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1.
Structure and Origin of Ordered Lipid Domains in Biological Membranes 总被引:16,自引:0,他引:16
2.
The aim of this study is to evaluate directly, using a reduced experimental system, the nature of interactions between voltage-gated
potassium channels and the resting membrane potential. Xenopus oocytes were injected with various concentrations of cRNA coding for a delayed-rectifier potassium channel Shaker-IR. The effects of the density and kinetics of the expressed channels on resting membrane potential is explored in isolated
(``inside-out') patches. The channel density is given in terms of maximal conductance (G
max), measured from the maximal slope of the I-V curve under voltage clamp conditions. The capacitance of the experimental setup is approximately 1 pF. At high channel densities
(G
max > 10 pA/mV) the mean membrane potential is stabilized at approximately −60 mV. This resting membrane potential is more than
35 mV positive to the reversal potential for potassium ions under the same experimental conditions. Analyses of voltage clamp
experiments indicate that at high channel densities the mean membrane potential is determined by the rates of channel activation
and deactivation, but is not affected by the rates involved in the process of slow (C-type) inactivation. In contrast, at
lower channel densities membrane potential is very unstable, and its mean value and amplitude of fluctuations are strongly
affected by the process of slow (C-type) inactivation.
Received: 21 March 1996/Revised: 6 August 1996 相似文献
3.
4.
O.V. Krasilnikov M.-F.P. Capistrano L.N. Yuldasheva R.A. Nogueira 《The Journal of membrane biology》1997,156(2):157-172
Replacement of an amino acid residue at position 130 -Gly by Cys- in the primary structure of Staphylococcus aureus alpha-toxin
decreases the single-channel conductance induced by the toxin in planar lipid bilayers. Concomitantly, the pH value at which
the channel becomes unable to discriminate between Cl− and K+ ions is also decreased. By contrast, the pH dependence of the efficiency of the mutant toxin to form ion channels in lipid
bilayers was unchanged (maximum efficiency at pH 5.5–6.0). The asymmetry and nonlinearity of the current-voltage characteristics
of the channel were increased by the point mutation but the diameter of the water pore induced by the mutant toxin, evaluated
in lipid bilayers and in erythrocyte membranes, was found to be indistinguishable from that formed by wild-type toxin and
equal to 2.4–2.6 nm.
Alterations at the ``trans mouth' were found to be responsible for all observed changes of the channel properties. This mouth is situated close to
the surface of the second leaflet of a bilayer lipid membrane. The data obtained allows us to propose that the region around
residue 130 in fact determines the main features of the ST-channel and takes part in the formation of the trans entrance of the channel.
Received: 8 September 1995/Revised: 20 November 1996 相似文献
5.
Fluxes of Na, Cl and volume were followed across Necturus small intestine under zero voltage clamp. 20 mm l-alanine doubles the net Na and fluid transfer. Although there is a ouabain-sensitive Na pump present in Necturus a major fraction of the net Na flux can be measured for an hour after application of 10−3
m ouabain. Collected fluid transferred by the epithelium is quasi-isotonic over a range of luminal osmolarities from 100 to
250 milliosmolar in alanine saline. The net Na fluxes account for the Na found in this transported fluid. Fluid transfer also
shows a large ouabain-insensitive fraction after the addition of alanine.
Compartmental analysis of 22Na-loaded epithelium was used to separate cellular and paracellular fluxes. The estimated Na concentration in the cell derived
from its Na content is 9–10 mm, in agreement with that determined with microelectrodes. The Na efflux from cell to serosa is stimulated by alanine, but
this increase accounts for only a quarter of the simultaneous rises in Na, fluid and current flow across the epithelium.
The increase of Na efflux from the cell induced by alanine is apparently insensitive to ouabain although the cell Na content
rises to circa 20 mm but no higher even after 20 hr. From the initial rate of rise of Na in the cell on treatment with ouabain the activity of
the Na pump can be estimated to be ∼92 pM/cm2· sec, a value much smaller than the transepithelial net flux.
The results are not consistent with the standard model in which Na-alanine influx stimulates the Na pump and enhances fluid
transport by osmotic coupling in the lateral interspace system. A scheme is proposed based upon that for absorption in Necturus gallbladder by which alanine stimulates an active paracellular fluid transfer driven by motile elements of the junction.
