共查询到20条相似文献,搜索用时 15 毫秒
1.
E. Modesto P. D. Lampe M. C. Ribeiro D. C. Spray A. C. Campos de Carvalho 《The Journal of membrane biology》1996,154(3):239-249
Membrane fractions highly enriched in chicken lens MIP (MIP28) were found to form ion channels when incorporated into planar
lipid bilayers. The channels displayed prominent unitary conductances of about 60 and 290 pS in symmetric 150 mm KCl solution and were slightly anion selective. For both depolarizing and hyperpolarizing voltages, voltage sensitivity of
the MIP28-induced conductance could be fit by a Boltzmann relation, symmetric around zero mV, with V
0
= 18.5 mV, n= 4.5 and g
min/g
max= 0.17. Channel properties were not appreciably altered by pH in the range of 5.8 to 7, although channel incorporation was
observed to occur more frequently at lower pH values. Calcium, at millimolar concentrations, decreased the channel mean open
time. Partial proteolysis of MIP28 to yield MIP21 did not appreciably affect single-channel conductance or voltage sensitivity
of the reconstituted channels. MIP28 was not phosphorylated by cAMP dependent protein kinase (PKA). Although unitary conductance
and selectivity of the chicken MIP channel are similar to those reported for the bovine MIP (MIP26), the voltage sensitivity
of MIP28 was higher than that of the bovine homologue, and voltage sensitivity of MIP28 was not modulated by treatments previously
shown to affect MIP26 voltage gating (partial proteolysis and protein phosphorylation by PKA: (Ehring et al., 1990). The existence
of such strikingly different functional properties in highly homologous channel isoforms may provide a useful system for exploration
of the structure-function relations of MIP channels.
Received: 27 March 1996/Revised: 5 August 1996 相似文献
2.
A.M. Feigin J.Y. Takemoto R. Wangspa J.H. Teeter J.G. Brand 《The Journal of membrane biology》1996,149(1):41-47
Using the planar lipid bilayer technique we demonstrate that the lipodepsipeptide antibiotic, syringomycin E, forms voltage-sensitive
ion channels of weak anion selectivity. The formation of channels in bilayers made from dioleoylglycerophosphatidylserine
doped with syringomycin E at one side (1–40 μg/ml) was greatly affected by cis-positive voltage. A change of voltage from a positive to a negative value resulted in (i) an abrupt increase in the single
channel conductance (the rate of increase was voltage dependent) simultaneous with (ii) a closing of these channels and an
exponential decrease in macroscopic conductance over time. The strong voltage dependence of multichannel steady state conductance,
the single channel conductance, the rate of opening of channels at positive voltages and closing them at negative voltages,
as well as the observed abrupt increase of single channel conductance after voltage sign reversal suggest that the change
of the transmembrane field induces a significant rearrangement of syringomycin E channels, including a change in the spacing
of charged groups that function as voltage sensors. The conductance induced by syringomycin E increased with the sixth power
of syringomycin E concentration suggesting that at least six monomers are required for channel formation.
Received: 3 April 1995/Revised: 24 August 1995 相似文献
3.
C.J. Giffard S. Ladha A.R. Mackie D.C. Clark D. Sanders 《The Journal of membrane biology》1996,151(3):293-300
Nisin, a prominent member of the lantibiotic family of antimicrobial agents, has wide application as a food preservative despite
poor understanding of its mode of action. Fluorescence recovery after photobleaching has been used with planar lipid bilayers
as a model membrane system to examine how nisin might interact with the surface of bacterial cells. Nisin associates with
planar lipid bilayers in the absence of an applied membrane potential causing an array of effects consistent with adsorption
of nisin onto the membrane surface which involves inhibition of the lateral diffusion and fluorescence of the lipid probe
N-(7--1,2,3-benzoxadiazol-4-yl) phosphatidylethanolamine (NBD-PE) and a reduction of the capacitance of the bilayer. Nisin
adsorption is dependent on phospholipid composition. In the presence of dioleoylphosphatidylcholine (PC): cardiolipin (CL)
4:1, the rate of lateral mobility of phospholipid is reduced to 61% of the control level which decreases to a value of 46%
when CL is replaced by 1-palmitoyl-2-oleoylphosphatidylserine (PS). These effects on bilayer parameters are transient, and
with time the values return to near original levels. High electrical conductivity is observed on application of a voltage
ramp suggesting that insertion into the membrane follows surface association. Results have been interpreted in terms of a
model in which nisin initially binds to the surface of the membrane causing a modulation of bilayer properties.
