共查询到20条相似文献,搜索用时 9 毫秒
1.
T. Rink H. Bartel G. Jung W. Bannwarth G. Boheim 《European biophysics journal : EBJ》1994,23(3):155-165
The effects of the peptide polycations salmon protamine (M
r = 4332,z = + 21) and poly-l-lysine (M
r 100,00,z + 775) on ion channels formed by synthetic alamethicin Alm-F30 (one negative charge), natural Alm-F50 (neutral) and phosphorylated Alm-F50 (two negative charges) reconstituted in planar lipid bilayers have been studied at the single channel level. It was observed that both polycations in micromolar concentrations transiently block ion permeation through the channels formed by each alamethicin analogue, although in case of the neutral Alm-F50 to a significantly lesser extent. Poly-l-lysine showed to be more effective than protamine in blocking these channels. If either polycation is present in the cis-compartment, blockade occurs only at cis positive membrane voltages. At constant polycation concentration, dwell times in the blocked state increase when salt concentration is lowered, and decrease at acidic pH with an apparent pK of 4.8. Mean lifetime of blockade events shortens when membrane voltage is increased, which suggests that both polycations may permeate through the oligomeric alamethicin channels if conductance levels are > 2. We suggest that blockade is caused by electrostatic binding of a single polycation molecule to the C-terminal channel mouth; in case of Alm-F30, Glu18 has to be considered as the putative binding site. Our results provide further evidence for the barrel-stave model and a parallel orientation of dipole monomers in the channel aggregate, the C-termini facing the membrane side with the more positive membrane potential. 相似文献
2.
Sylvie Rebuffat Hervé Duclohier Catherine Auvin-Guette Gérard Molle Gérard Spach Bernard Bodo 《FEMS microbiology letters》1992,105(1-3):51-60
Abstract Harzianin HA V and saturnisporin SA IV are α-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 α-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. 相似文献
3.
Zervamicin-IIB (Zrv-IIB) is a 16 residue peptaibol which forms voltage-activated, multiple conductance level channels in planar lipid bilayers. A molecular model of Zrv-IIB channels is presented. The structure of monomerc Zrv-II3 is based upon the crystal structure of Zervamicin-Leu. The helical backbone is kinked by a hydroxyproline residue at position 10. Zrv-IIB channels are modelled as helix bundles of from 4 to 8 parallel helices surrounding a central pore. The monomers are packed with their C-terminal helical segments in close contact, and the bundles are stabilized by hydrogen bonds between glutamine 11 and hydroxyproline 10 of adjacent helices. Interaction energy profiles for movement of three different probes species (K+, Cl– and water) through the central pore are analyzed. The conformations of: (a) the sidechain of glutamine 3; (b) the hydroxyl group of hydroxyproline 10; and (c) the C-terminal hydroxyl group are optimized in order to maximize favourable interactions between the channel and the probes, resulting in favourable interaction energy profiles for all three. This suggests that conformational flexibility of polar sidechains enables the channel lining to mimic an aqueous environment. 相似文献
4.
The appearance of ion channels was induced in phospholipid bilayers by acidification of the bulk solution on one side of the bilayer. by addition of HCl. acetic acid or by hydrolytic production of protons using purified acetylcholinesierase. Further acidification below an apparent critical pH range led to restoration of a low conductance state similar to that seen at neutral pH. Such experiments were performed with a heterogeneous soybean lecithin extract, with homogeneous synthetic di-phytanoylphosphatidylcholine, and with a mixture of cholesterol and synthetic dioleoylphosphatdylcholine. It is proposed that the physical mechanism for this phenomenon involves fluctuations of lipid order induced by fluctuations in protnation of phospholipid head groups within a critical pH range; these, in turn, create conductive defect in the two-dimensional lattice of the lipid bilayer. 相似文献
5.
Channel forming peptides (CFPs) are amphipathic peptides, of length ca. 20 residues, which adopt an -helical conformation in the presence of lipid bilayers and form ion channels with electrophysiological properties comparable to those of ion channel proteins. We have modelled CFP channels as bundles of parallel trans-bilayer helices surrounding a central ion-permeable pore. Ion-channel interactions have been explored via accessible surface area calculations, and via evaluation of changes in van der Waals and electrostatic energies as a K+ ion is translated along the length of the pore. Two CFPs have been modelled: (a) zervamicin-A1-16, a synthetic apolar peptaibol related to alamethicin, and (b) -toxin from Staphylococcus aureus. Both of these CFPs have previously been shown to form ion channels in planar lipid bilayers, and have been shown to have predominantly helical conformations. Zervamicin-A1-16 channels were modelled as bundles of 4 to 8 parallel helices. Two related helix bundle geometries were explored. K+channel interactions have been shown to involve exposed backbone carbonyl oxygen atoms. -Toxin channels were modelled as bundles of 6 parallel helices. Residues Q3, D11 and D18 generate favourable K+-channel interactions. Rotation of W15 about its C-C bond has been shown to be capable of occluding the central pore, and is discussed as a possible model for sidechain conformational changes in relation to ion channel gating. 相似文献
6.