Received: 5 August 1996/Revised: 7 February 1997 相似文献
6.
Structure-Function Relationship of the Ion Channel Formed by Diphtheria Toxin in Vero Cell Membranes
Diphtheria toxin (DT) forms cation selective channels at low pH in cell membranes and planar bilayers. The channels formed
by wild-type full length toxin (DT-AB), wild-type fragment B (DT-B) and mutants of DT-B were studied in the plasma membrane
of Vero cells using the patch-clamp technique. The mutations concerned certain negatively charged amino acids within the channel-forming
transmembrane domain (T-domain). These residues might interact electrostatically with cations flowing through the channel,
and were therefore exchanged for uncharged amino acids or lysine. The increase in whole-cell conductance induced by toxin,
Δg
m
, was initially determined. DT-AB induced a ∼10-fold lower Δg
m
than DT-B. The mutations DT-B E327Q, DT-B D352N and DT-B E362K did not affect Δg
m
, whereas DT-B D295K, DT-B D352K and DT-B D318K drastically reduced Δg
m
. Single channel analysis of DT-B, DT-AB, DT-B D295K, DT-B D318K and DT-B E362K was then performed in outside-out patches.
No differences were found for the single-channel conductances, but the mutants varied in their gating characteristics. DT-B
D295K exhibited only a very transient channel activity. DT-AB as well as DT-B D318K displayed significantly lower open probability
and mean dwell times than DT-B. Hence, the lower channel forming efficiency of DT-AB and DT-B D318K as compared to DT-B is
reflected on the molecular level by their tendency to spend more time in the closed position and the fast flickering mode.
Altogether, the present work shows that replacements of single amino acids distributed throughout a large part of the transmembrane
domain (T-domain) strongly affect the overall channel activity expressed as Δg
m
and the gating kinetics of single channels. This indicates clearly that the channel activity observed in DT-exposed Vero
cells at low pH is inherent to DT itself and not due to DT-activation of an endogenous channel.
Received: 20 June 1996/Revised: 8 November 1996 相似文献
7.
A fractal renewal point process (FRPP) is used to model molecular evolution in agreement with the relationship between the
variance and the mean numbers of nonsynonymous and synonymous substitutions in mammals. Like other episodic models such as
the doubly stochastic Poisson process, this model accounts for the large variances observed in amino acid substitution rates,
but unlike certain other episodic models, it also accounts for the increase in the index of dispersion with the mean number
of substitutions in Ohta's (1995) data. We find that this correlation is significant for nonsynonymous substitutions at the
1% level and for synonymous substitutions at the 10% level, even after removing lineage effects and when using Bulmer's (1989)
unbiased estimator of the index of dispersion. This model is simpler than most other overdispersed models of evolution in
the sense that it is fully specified by a single interevent probability distribution. Interpretations in terms of chaotic
dynamics and in terms of chance and selection are discussed.
Received: 12 January 1998 / Accepted: 19 May 1998 相似文献
8.
Permeabilization of Model Lipid Membranes by Bacillus sphaericus Mosquitocidal Binary Toxin and its Individual Components 总被引:3,自引:0,他引:3
J.-L. Schwartz L. Potvin F. Coux J.-F. Charles C. Berry M.J. Humphreys A.F. Jones I. Bernhart M. Dalla Serra G. Menestrina 《The Journal of membrane biology》2001,184(2):171-183
The high larvicidal effect of Bacillus sphaericus (Bs), a mosquito control agent, originates from the presence of a binary toxin (Bs Bin) composed of two proteins (BinA and BinB) that work together to lyse gut cells of susceptible larvae. We demonstrate
for the first time that the binary toxin and its individual components permeabilize receptor-free large unilamellar phospholipid
vesicles (LUVs) and planar lipid bilayers (PLBs) by a mechanism of pore formation. Calcein-release experiments showed that
LUV permeabilization was optimally achieved at alkaline pH and in the presence of acidic lipids. BinA was more efficient than
BinB, BinB facilitated the BinA effect, and their stoichiometric mixture was more effective than the full Bin toxin. In PLBs,
BinA formed voltage-dependent channels of ≈100–200 pS with long open times and a high open probability. Larger channels (≥400
pS) were also observed. BinB, which inserted less easily, formed smaller channels (≤100 pS) with shorter mean open times.
