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1.
In the giant-celled marine algae Valonia utricularis the turgor-sensing mechanism of the plasmalemma and the role of the tonoplast in turgor regulation is unknown because of
the lack of solid data about the individual electrical properties of the plasmalemma and the vacuolar membrane. For this reason,
a vacuolar perfusion technique was developed that allowed controlled manipulation of the vacuolar sap under turgescent conditions
(up to about 0.3 MPa). Charge-pulse relaxation studies on vacuolarly perfused cells at different turgor pressure values showed
that the area-specific resistance of the total membrane barrier (tonoplast and plasmalemma) exhibited a similar dependence
on turgor pressure as reported in the literature for nonperfused cells: the resistance assumed a minimum value at the physiological
turgor pressure of about 0.1 MPa. The agreement of the data suggested that the perfusion process did not alter the transport
properties of the membrane barrier.
Addition of 16 μm of the H+-carrier FCCP (carbonylcyanide p-trifluoromethoxyphenyhydrazone) to the perfusion solution resulted in a drop of the total membrane potential from +4 mV to
−22 mV and in an increase of the area-specific membrane resistance from 6.8 × 10−2 to 40.6 × 10−2Ωm2. The time constants of the two exponentials of the charge pulse relaxation spectrum increased significantly. These results
are inconsistent with the assumption of a high-conductance state of the tonoplast (R. Lainson and C.P. Field, J. Membrane Biol.
29:81–94, 1976).
Depending on the site of addition, the pore-forming antibiotics nystatin and amphotericin B affected either the time constant
of the fast or of the slow relaxation (provided that the composition of the perfusion solution and the artificial sea water
were replaced by a cytoplasma-analogous medium). When 50 μm of the antibiotics were added externally, the fast relaxation process disappeared. Contrastingly, the slow relaxation process
disappeared upon vacuolar addition. The antibiotics cannot penetrate biomembranes rapidly, and therefore, the findings suggested
that the fast and slow relaxations originated exclusively from the electrical properties of the plasmalemma and the tonoplast
respectively. This interpretation implies that the area-specific resistance of the tonoplast is significantly larger than
that of the plasmalemma (consistent with the FCCP data) and that the area-specific capacitance of the tonoplast is unusually
high (6.21 × 10−2 Fm−2 compared to 0.77 × 10−2 Fm−2 of the plasmalemma). Thus, we have to assume that the vacuolar membrane of V. utricularis is highly folded (by a factor of about 9 in relation to the geometric area) and/or contains a fairly high concentration of
mobile charges of an unknown electrogenic ion carrier system.
Received: 22 October 1996/Revised: 16 January 1997 相似文献
2.
V.L. Sukhorukov R. Benkert G. Obermeyer F.-W. Bentrup U. Zimmermann 《The Journal of membrane biology》1998,161(1):21-32
The dielectric structure of mature pollen of the angiosperm Lilium longiflorum was studied by means of single-cell electrorotation. The use of a microstructured four-electrode chamber allowed the measurements
to be performed over a wide range of medium conductivity from 3 to 500 mS m−1. The rotation spectra of hydrated pollen grains exhibited at least three well-resolved peaks in the kHz-MHz frequency range,
which obviously arise due to the multilayered structure of pollen grains. The three-shell model can explain the complex rotational
behavior of pollen grains in terms of conductivities, permittivities and thicknesses of the following compartments: the exine
and intine of the pollen grain wall as well as the membrane and cytoplasm of the vegetative cell. However, the number of unknown
parameters (more than 8) was too large to allow unambiguous values to be assigned to any of them. Therefore, to facilitate
the evaluation of the pollen grain parameters, additional rotational measurements were made on isolated vegetative and generative
cells. The rotation spectra of these cells could be fitted very accurately on the basis of the single-shell model by assuming
a dispersion of the cytoplasm. The data on the membrane and cytoplasmic properties of isolated vegetative cells were then
used for modeling the rotation spectra of pollen grains. This greatly facilitated the fitting of the theoretical model to
the experimental data and allowed the dielectric properties of the major structural units to be determined. The dielectric
characterization of pollen is of enormous interest for plant biotechnology, where pollen and isolated germ cells are successfully
used for production of transgenic crop and drug plants of economic importance by means of electromanipulation techniques.
Received: 9 June 1997/Revised: 4 August 1997 相似文献
3.