Received: 14 August 1995/Revised: 22 February 1996 相似文献
4.
A novel method was developed for the direct examination of pairwise encounters between positively and negatively charged
phospholipid bilayer vesicles. Giant bilayer vesicles (unilamellar, 4–20 μm in diameter) prepared from 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, a new cationic phospholipid derivative, were electrophoretically maneuvered into contact with
individual anionic phospholipid vesicles. Fluorescence video microscopy revealed that such vesicles commonly underwent fusion
within milliseconds (1 video field) after contact, without leakage. Fusion occurred at constant volume and, since flaccid
vesicles were rare, the excess membrane was not available after fusion. Hemifusion (the outer monolayers of each vesicle fused
while the inner monolayers remained intact) was inferred from membrane-bound dye transfer and a change in the contact area.
Hemifusion was observed as a final stable state and as an intermediate to fusion of vesicles composed of charged phospholipids
plus zwitterionic phospholipids. Hemifusion occurred in one of three ways following adhesion: either delayed with an abrupt
increase in area of contact, immediately with a gradual increase in area of contact, or with retraction during which adherent
vesicles dissociated from a flat contact to a point contact. Phosphatidylethanolamine strongly promoted immediate hemifusion;
the resultant hemifused state was stable and seldom underwent complete fusion. Although sometimes single contacts between
vesicles led to rupture of both, in other cases, a single vesicle underwent multiple fusion events. Direct observation has
unequivocally demonstrated the fusion of two, isolated bilayer-bounded bodies to yield a stable, non-leaky product, as occurs
in cells, in the absence of proteins.
Received: 25 November 1998/Revised: 23 March 1999 相似文献
5.
Boron enters plant roots as undissociated boric acid (H3BO3). Significant differences in B uptake are frequently observed even when plants are grown under identical conditions. It has
been theorized that these differences reflect species differences in permeability coefficient of H3BO3 across plasma membrane. The permeability coefficient of boric acid however, has not been experimentally determined across
any artificial or plant membrane. In the experiments described here the permeability coefficient of boric acid in liposomes
made of phosphatidylcholine was 4.9 × 10−6 cm sec−1, which is in good agreement with the theoretical value. The permeability coefficient varied from 7 × 10−6 to 9.5 × 10−9 cm sec−1 with changes in sterols (cholesterol), the type of phospholipid head group, the length of the fatty acyl chain, and the pH
of the medium. In this study we also used Arabidopsis thaliana mutants which differ in lipid composition to study the effect of lipid composition on B uptake. The chs1-1 mutant which has lower proportion of sterols shows 30% higher B uptake compared with the wild type, while the act1-1 mutant which has an increased percentage of longer fatty acids, exhibited 35% lower uptake than the wild type. Lipid composition
changes in each of the remaining mutants influenced B uptake to various extents. These data suggest that lipid composition
of the plasma membrane can affect total B uptake.
Received: 15 October 1999/Revised: 11 February 2000 相似文献
6.
A 107-pS (symmetrical 150 mm KCl), nonselective cation channel was reconstituted from a microsomal membrane fraction of the larval stage of the tapeworm
Echinococcus granulosus. Most of the time, it displayed a high open probability (>0.95) irrespective of either the applied voltage, Ca2+, Ba2+, or tetraethylammonium concentration. Nevertheless, in contrast with this ``leaklike' behavior, less frequently this ``all-the-time-open'
channel reversibly entered two different kinetic modes. One of them was characterized by lower P
o
values and some voltage sensitivity (V
?≅ 129 mV, and an equilibrium constant for channel closing changing e-fold per 63-mV change) the kinetic analysis revealing that it resulted from the appearance of voltage-sensitivity in the
mean closed times and a sixfold increase in the equilibrium constant for channel closing at 0 mV. The other mode was characterized
by a very fast open-close activity leading to poorly resolved current levels and a P
o
around 0.6–0.7 which, occasionally and in a voltage-sensitive manner, entered a long-lived nonconducting state. However,
the rare nature of these mode-shifting transitions precluded a more detailed analysis of their kinetics. The conductive properties
of the channel were not affected by these switches. Model gating alone does not seem to ensure any physiological role of this
channel and, instead, some other channel changes must occur if this phenomenon were to be of regulatory importance in vivo.