The refractive indices of the bilayer-electrolyte system allow the membrane to operate as a light-guide. This system is then able to monitor, optically, the flow of ions across the bilayer. The light is coupled into and decoupled from a spherically bulged bilayer by means of optical, single mode fibers. The light wave travels along the curved bilayer for several millimeters. This light transmission depends critically on the angle of incidence between the fiber axis and the tangent to the film. Three transmission peaks were observed when the angle of incidence was varied between 0° and 90°. The transmitted light intensity can be modulated by the application of an electric potential upon the bilayer. The center peak, with maximum light transmission, appears at an angle of incidence which is defined by the launching geometry. A quadratic field dependence (independent of the polarity) is observed, which originates from changes in the shape of the torus transition region. The transmission of the satellite peaks, which appear just before and after the central peak, can also be modulated by an external potential. This modulation signal reflects a linear dependence on the polarity of the external voltage. The phase of the modulation signal changes its sign at each satellite peak. It is shown that this modulation signal originates from the bimolecular area of the lipid film. We present evidence that this transmission modulation occurs as a result of ion transport through the lipid film. This provides the basis for the use of wave-guide spectroscopy to investigate membrane ionic fluxes. 相似文献
7.
M. S. P. Sansom 《European biophysics journal : EBJ》1993,22(2):105-124
Peptaibols are considered as models of those ion channels which consist of a bundle of transbilayer helices surrounding a central pore. X-Ray diffraction and NMR studies have yielded high resolution structures for several peptaibols. In conjunction with other spectroscopic investigations and molecular dynamics simulations, these studies suggest that peptaibols form proline-kinked -helices, and that there may be hinge-bending movement of the helix in the region of the central proline residue. The amphipathicity of peptaibol helices is analyzed in relation to their channel-forming properties. Studies of the interactions of peptaibols with lipid bilayers suggest that they are helical when in a membrane-like environment, and that the helix orientation relative to the bilayer is sensitive to the peptaibol: lipid ratio, and to the degree of hydration of the bilayer. Electrical studies reveal that many peptaibols form multiple-conductance level channels in a voltage-dependent fashion. Analysis of conductance levels provides support for the barrel stave model of channel formation, whereby different conductance levels correspond to different numbers of monomers in a helix bundle. Alternative models for voltage-activation are discussed, and the roles of molecular dipoles and of hinge-bending in this process are considered. Two molecular models for an N = 6 bundle of alamethicin helices are presented and their electrostatic properties analyzed. The relevance of studies of peptaibols to channel and transport proteins in general is considered.Abbreviations Aib
-amino-isobutyric acid
- Alm
alamethicin
- ATR-FTIR
attenuated total reflection Fourier transform infrared
- CD
circular dichroism
- CFP
channel-forming peptide
- Chol
cholesterol
- diPhyPC
diphytanoyl phosphatidylcholine
- DMPC
dimyristoyl phosphatidylcholine
- DOPC
dioleoyl phosphatidylcholine
- DOPE
dioleoyl phosphatidylethanolamine
- DPPS
dipahnitoyl phosphatidylserine
- DTPC
ditetradecyl phosphatidylcholine
- HSM
hydrophilic surface map
- I-V
current-voltage
- MLV
multilamellar vesicle
- nAChR
nicotinic acetylcholine receptor
- P:L
protein-to-lipid ratio
- POPC
palmitoyloleoyl phosphatidylcholine
- SUV
small unilamellar vesicle
- Zrv
zervamicin 相似文献
8.
The aromatic polyene antibiotic levorin A2 forms ion channels permeable to monovalent cations, in lipid membranes containing cholesterol or ergosterol. Channel conductivity is in the range 0.3–0.5 pS. The channel has two main states: conducting (open) and nonconducting (closed). The potential-dependent formation of levorin A2 channels is observed in lipid membranes. The system responsible for the ion-channel selectivity is localized on the hydrophilic side of the lactone ring of the polyene molecule. 相似文献
9.