Fresh-water plants generate extraordinarily high electric potential differences at the plasma membrane. For a deeper understanding
of the underlying transport processes a mathematical model of the electrogenic plasmalemma ion transport was developed based
on experimental data mainly obtained from Egeria densa. The model uses a general nonlinear network approach and assumes coupling of the transporters via membrane potential. A proton pump, an outward-rectifying K+ channel, an inward-rectifying K+ channel, a Cl− channel and a (2H-Cl)+ symporter are considered to be elements of the system. The model takes into consideration the effects of light, external
pH and ionic content of the bath medium on ion transport. As a result it does not only satisfactorily describe the membrane
potential as a function of these external physiological factors but also succeeds in simulating the effects of specific inhibitors
as well as I-V-curves obtained with the patch-clamp technique in the whole cell mode. The quality of the model was checked by stability and
sensitivity analyses.
Received: 18 March 1996/Revised: 17 July 1996 相似文献
4.
Photofrin II is a photosensitizer frequently applied in photodynamic therapy. Light-induced tumor cell inactivation observed
in the presence of this substance has been suggested to start with modifications at the level of cellular membranes. In the
present study electrophysiological techniques are applied in order to investigate the action of photofrin II on functional
properties of the plasma membrane of opossum kidney (OK) cells (as an epithelial model system) and of fibroblasts. Illumination
of the cells in the presence of photofrin II (or Zn-phthalocyanine) leads to comparatively fast depolarization of the membrane
potential. It is caused by a strong change of the membrane conductance which proceeds in two phases. Both phases contribute
to a loss of ion selectivity of the plasma membrane between K+ and Na+. In the first phase, specific pathways for K+, which determine the resting potential under physiological conditions, are inactivated. The second phase is distinguished
by a marked increase of a nonselective conductance. The increase of the latter — after light-induced initiation — continues
in the dark. The conclusions are derived from light-induced, time-dependent changes of the membrane conductance and of the
shape of the current-voltage relationship detected under different experimental conditions.
Received: 26 May 1998/Revised: 8 September 1998 相似文献
5.
H. Ramos E. Valdivieso M. Gamargo F. Dagger B.E. Cohen 《The Journal of membrane biology》1996,152(1):65-75
The polyene antibiotic amphotericin B (AmB) is known to form two types of ionic channels across sterol-containing liposomes,
depending on its concentration and time after mixing (Cohen, 1992). In the present study, it is shown that AmB only kills
unicellular Leishmania promastigotes (LPs) when aqueous pores permeable to small cations and anions are formed. Changes of membrane potential across
ergosterol-containing liposomes and LPs were followed by fluorescence changes of 3,3′ dipropylthiadicarbocyanine (DiSC3(5)). In KCl-loaded liposomes suspended in an iso-osmotic sucrose solution, low AmB concentrations (≤0.1 μm) induced a polarization potential, indicating K+ leakage, but no movement of cations and anions was allowed until AmB concentrations greater than 0.1 μm were added. In agreement with these data, it was found that AmB altered the negative membrane potential held across LPs in
a manner consistent with the differential cation/anion selectivity exhibited by the channels formed in liposomes. Thus, LPs
suspended in an iso-osmotic sucrose solution did not exhibit any AmB-induced membrane depolarization effect brought about
by efflux of anions until 0.1 μm or higher AmB concentrations were added. By contrast, LPs suspended in an iso-osmotic NaCl solution and exposed to 0.05 μm AmB exhibited a nearly total collapse of the negative membrane potential, indicating Na+ entry into the cells.
The concentration dependence of the AmB-induced permeability to different salts was also measured across vesicles derived
from the plasma membrane of leishmanias (LMVs), by using a rapid mixing technique. At concentrations above 0.1 μm, AmB induced the formation of aqueous pores across LMVs with a positive cooperativity, yielding Hill coefficients between
2 to 3. Measured anion selectivity across such aqueous pores followed the sequence: SCN > NO3 > Cl > I > Br > acetate (SO2−
4 being impermeable). Cell killing by AmB was followed by fluorescence changes of the DNA-binding compound ethidium bromide
(EB). At low concentrations (≤0.1 μm), AmB was found to be nonlethal against LPs but, above this concentration, leishmanias were rapidly killed. The rate and
extent of such an effect were found to be dependent on the type of cation and anion present in the external aqueous solution.
