-S45 assessed by circular dichroism was found mainly helical, both in organic solvents and in lipid vesicles, especially negatively-charged ones. The macroscopic conductance properties of neutral and negatively-charged Montal-Mueller planar lipid bilayers doped with S4-S45 were studied and compared with those of S4. With regard to voltage-dependence, the most efficient system was S4-S45 in neutral bilayers. Voltage thresholds for exponential conductance development were found to correlate with the background or "leak" conductance. Assuming that the latter reflects interfacial peptide concentration, the mean apparent number of monomers per conducting aggregate could be estimated to be 3-5. In single-channel experiments, the most probable events had amplitudes of 8 pS and 5 pS in neutral and negatively-charged bilayers respectively. Ionic selectivity under salt gradients conditions, both at macroscopic and single-channel levels, was in favour of sodium ions (PNa/PK = 3). These properties compare favourably to previous reports dealing with peptide modelling transmembrane segments of voltage-dependent ionic channels. Specifically, when compared to S4 alone, the reduced unit conductance and the increased selectivity for sodium support the implication of the S45 region in the inner lining of the open configuration of sodium channels.

Amino Acid Sequence↗

PubMed1993

Purification and characterization of protein H, the major porin of Pasteurella multocida.

Protein H (B. Lugtenberg, R. van Boxtel, D. Evenberg, M. de Jong, P. Storm, and J. Frik, Infect. Immun. 52:175-182, 1986) is the major polypeptide of the outer membrane of Pasteurella multocida, a bacterium pathogenic for humans and animals. We have purified this protein to homogeneity by size exclusion chromatography after selective extraction with surfactants and demonstrated its pore-forming ability after reincorporation into planar lipid bilayers. In these experiments, the current through the pores was a linear function of the applied voltage in the range of -50 to +50 mV. Voltages beyond +/- 50 mV tended to partially close the channels, giving rise to apparent negative resistances. These observations suggest that protein H channels are probably not voltage regulated in vivo. With the patch clamp technique, single-channel conductance fluctuations of 0.33 nS were recorded in 1 M KCl. Electrophoretic and circular dichroism analyses showed that protein H forms homotrimers stable in sodium dodecyl sulfate at room temperature, with a high content of beta-sheet secondary structure. Upon boiling, the trimers were fully dissociated into monomers with an increase of alpha helix and irregular structure, at the expense of beta sheets. The apparent molecular mass of fully denatured monomers ranged between 37 and 41.8 kDa, depending on the electrophoretic system used for analysis. The trimeric arrangement of protein H was confirmed by image analysis of negatively stained, two-dimensional crystal arrays. This morphological study revealed, in agreement with electrophoretical data, a trimeric structure with an overall diameter of 7.7 nm. Each monomer appeared to contain a pore with an average diameter of 1 nm. Quantitative comparisons revealed that the amino acid composition (hydropathy index of -0.40) and the N-terminal sequence (determined over 36 residues) of protein H are similar to those of bacterial general porins, notably porin P2 of Haemophilus influenzae. We conclude from this set of structural and functional data that protein H of P. multocida is a pore-forming protein related to the superfamily of the nonspecific bacterial porins.

Amino Acid Sequence↗

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↗