Single anion channels reconstituted from cardiac mitoplasts 总被引:4,自引:0,他引:4
Ion channels from sheep cardiac mitoplast (inverted inner mitochondrial membrane vesicle) preparations were incorporated into
voltage-clamped planar lipid bilayers. The appearance of anion rather than cation channels could be promoted by exposing the
bilayers to osmotic gradients formed by Cl− salts of large, relatively imperment, cations at a pH of 8.8. Two distinct activities were identified. These comprised a
multisubstate anion channel of intermediate conductance (∼60 pS in 300vs. 50mm choline Cl, ∼100 pS in symmetric 150mm KCl), and a lower-conductance anion channel (∼25 or ∼50 pS in similar conditions), which only displayed two well-defined
substates, at ∼25 and ∼50% of the fully open state. The larger channels were not simple multiples of the lower-conductance
channels, but both discriminated poorly, and to a similar extent, between anions and cations (PCl
−/Pcholine
+ ∼12, PCl
−/PK
+∼8). The lower-conductance channel was only minimally selective between different anions (PNO
3
−
(1.0)=PCl
−>PBr
−>PI
−>PSCN
−(0.8)), and its conductance failed to saturate even in high (>1.0 M) activities of KCl. The channels were not obviously voltage
dependent, and they were unaffected by 0.5 mM SITS, H2O2, propranolol, quinine or amitriptyline, or by 2 mM ATP, or by variations in pH (5.5–8.8). Ca2+ and Mg2+ did not alter single channel activity, but did modify single current amplitudes in the lower-conductance channel. This effect,
together with voltage-dependent substate behavior, is described in the following paper. 相似文献
10.
Ceratotoxins are α-helical cationic peptides isolated from the medfly Ceratitis capitata. These amphipathic peptides were found to display strong antibacterial activity and weak hemolytic activity. When reconstituted into planar lipid bilayers, ceratotoxins developed highly asymmetric I/V curves under voltage ramps and formed, in single-channel experiments, well-defined voltage-dependent ion channels according to the barrel stave model. The antibacterial activity and pore-forming properties of these molecules were well correlated. Similar experiments performed with synthesized truncated fragments showed that the C-terminal domain of ceratotoxins is strongly implicated in the formation of helical bundles in the membrane whereas the largely cationic N-terminal region is likely to anchor ceratotoxins on the lipid surface. 相似文献
11.
Ion channels may be formed by self-assembly of amphipathic α-helical peptides into parallel helix bundles. The transbilayer pores formed by such peptides contain extended columns of
water molecules, the properties of which may differ from those of water in its bulk state. The de novo designed peptides of
DeGrado et al., which contain only leucine and serine residues, are considered as a simple example of such channels. Molecular
dynamics simulations of peptide helix bundles with water molecules within and at the mouths of their pores are used to refine
such models and to investigate the properties of intra-pore water. The translational and rotational mobility of water molecules
within the pores are reduced relative to bulk water. Furthermore, intra-pore waters orient themselves with their dipoles anti-parallel
to the helix dipoles, as do the hydroxyl groups of serine residues. Comparison of approximate predictions of ionic conductances
with experimental values provides support for the validity of these models.
Received: 23 April 1996 / Accepted: 7 August 1996 相似文献
12.
Crary JI Dean DM Nguitragool W Kurshan PT Zimmerman AL 《The Journal of general physiology》2000,116(6):755-768
Cyclic nucleotide-gated (CNG) channels are critical components in the visual and olfactory signal transduction pathways, and they primarily gate in response to changes in the cytoplasmic concentration of cyclic nucleotides. We previously found that the ability of the native rod CNG channel to be opened by cGMP was markedly inhibited by analogues of diacylglycerol (DAG) without a phosphorylation reaction (Gordon, S.E., J. Downing-Park, B. Tam, and A.L. Zimmerman. 1995. Biophys. J. 69:409-417). Here, we have studied cloned bovine rod and rat olfactory CNG channels expressed in Xenopus oocytes, and have determined that they are differentially inhibited by DAG. At saturating [cGMP], DAG inhibition of homomultimeric (alpha subunit only) rod channels was similar to that of the native rod CNG channel, but DAG was much less effective at inhibiting the homomultimeric olfactory channel, producing only partial inhibition even at high [DAG]. However, at low open probability (P(o)), both channels were more sensitive to DAG, suggesting that DAG is a closed state inhibitor. The Hill coefficients for DAG inhibition were often greater than one, suggesting that more than one DAG molecule is required for effective inhibition of a channel. In single-channel recordings, DAG decreased the P(o) but not the single-channel conductance. Results with chimeras of rod and olfactory channels suggest that the differences in DAG inhibition correlate more with differences in the transmembrane segments and their attached loops than with differences in the amino and carboxyl termini. Our results are consistent with a model in which multiple DAG molecules stabilize the closed state(s) of a CNG channel by binding directly to the channel and/or by altering bilayer-channel interactions. We speculate that if DAG interacts directly with the channel, it may insert into a putative hydrophobic crevice among the transmembrane domains of each subunit or at the hydrophobic interface between the channel and the bilayer. 相似文献
13.