Channels observed after sequential addition of the two components, or formed by their 1:1 mixture (w/w), displayed BinA-like
activity. Bs Bin toxin was less efficient at forming channels than the BinA/BinB mixture, with channels displaying the BinA channel behavior.
Our data support the concept of BinA being principally responsible for pore formation in lipid membranes with BinB, the binding
component of the toxin, playing a role in promoting channel activity.
Received: 29 March 2001/Revised: 20 July 2001 相似文献
9.
M. Münstermann A. Wiese K. Brandenburg U. Zähringer L. Brade K. Kawahara U. Seydel 《The Journal of membrane biology》1999,167(3):223-232
Planar asymmetric glycolipid/phospholipid bilayer membranes were used as a reconstitution model of the lipid matrix of the
outer membrane of Gram-negative bacteria to study complement (C) activation by various bacterial surface glycolipids with
the aim of defining the C activation pathway. As glycolipids the lipopolysaccharides of Salmonella enterica serovar Minnesota R mutant strains R595 (Re LPS) and R4 (Rd2 LPS), pentaacyl lipid A from the LPS of the Escherichia coli Re mutant F515, and glycosphingolipid GSL-1 of Sphingomonas paucimobilis IAM 12576 were used. Methylester and carboxyl-reduced derivatives of GSL-1 were used to elucidate the role of the carboxyl
group as common functional group of LPS and GSL-1 for C activation. The formation of lytic pores was monitored via the measurement of changes in membrane current. For all glycolipids we observed a considerable increase in membrane current
soon after addition of whole human serum due to the formation of lytic pores in the membranes. Pore formation was dependent
on the presence of C9, indicating that the observed current changes were due to C activation.
We found that in our reconstitution system of the outer membrane lipid A, Re LPS, and Rd2 LPS activated the classical pathway, the activation being independent of specific anti-LPS antibodies. In contrast, GSL-1
and the methylester derivative of GSL-1 activated the alternative pathway even at the low serum concentrations used in this
study (about 0.2% v/v). Interestingly, the carboxyl reduced GSL-1 activated the classical pathway.
Received: 16 July 1998/Revised: 28 October 1998 相似文献
10.
U. Yermiyahu G. Rytwo D.K. Brauer T.B. Kinraide 《The Journal of membrane biology》1997,159(3):239-252
A general model for the sorption of trivalent cations to wheat-root (Triticum aestivum L cv. Scout 66) plasma membranes (PM) has been developed and includes the first published coefficients for La3+ and Al3+ binding to a biological membrane. Both ions are rhizotoxic, and the latter ion is the principal contributor to the toxicity
of acidic soils around the world. The model takes into account both the electrostatic attraction and the binding of cations
to the negatively charged PM surface. Ion binding is modeled as the reaction P
−+I
Z⇌PI
Z
−1 in which P
− represents a negatively charged PM ligand, located in an estimated area of 540 ?2, and I
Z represents an ion of charge Z. Binding constants for the reaction were assigned for K+ (1 m
−1) and Ca2+ (30 m
−1) and evaluated experimentally for La3+ (2200 m
−1) and H+ (21,500 m
−1). Al sorption is complicated by Al3+ hydrolysis that yields hydroxoaluminum species that are also sorbed. Binding constants of 30 and 1 m
−1 were assigned for AlOH2+ and Al(OH)+
2, respectively, then a constant for Al3+ (20,000 m
−1) was evaluated experimentally using the previously obtained values for K+, Ca2+ and H+ binding. Electrostatic attraction was modeled according to Gouy-Chapman theory. Evaluation of parameters was based upon the
sorption of ions to PM vesicles suspended in solutions containing variable concentrations of H+, Ca2+ and La3+ or Al3+. Use of small volumes, and improved assay techniques, allowed the measurement of concentration depletions caused by sorption
to vesicles. Some independent confirmation of our model is provided by substantial agreement between our computations and
two published reports of La3+ effects upon zeta potentials of plant protoplasts. The single published report concerning the electrostatic effects of Al
on cell membranes is in essential agreement with the model.