Thus, mode-shifting might constitute an alternative target for channel activity modulation also in tapeworms.
Received: 30 August 1996/Revised: 31 January 1997 相似文献
7.
O.V. Krasilnikov J.B. Da Cruz L.N. Yuldasheva W.A. Varanda R.A. Nogueira 《The Journal of membrane biology》1998,161(1):83-92
This paper describes a new approach to evaluate the inner structure (including a main constriction and its localization)
of the water lumen of an ion channel. The method is based on the determination of channel filling by different nonelectrolyte
molecules through each side of an ion channel. The method has two characteristic features that make its use attractive: (i)
the possibility to ascertain the existence, localization and size of a narrow part inside an ion channel water lumen and (ii)
the chances to determine the maximal size of both entrances of an ion channel and to obtain additional information about the
geometry of its water lumen at the same time. Determinations were made on colicin Ia ion channels inserted into planar lipid
bilayers. This channel was chosen because there is an apparent contradiction between its low single channel conductance and
the large diameter of its water lumen. Our results show that the water lumen of the colicin Ia channel has a funnel-like structure
with a small trans-entrance, with a diameter of about 1.0 nm, and a large cis-entrance, with a diameter of approximately 1.8 nm. A constriction with a diameter of approximately 0.7 nm is shown to be
located close to the trans-entrance of the channel. The method can also be applied to patch clamp studies of single ion channels.
Received: 20 February 1997/Revised: 19 August 1997 相似文献
8.
A K+ channel with a main conductance of 29 pS was recorded after the incorporation of coronary artery membrane vesicles into lipid
bilayers. This channel was identified as an ATP-sensitive K+ channel (KATP) because its activity was diminished by the internal application of 50–250 μm ATP-Na2. Moreover, it was opened when 10–50 μm pinacidil was externally applied. Single-channel records revealed the existence of several (sub)conductance states. At 0
mV and with a 5/250 KCl gradient, the main conductance of the KATP channel was 29 pS. The other (sub)conductance states were less frequent and had discrete values of 12, 17 and 22 pS. Pinacidil
stabilized the channel open state primarily in the 29 pS conductance level; whereas ATP inhibited all the conductance levels.
In general, KATP channels were characterized by brief openings followed by long closings (open probability, P
o
≈ 0.02); only occasionally (3 out of 12 experiments) did the KATP channels have a high open probability (P
o
≥ 0.7). Channel activity could be increased or rescued by adding 2.5–10 mm UDP-TRIS and 0.5–2 mm MgCl2 to the internal side of the channel.
Received: 7 November 1995/Revised: 10 June 1996 相似文献
9.
The apical brush border membrane, the main target site of Bacillus thuringiensis toxins, was isolated from gypsy moth (Lymantria dispar) larval midguts and fused to artificial planar lipid bilayer membranes. Under asymmetrical N-methyl-d-glucamine-HCl conditions (450 mm
cis/150 mm
trans, pH 9.0), which significantly reduce endogenous channel activity, trypsin-activated Cry1Aa, a B. thuringiensis insecticidal protein active against the gypsy moth in vivo, induced a large increase in bilayer membrane conductance at much
lower concentrations (1.1–2.15 nm) than in receptor-free bilayer membranes. At least 5 main single-channel transitions with conductances ranging from 85 to
420 pS were resolved. These Cry1Aa channels share similar ionic selectivity with P
Cl/P
NMDG permeability ratios ranging from 4 to 8. They show no evidence of current rectification. Analysis of the macroscopic current
flowing through the composite bilayer suggested voltage-dependence of several channels. In comparison, the conductance of
the pores formed by 100–500 nm Cry1Aa in receptor-free bilayer membranes was significantly smaller (about 8-fold) and their P
Cl/P
NMDG permeability ratios were also reduced (2- to 4-fold). This study provides a detailed demonstration that the target insect
midgut brush border membrane material promotes considerably pore formation by a B. thuringiensis Cry toxin and that this interaction results in altered channel properties.