J. O. Bullock S. K. Armstrong J. L. Shear D. P. Lies M. A. McIntosh 《The Journal of membrane biology》1990,114(1):79-95
Summary The gene for the antibacterial peptide colicin B was cloned and transformed into a host background where it was constitutively overexpressed. The purified gene product was biologically active and formed voltage-dependent, ion-conducting channels in planar phospholipid bilayers composed of asolectin. Colicin B channels exhibited two distinct unitary conductance levels, and a slight preference for Na+ over Cl–. Kinetic analysis of the voltage-driven opening and closing of colicin channels revealed the existence of at least two conducting states and two nonconducting states of the protein. Both the ion selectivity and the kinetics of colicin B channels were highly dependent on pH. Excess colicin protein was readily removed from the system by perfusing the bilayer, but open channels could be washed out only after they were allowed to close. A monospecific polyclonal antiserum generated against electrophoretically purified colicin B eliminated both the biological and in vitro activity of the protein. Membrane-associated channels, whether open or closed, remained functionally unaffected by the presence of the antiserum. Taken together, our results suggest that the voltage-independent binding of colicin B to the membrane is the rate-limiting step for the formation of ion channels, and that this process is accompanied by a major conformational rearrangement of the protein. 相似文献
14.
G Prestipino H H Valdivia A Liévano A Darszon A N Ramírez L D Possani 《FEBS letters》1989,250(2):570-574
Voltage-dependent K+ channels are responsible for repolarization of the cell membrane during the late phase of the action potential. Here we report the purification of proteins from squid axon membranes which bind the K+-channel blocker noxiustoxin (NTX), and their subsequent functional reconstitution in planar bilayers. The NXT-affinity purified proteins had Mr values of 60000 ± 6000, 160000 ± 15000 and 220000 ± 20000. Their incorporation into bilayers resulted in single-channel currents with three conductances, the most frequent one of 11 pS in 300/100 mM KCl (cis/trans). The voltage dependence, reversal potential and bursting behavior suggest that these are the K+ channels involved in the squid axon action potential. 相似文献
15.
Hiroshi Monoi 《生物化学与生物物理学报:生物膜》1982,693(1):159-164
An electrostatic calculation suggests that when an ion is bound near the mouth of a channel penetrating a low-dielectric membrane, a counter ion may form an ion pair with this ion. The tendency towards ion-pair formation is remarkably enhanced at channel mouths by forces (image forces) arising from the charges induced on the boundaries between different dielectrics. The binding constant for the formation of ion-pairs of monovalent ions is estimated under the assumption that local interactions between the counter ion and the channel wall are negligibly small. It is of the order of 1–10 molal?1 or more for the binding of a Cl? (F?) counter ion to an () ion if appropriate conditions are fulfilled. The binding constant depends on the position of the binding site, the dimensions and geometries of the channel and channel mouth, and the state of ion loading of the channel, as well as the ionic species. The present results also indicate that when cation (anion) channels have anionic (cationic) groups as integrant parts of their channel walls, interactions between these charged groups and permeant ions are markedly enhanced by the image forces. 相似文献
16.
The magnesium ion (Mg2 +) is the most abundant divalent cation within cells. In man, Mg2 +-deficiency is associated with diseases affecting the heart, muscle, bone, immune, and nervous systems. Despite its impact on human health, little is known about the molecular mechanisms that regulate magnesium transport and storage. Complete structural information on eukaryotic Mg2 +-transport proteins is currently lacking due to associated technical challenges. The prokaryotic MgtE and CorA magnesium transport systems have recently succumbed to structure determination by X-ray crystallography, providing first views of these ubiquitous and essential Mg2 +-channels. MgtE and CorA are unique among known membrane protein structures, each revealing a novel protein fold containing distinct arrangements of ten transmembrane-spanning α-helices. Structural and functional analyses have established that Mg2 +-selectivity in MgtE and CorA occurs through distinct mechanisms. Conserved acidic side-chains appear to form the selectivity filter in MgtE, whereas conserved asparagines coordinate hydrated Mg2 +-ions within the selectivity filter of CorA. Common structural themes have also emerged whereby MgtE and CorA sense and respond to physiologically relevant, intracellular Mg2 +-levels through dedicated regulatory domains. Within these domains, multiple primary and secondary Mg2 +-binding sites serve to staple these ion channels into their respective closed conformations, implying that Mg2 +-transport is well guarded and very tightly regulated. The MgtE and CorA proteins represent valuable structural templates to better understand the related eukaryotic SLC41 and Mrs2–Alr1 magnesium channels. Herein, we review the structure, function and regulation of MgtE and CorA and consider these unique proteins within the expanding universe of ion channel and transporter structural biology. 相似文献
17.