Received: 6 January 1997/Revised: 6 June 1997 相似文献
11.
Ion Channel Permeable for Divalent and Monovalent Cations in Native Spinach Thylakoid Membranes 总被引:6,自引:0,他引:6
A cation-selective channel was characterized in isolated patches from osmotically swollen thylakoids of spinach (Spinacea oleracea). This channel was permeable for K+ as well as for Mg2+ and Ca2+ but not for Cl−. When K+ was the main permeant ion (symmetrical 105 mm KCl) the conductance of the channel was about 60 pS. The single channel conductance for different cations followed a sequence
K+ > Mg2+≥ Ca2+. The permeabilities determined by reversal potential measurements were comparable for K+, Ca2+, and Mg2+. The cation channel displayed bursting behavior. The total open probability of the channel increased at more positive membrane
potentials. Kinetic analysis demonstrated that voltage dependence of the total open probability was determined by the probability
of bursts formation while the probability to find the channel in open state within a burst of activity was hardly voltage-dependent.
The cation permeability of intact spinach thylakoids can be explained on the single channel level by the data presented here.
Received: 26 December 1995/Revised: 17 April 1996 相似文献
12.
The influence of the nonchannel conformation of the transmembrane protein gramicidin A on the permeability coefficients of
neutral and ionized α-X-p-methyl-hippuric acid analogues (XMHA) (X = H, OCH3, CN, OH, COOH, and CONH2) across egg-lecithin membranes has been investigated in vesicle efflux experiments. Although 10 mol% gramicidin A increases
lipid chain ordering, it enhances the transport of neutral XMHA analogues up to 8-fold, with more hydrophilic permeants exhibiting
the greatest increase. Substituent contributions to the free energies of transfer of both neutral and anionic XMHA analogues
from water into the bilayer barrier domain were calculated. Linear free-energy relationships were established between these
values and those for solute partitioning from water into decadiene, chlorobutane, butyl ether, and octanol to assess barrier
hydrophobicity. The barrier domain is similar for both neutral and ionized permeants and substantially more hydrophobic than
octanol, thus establishing its location as being beyond the hydrated headgroup region and eliminating transient water pores
as the transport pathway for these permeants, as the hydrated interface or water pores would be expected to be more hydrophilic
than octanol. The addition of 10 mol% gramicidin A alters the barrier domain from a decadiene-like solvent to one possessing
a greater hydrogen-bond accepting capacity. The permeability coefficients for ionized XMHAs increase with Na+ or K+ concentration, exhibiting saturability at high ion concentrations. This behavior can be quantitatively rationalized by Gouy-Chapman
theory, though ion-pairing cannot be conclusively ruled out. The finding that transmembrane proteins alter barrier selectivity,
favoring polar permeant transport, constitutes an important step toward understanding permeability in biomembranes.
Received: 12 July 1999/Revised: 20 October 1999 相似文献
13.
Van Mau N Missé D Le Grimellec C Divita G Heitz F Veas F 《The Journal of membrane biology》2000,177(3):251-257
Increasing evidence suggests that the HIV envelope binds through its surface (SU) gp120 not only to receptors and coreceptors,
but also to other components of the cellular membrane where the glycolipids appear to be good candidates. To assess the ability
of HIV-1 SU gp120 to penetrate into phospholipid membranes, we carried out a study of the interactions between a recombinant
SU gp120 from HIV-1/HXB2 and artificial lipid monolayers mimicking the composition of the outer leaflet of the lymphocytes
and which were spread at the air-water interface. We show that the protein, in its aggregated form, has amphipathic properties
and that the insertion of this amphipathic species into lipids is favored by the presence of sphingomyelin. Furthermore, cholesterol
enhances the penetration into mixed phosphatidylcholine-sphingomyelin monolayers. Coexistence of different physical states
of the lipids and thus of domains appears to play a major role for protein penetration independently of the presence of receptors
and coreceptors.
Received: 24 April 2000/Revised: 11 July 2000 相似文献
14.
15.