Received: 23 February 2001/Revised: 15 June 2001 相似文献
10.
11.
Membrane-related processes in archaea, the third and most-recently described domain of life, are in general only poorly understood.
One obstacle to a functional understanding of archaeal membrane-associated activities corresponds to a lack of archaeal model
membrane systems. In the following, characterization of inverted archaeal membrane vesicles, prepared from the halophilic
archaeon Haloferax volcanii, is presented. The inverted topology of the vesicles was revealed by defining the orientation of membrane-bound enzymes that
in intact cells normally face the cytoplasm or of other protein markers, known to face the exterior medium in intact cells.
Electron microscopy, protease protection assays and lectin-binding experiments confirmed the sealed nature of the vesicles.
Upon alkalinization of the external medium, the vesicles were able to generate ATP, reflecting the functional nature of the
membrane preparation. The availability of preparative scale amounts of inverted archaeal membrane vesicles provides a platform
for the study of various membrane-related phenomena in archaea.
Received: 27 March 2001/Revised: 13 June 2001 相似文献
12.
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
13.
Timothy L. Porter Michael P. Eastman Michael E. Hagerman Lance B. Price Richard F. Shand 《Journal of molecular evolution》1998,47(4):373-377
Condensation reactions of the amino acid glycine on the surface of Cu(II)-exchanged hectorite are investigated using the
technique of scanning force microscopy. Prebiotic conditions are simulated using alternate wetting and heating cycles. Concentration,
immobilization, and subsequent polymerization resulting in glycine oligomers are seen to occur primarily at step edges or
faults in the topmost layer. Condensation reactions also occur within tiny micropores or defects in the topmost layer. These
reactions are facilitated by the availability of intergallery metal cations at the step edges or pores in the surface region.
Received: 19 January 1998 / Accepted: 24 April 1998 相似文献
14.
How thyroid hormones move across biological or model membranes is a subject of controversy. The passage of the 3,5,3′triiodo
l-thyronine and 3,5,3′,5′ tetraiodo l-thyronine across model membranes was evaluated by the addition of the hormones to liposomes containing 2,4,6-trinitrobenzene
sulfonic acid. Results indicate that hormones can react with an amino-reactive compound pre-encapsulated into phosphatidylcholine
liposomes. The transversal motions of thyroid hormones were characterized by using physiological concentration levels of (125I) 3,5,3′triiodo l-thyronine and (125I) 3,5,3′,5′ tetraiodo l-thyronine. The hormone distribution between the two monolayers was time-dependent and kinetic data were fitted to a single
exponential. Results obtained show that 3,5,3′ triiodo l-thyronine can permeate phospholipid membranes and the diffusion time increases in the gel and liquid-ordered phase. On the
contrary, 3,5,3′, 5′ tetraiodo l-thyronine could not diffuse the liposomal membrane from dimyristoyl and dipalmitoyl phosphatidylcholine in gel phase and
egg yolk phosphatidylcholine:cholesterol in the liquid-ordered phase. Our results in the liquid-ordered phase suggest that
diffusion movement of thyroid hormones across cell membranes depends on the amount of cholesterol in the bilayer.
Received: 1 June 1998/Revised: 14 October 1998 相似文献
15.