Feeding insects introduce oral secretions (OS) into the wounded tissue of the attacked plant. Various OS-derived molecules must be involved in subsequent processes including the induction of plant defence reactions. Using the planar lipid bilayer membrane technique, isolated OS were analyzed with respect to their membrane activities. Transmembrane ion fluxes were generated by OS of eight different lepidopteran larvae, all of which form comparable ion channels in artificial membranes. Currents were characterized by long lasting open times and conductivities from 250pS up to 1100pS. Channels formed by Spodoptera exigua secretions showed a preference for cations over anions. OS also induced a transient increase of the cytosolic calcium concentration in soybean cells, determined by the aequorin technique. Known compounds of the OS, fatty-acid-glutamine conjugates, also interfered with the membrane but were unable to form stable channels. Since ion fluxes and depolarization are early responses upon insect feeding, OS-derived components may be involved in the elicitation process by direct interaction with the plant membranes. 相似文献
18.
As a step towards an automated and operator-free ion channel measurement platform we have previously demonstrated a solution formulation for artificial lipid bilayers that enabled the indefinite storage and shipping of frozen bilayer precursors. In this work, the solutions were deposited by hand. Here, we have adapted pin tools to deposit the bilayer precursor solutions onto multi-element arrays, a popular method for microarray solution deposition. The pin tools have enabled the deposited volume to be applied highly repeatably and controllably, resulting in reduction of bilayer formation times to <1 h. The pin tools are also compatible with computerized motion control platforms, enabling automated and high throughput production. We discuss these results and the prospects of this technology to produce high density bilayer arrays for high throughput measurement of ion channels incorporated into artificial bilayers. 相似文献
19.
Tomaskova Z Gaburjakova J Brezova A Gaburjakova M 《Journal of bioenergetics and biomembranes》2007,39(4):301-311
The objective of this work was to characterize in more detail the inhibition effect of diisothiocyanatostilbene-2′,2-disulfonic
acid (DIDS) on anion channels isolated from the rat heart mitochondria. The channels reconstituted into a planar lipid membrane
displayed limited powers of discrimination between anions and cations and the ion conductance measured under asymmetric (250/50 mM
KCl, cis/trans) and symmetric (150 mM KCl) conditions was ∼100 pS. DIDS caused a dramatic decrease in the channel activity (IC50 = 11.7 ± 3.1 μM) only when it was added to the cis side of a planar lipid membrane. The inhibition was accompanied by the significant prolongation of closings and the shortening
of openings within the burst as well as gaps between bursts were prolonged and durations of bursts were reduced. The blockade
was complete and irreversible when concentration of DIDS was increased up to 200 μM. Our data indicate that DIDS is an allosteric
blocker of mitochondrial anion channels and this specific effect could be used as a tool for reliable identification of anion
channels on the functional level. 相似文献
20.
Macroscopic ion channel current can be derived by summation of the stochastic records of individual channel currents. In this paper, we present two probability density functions of single channel records that can uniquely determine the macroscopic current regardless of other statistical properties of records or the stochastic model of channel gating (presented often with stationary Markov models). We show that H(t), probability density function of channel opening events (introduced explicitly in this paper), and D(t), probability density function of the open duration (sometimes has named dwell time distribution as well), determine the normalized macroscopic current, G(t), through G(t) = P(t) - H(t) * Q(t) where P(t) is the cumulative density function of H(t), Q(t) is the cumulative density function of D(t), * is the symbol of convolution integral and G(t) is the macroscopic current divided by the amplitude of single channel current and the number of single channel sweeps. Compared to other equations for the macroscopic current, here the macroscopic current is expressed only in terms of the statistical properties of single channel current and not the stochastic model of ion channel gating or a conditioned form of macroscopic current. Single channel currents of an inactivating BK channel were used to validate this relationship experimentally too. In this paper, we used median filters as they can remove the unwanted noise without smoothing the transitions between open and closed states (compare to low pass filters). This filtering leads to more accurate measurement of transition times and less amount of missed events. 相似文献