We combined widely different biochemical methods to analyze proteins of the cell surface of P. tetraurelia since so far one can isolate only a subfraction of cell membrane vesicles enriched in the GPI-anchored surface antigens (``immoblization'
or ``i-AGs'). We also found that i-AGs may undergo partial degradation by endogenous proteases. Genuine intrinsic membrane
proteins were recognized particularly with lipophilic 5-[125I]-iodonaphthalene-1-azide (INA) labeling which reportedly ``sees' integral proteins and cytoplasmic cell membrane-associated
proteins. With INA (+DTT), bands of ≤55 kDa were similar as with hydrophilic iodogen (+DTT), but instead of large size bands
including i-AGs, a group of 122, 104 and 94 kDa appeared. Several bands of the non i-AG type are compatible with integral
(possibly oligomeric) or associated proteins of the cell membrane of established molecular identity, as we discuss. In summary,
we can discriminate between i-AGs and some functionally important minor cell membrane components. Our methodical approach
might be relevant also for an analysis of some related protozoan parasites.
Received: 5 April 1999/Revised: 19 July 1999 相似文献
16.
Steinmetz PN Manwani A Koch C London M Segev I 《Journal of computational neuroscience》2000,9(2):133-148
Voltage-gated ion channels in neuronal membranes fluctuate randomly between different conformational states due to thermal agitation. Fluctuations between conducting and nonconducting states give rise to noisy membrane currents and subthreshold voltage fluctuations and may contribute to variability in spike timing. Here we study subthreshold voltage fluctuations due to active voltage-gated Na+ and K+ channels as predicted by two commonly used kinetic schemes: the Mainen et al. (1995) (MJHS) kinetic scheme, which has been used to model dendritic channels in cortical neurons, and the classical Hodgkin-Huxley (1952) (HH) kinetic scheme for the squid giant axon. We compute the magnitudes, amplitude distributions, and power spectral densities of the voltage noise in isopotential membrane patches predicted by these kinetic schemes. For both schemes, noise magnitudes increase rapidly with depolarization from rest. Noise is larger for smaller patch areas but is smaller for increased model temperatures. We contrast the results from Monte Carlo simulations of the stochastic nonlinear kinetic schemes with analytical, closed-form expressions derived using passive and quasi-active linear approximations to the kinetic schemes. For all subthreshold voltage ranges, the quasi-active linearized approximation is accurate within 8% and may thus be used in large-scale simulations of realistic neuronal geometries. 相似文献
17.
Kummerow D Hamann J Browning JA Wilkins R Ellory JC Bernhardt I 《The Journal of membrane biology》2000,176(3):207-216
The change of intracellular pH of erythrocytes under different experimental conditions was investigated using the pH-sensitive
fluorescent dye BCECF and correlated with (ouabain + bumetanide + EGTA)-insensitive K+ efflux and Cl− loss. When human erythrocytes were suspended in a physiological NaCl solution (pH
o
= 7.4), the measured pH
i
was 7.19 ± 0.04 and remained constant for 30 min. When erythrocytes were transferred into a low ionic strength (LIS) solution,
an immediate alkalinization increased the pH
i
to 7.70 ± 0.15, which was followed by a slower cell acidification. The alkalinization of cells in LIS media was ascribed
to a band 3 mediated effect since a rapid loss of approximately 80% of intracellular Cl− content was observed, which was sensitive to known anion transport inhibitors. In the case of cellular acidification, a comparison
of the calculated H+ influx with the measured unidirectional K+ efflux at different extracellular ionic strengths showed a correlation with a nearly 1:1 stoichiometry. Both fluxes were
enhanced by decreasing the ionic strength of the solution resulting in a H+ influx and a K+ efflux in LIS solution of 108.2 ± 20.4 mmol (l
cells
hr)−1 and 98.7 ± 19.3 mmol (l
cells
hr)−1, respectively. For bovine and porcine erythrocytes, in LIS media, H+ influx and K+ efflux were of comparable magnitude, but only about 10% of the fluxes observed in human erythrocytes under LIS conditions.
Quinacrine, a known inhibitor of the mitochondrial K+(Na+)/H+ exchanger, inhibited the K+ efflux in LIS solution by about 80%. Our results provide evidence for the existence of a K+(Na+)/H+ exchanger in the human erythrocyte membrane.