The charge-pulse relaxation spectrum of nonperfused and perfused (turgescent) cells of the giant marine alga Ventricaria ventricosa showed two main exponential decays with time constants of approximately 0.1 msec and 10 msec, respectively, when the cells were bathed in artificial sea water (pH 8). Variation of the external pH did not change the relaxation pattern (in contrast to other giant marine algae). Addition of nystatin (a membrane-impermeable and pore-forming antibiotic) to the vacuolar perfusion solution resulted in the disappearance of the slow exponential, whereas external nystatin decreased dramatically the time constant of the fast one. This indicated (by analogy to corresponding experiments with Valonia utricularis, J. Wang, I. Spiess, C. Ryser, U. Zimmermann, J. Membrane Biol. 157: 311-321, 1997) that the fast relaxation must be assigned to the RC-properties of the plasmalemma and the slow one to those of the tonoplast. Consistent with this, external variation of [K+]o or of [Cl-]o as well as external addition of K+- or Cl--channel/carrier inhibitors (TEA, Ba2+, DIDS) affected only the fast relaxation, but not the slow one. In contrast, addition of these inhibitors to the vacuolar perfusion solution had no measurable effect on the charge-pulse relaxation spectrum. The analysis of the data in terms of the "two membrane model" showed that K+- and (to a smaller extent) Cl--conducting elements dominated the plasmalemma conductance. The analysis of the charge-pulse relaxation spectra also yielded the following area-specific data for the capacitance and the conductance for the plasmalemma and tonoplast (by assuming that both membranes have a planar surface): (plasmalemma) Cp = 0.82 * 10(-2) F m-2, Rp = 1.69 * 10(-2) Omega m2, Gp = 5.9 * 10(4) mS m-2, (tonoplast) Ct = 7. 1 * 10(-2) F m-2, Rt = 14.9 * 10(-2) Omega m2 and Gt = 0.67 * 10(4) mS m-2. The electrical data for the tonoplast show that (in contrast to the literature) the area-specific membrane resistance of the tonoplast of these marine giant algal cells is apparently very high as reported already for V. utricularis. The exceptionally high value of the area-specific capacitance could be explained - among other interpretations - by assuming a 9-fold enlargement of the tonoplast surface. The hypothesis of a multifolded tonoplast was supported by transmission electronmicroscopy of cells fixed under maintenance of turgor pressure and of the electrical parameters of the membranes. This finding indicates that the tonoplast of this species exhibited a sponge-like appearance. Taking this result into account, it can be easily shown that the tonoplast exhibits a high-resistance (1.1 Omega m2). Vacuolar membrane potential measurements (performed in parallel with charge-pulse relaxation studies) showed that the potential difference across the plasmalemma was mainly controlled by the external K+-concentration which suggested that the resting membrane potential of the plasmalemma is largely a K+-diffusion potential. After permeabilization of the tonoplast with nystatin the potential of the intact membrane barrier dropped from about slightly negative or positive (-5.1 to +18 mV, n = 13) to negative values (-15 up to -68 mV; n = 8). This indicated that the cytoplasm of V. ventricosa was apparently negatively charged relative to the external medium. Permeabilization of the plasmalemma by addition of external nystatin resulted generally in an increase in the potential to slightly more positive values (-0.8 to +4.3 mV; n = 5), indicating that the vacuole is positively charged relative to the cytoplasm. These findings apparently end the long-term debate about the electrical properties of V. ventricosa. The results presented here support the findings of Davis (Plant Physiol. 67: 825-831, 1981), but are contrary to the results of Lainson and Field (J. Membrane Biol. 29: 81-94, 1976). 相似文献
16.
Unilamellar liposomes with native phospholipid fatty acid composition were prepared from rat liver mitochondrial inner membrane
phospholipids by extrusion in medium containing 50 mm potassium. They were diluted into low potassium medium to establish a transmembrane potassium gradient. A known membrane
potential was imposed by addition of valinomycin, and proton flux into liposomes was measured. Valinomycin in the range 10
pm–1nm was sufficient to fully establish membrane potential. Valinomycin concentrations above 3 nm catalyzed additional proton flux and were avoided. At 300 pm valinomycin, proton flux depended nonlinearly on membrane potential. At 160 mV membrane potential the flux was 30 nmol H+/min/mg phospholipid—approximately 5% of the proton leak flux under comparable conditions in isolated mitochondria, indicating
that leak pathways through bulk phospholipid bilayer account for only a small proportion of total mitochondrial proton leak.
Received: 5 August 1996/Revised: 1 October 1996 相似文献
17.
Duda VI Suzina NE Severina LO Dmitriev VV Karavaiko GI 《The Journal of membrane biology》2001,180(1):33-48
Analysis of freeze-fracture replicas and thin sections of cells of the bacteria Sulfobacillus thermosulfidooxidans and Anaerobacter polyendosporus showed that their cytoplasmic membranes contain some regions in the form of flat lamellar inverted lipid membranes a few
tenths of nanometers to a few microns in size. The specific features of these membrane structures are as follows: (i) they
contain no familiar intramembrane particles commonly present on freeze-fracture replicas; (ii) in cross thin sections, intramembrane
structures are bifurcate on the periphery and look like thylakoids; and (iii) the leaflets of intramembrane structures in
S. thermosulfidooxidans cells are corrugated. These structures were revealed in bacterial cells cultivated under normal growth conditions. The data
obtained suggest the occurrence of a complex type of compartmentalization in biological membranes.