Received: 22 December 1999/Revised: 10 April 2000 相似文献
18.
Electrical breakdown of erythrocytes induces hemoglobin release which increases markedly with decreasing conductivity of
the pulse medium. This effect presumably results from the transient, conductivity-dependent deformation forces (elongation
or compression) on the cell caused by Maxwell stress. The deformation force is exerted on the plasma membrane of the cell,
which can be viewed as a transient dipole induced by an applied DC electric field pulse. The induced dipole arises from the
free charges that accumulate at the cell interfaces via the Maxwell-Wagner polarization mechanism. The polarization response
of erythrocytes to a DC field pulse was estimated from the experimental data obtained by using two complementary frequency-domain
techniques. The response is very rapid, due to the highly conductive cytosol. Measurements of the electrorotation and electrodeformation
spectra over a wide conductivity range yielded the information and data required for the calculation of the deformation force
as a function of frequency and external conductivity and for the calculation of the transient development of the deformation
forces during the application of a DC-field pulse. These calculations showed that (i) electric force precedes and accompanies
membrane charging (up to the breakdown voltage) and (ii) that under low-conductivity conditions, the electric stretching force
contributes significantly to the enlargement of ``electroleaks' in the plasma membrane generated by electric breakdown.
Received: 12 December 1997/Revised: 13 March 1998 相似文献
19.
The presence of proteins in lipid bilayers always decreases the excimer formation rate of pyrene and pyrene lipid analogues
in a way that is related to the protein-to-lipid ratio. Energy transfer measurements from intrinsic tryptophans to pyrene
have shown (Engelke et al., 1994), that in microsomal membranes, the excimer formation rate of pyrene and pyrene fatty acids
is heterogeneous within the membrane plane, because a lipid layer of reduced fluidity surrounds the microsomal proteins. This
study investigates whether of not liposomes prepared from egg yolk phosphatidylcholine with incorporated gramicidin A give
results comparable to those from microsomal membranes. The results indicate that the influence of proteins on the lipid bilayer
cannot be described by one unique mechanism: Small proteins such as gramicidin A obviously reduce the excimer formation rate
by occupying neighboring positions of the fluorescent probe and thus decrease the pyrene collision frequency homogeneously
in the whole membrane plane, while larger proteins are surrounded by a lipid boundary layer of lower fluidity than the bulk
lipid.
The analysis of the time-resolved tryptophan fluorescence of gramicidin A incorporated liposomes reveals, that the tryptophan
quenching by pyrene is stronger for tryptophans located closely below the phospholipid headgroup region because of the pyrene
enrichment in this area of the lipid bilayer.
Received: 29 December 1996/Revised: 15 May 1996 相似文献
20.
We harvested canalicular-enriched plasma membranes of hepatocytes and collected fistula bile from male rats and isolated
the sphingomyelins. Following sphingomyelinase hydrolysis, we identified the sphingomyelin molecular species on the basis
of their benzoylated ceramide derivatives employing high performance liquid chromatography. Sphingomyelin constitutes ≤3%
of total biliary phospholipids (which are mostly sn-1 16:0 long-chain phosphatidylcholines) and approximately 30% of canalicular-enriched
membranes. In both cases, the principal molecular species were composed of 16:0, 18:0, 20:0, 22:0, 23:0, 24:0, 24:1 and 24:2
fatty acid classes. However, the 16:0 fatty acid species was enriched in biliary sphingomyelin to a significantly greater
degree than in sphingomyelins of canalicular-enriched plasma membranes (46% vs. 25% of total). We argue a physical-chemical case for laterally separated domains of very long chain sphingomyelins on the
exoplasmic leaflet of the canalicular membrane. We bolster our hypothesis by the likelihood that the least hydrophobic, e.g.,
16:0 sphingomyelin molecular species, are miscible with biliary phosphatidylcholines, and are secreted into bile. Laterally
separated domains of very long chain sphingomyelins on the exoplasmic leaflet of the canalicular membrane could provide a
means of sequestering cholesterol molecules prior to secretion into bile.
Received: 19 March 1998/Revised: 8 October 1998 相似文献