Received: 17 July 2000/Revised: 22 November 2000 相似文献
18.
L-lactate transport mechanism across rat jejunal enterocyte was investigated using isolated membrane vesicles. In basolateral
membrane vesicles l-lactate uptake is stimulated by an inwardly directed H+ gradient; the effect of the pH difference is drastically reduced by FCCP, pCMBS and phloretin, while furosemide is ineffective.
The pH gradient effect is strongly temperature dependent. The initial rate of the proton gradient-induced lactate uptake is
saturable with respect to external lactate with a K
m
of 39.2 ± 4.8 mm and a J
max of 8.9 ± 0.7 nmoles mg protein−1 sec−1. A very small conductive pathway for l-lactate is present in basolateral membranes. In brush border membrane vesicles both Na+ and H+ gradients exert a small stimulatory effect on lactate uptake. We conclude that rat jejunal basolateral membrane contains
a H+-lactate cotransporter, whereas in the apical membrane both H+-lactate and Na+-lactate cotransporters are present, even if they exhibit a low transport rate.
Received: 22 October 1996/Revised: 11 March 1997 相似文献
19.
Influence of membrane physical state on the proton permeability of isolated lysosomes was assessed by measuring the membrane
potential with 3,3′-dipropylthiadicarbocyanine iodide and monitoring their proton leakage with p-nitrophenol. Changes in the membrane order were examined by the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene.
Both the membrane potential and proton leakage increased with fluidizing the lysosomal membranes by benzyl alcohol and decreased
with rigidifying the membranes by cholesteryl hemisuccinate. The proton permeability increased to the maximum of 42% by the
benzyl alcohol treatment and decreased to the minimum of 38.1% by the cholesteryl hemisuccinate treatment. Treating the lysosomes
with protonophore CCCP increased the proton permeability by 58%. The effects of the membrane fluidization and rigidification
can be reversed by rigidifying the fluidized membranes and fluidizing the rigidified membranes, respectively. The results
indicate that the proton permeability of lysosomes increased and decreased with increasing and decreasing their membrane fluidity,
respectively. Moreover, the lysosomal proton permeability did not alter further if the changes, either an increase or a decrease,
in the fluidity exceeded some amount. The results suggest that the proton permeability of lysosomes can be modulated finitely
by the alterations in their membrane physical state.
Received: 27 September 1999 / Revised: 27 December 1999 相似文献
20.
C. Grosman M. I. Mariano J. P. Bozzini I. L. Reisin 《The Journal of membrane biology》1997,157(1):83-95
We describe the first successful reconstitution of placental ionic channels on planar lipid bilayers. An apical plasma membrane-enriched
vesicle fraction from human syncytiotrophoblast at term was prepared by following isotonic agitation, differential centrifugation,
and Mg2+-induced selective precipitation of nonapical membranes, and its purity was assessed by biochemical and morphological marker
analysis. We have already reported that, unlike previous patch-clamp studies, nonselective cation channels were incorporated
in most cases, a result consistent with the higher permeability for cations as compared with Cl− and with the low apical membrane potential difference at term revealed by fluorescent probe partition studies, and microelectrode
techniques. In this paper, we report that Cl−-selective channels were incorporated in 4% of successful reconstitutions (14 out of 353) and that their analysis revealed
two types of activity. One of them was consistent with a voltage-dependent, 100-pS channel while the other was consistent
with the lateral association of 47-pS conductive units, giving rise to multibarrelled, DIDS-sensitive channels of variable
conductance (300 to 650 pS). The latter displayed a very complex behavior which included cooperative gating of conductive
units, long-lived substates, voltage-dependent entry into an apparent inactivated state, and flickering activity. The role
of the reported Cl− channels in transplacental ion transport and/or syncytium homeostasis remains to be determined.
Received: 17 September/Revised: 12 December 1996 相似文献