For both NH+
4 and Na+ salts, the measured rank order of AmB cell killing followed the same sequence that was determined for AmB-induced salt permeation
across LMVs. Further, replacement of either extracellular Na+ by choline or Cl− by SO2−
4, or its partial substitution by sucrose, in iso-osmotic conditions, led to a complete inhibition of the killing effect exerted
by otherwise lethal AmB concentrations. Finally, it was shown that tetraethylammonium (TEA+), an organic cation that is known to block AmB-induced salt permeation across LMVs was able to retard the time lag observed
for EB incorporation across LPs, indicating that this parameter can be taken to represent the time taken for salt accumulation
inside the parasites. The present results thus indicate clearly that low AmB concentrations (≤0.1 μm) were able to form across LPs, cation channels that collapsed the parasite membrane potential but are not lytic. At high
concentrations (<≥0.1 μm), a salt influx via the aqueous pores formed by the antibiotic was followed by osmotic changes leading to cell lysis. This
last stage is supported by electron microscopy observations of the changes of parasite morphology immediately upon addition
of AmB, which indicated that the typical elongated promastigote cell forms became rounded and the flagella swells and round
up. The present work is the first demonstration of the in vitro sensitivity of Leishmania promastigotes to osmotic lysis by AmB.
Received: 25 September 1995/Revised: 11 March 1996 相似文献
6.
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
7.
I.T. Arkin P.D. Adams A.T. Brünger S. Aimoto D.M. Engelman S.O. Smith 《The Journal of membrane biology》1997,155(3):199-206
Phospholamban, a 52-residue membrane protein, associates to form a pentameric complex of five long α-helices traversing the
sarcoplasmic reticulum membrane of cardiac muscle cells. The transmembrane domain of the protein is largely hydrophobic, with
only three cysteine residues having polar side chains, yet it functions as a Ca2+-selective ion channel. In this report, infrared spectroscopy is used to probe the conformation of the three cysteine side
chains and to establish whether the free S-H groups form intrahelical hydrogen bonds in the pentameric complex. Vibrational
spectra of a transmembrane peptide were obtained which corresponded to the transmembrane domain of wild-type phospholamban
and three peptides each containing a cysteine ⇒ alanine substitution. The observed S-H frequencies argue that each of the
sulfhydryl groups is hydrogen-bonded to an i-4 backbone carbonyl oxygen. Electrostatic calculations on a model of phospholamban
based on molecular dynamics and mutagenesis studies, show that the sulfhydryl groups may significantly contribute to the electrostatic
potential field of the protein.
Received: 22 July 1996/Revised: 10 October 1996 相似文献
8.
Membrane trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) is supposed to be an important mechanism
controlled by the intracellular messenger cAMP. This has been shown with fluorescence techniques, electron microscopy and
membrane capacitance measurements. In order to visualize protein insertion we applied atomic force microscopy (AFM) to inside-out
oriented plasma membrane patches of CFTR-expressing Xenopus laevis oocytes before and after cAMP-stimulation. In a first step, oocytes injected with CFTR-cRNA were voltage-clamped, verifying
successful CFTR expression. Water-injected oocytes served as controls. Then, plasma membrane patches were excised, placed
(inside out) on glass and scanned by AFM. Before cAMP-stimulation plasma membranes of both water-injected and CFTR-expressing
oocytes contained about 200 proteins per μm2. Molecular protein masses were estimated from molecular volumes measured by AFM. Before cAMP-stimulation, protein distribution
showed a peak value of 11 nm protein height corresponding to 475 kDa. During cAMP-stimulation with 1 mm isobutylmethylxanthine (IBMX) plasma membrane protein density increased in water-injected oocytes to 700 proteins per μm2 while the peak value shifted to 7 nm protein height corresponding to 95 kDa. In contrast, CFTR-expressing oocytes showed
after cAMP-stimulation about 400 proteins per μm2 while protein distribution exhibited two peak values, one peak at 10 nm protein height corresponding to 275 kDa and another
one at 14 nm corresponding to 750 kDa. They could represent heteromeric protein clusters associated with CFTR. In conclusion,
we visualized plasma membrane protein insertion upon cAMP-stimulation and quantified protein distribution with AFM at molecular
level. We propose that CFTR causes clustering of plasma membrane proteins.
Received: 11 September 2000/Revised: 13 December 2000 相似文献
9.
The possible correlation between P-glycoprotein (PGP) and volume-sensitive Cl− channel was examined in a pair of cell lines: a subline of the human epidermoid KB cell (KB-3-1) and the corresponding MDR1-transfected cell line (KB-G2). Western blot analysis and indirect immunofluorescence studies indicated that KB-G2, but not
KB-3-1, exhibits the PGP expression. Patch-clamp whole-cell recordings showed that osmotic swelling activates Cl− currents not only in PGP-expressing but also in PGP-lacking cells. The amplitude of the maximal current was indistinguishable
between both cells. Activation of protein kinase C (PKC) or loading with a PKC inhibitor failed to affect the swelling-induced
activation of the Cl− currents in both cells. The relation between whole-cell Cl− currents and cell size measured simultaneously showed that volume sensitivity of the Cl− channel was augmented by the PGP expression irrespective of the activity of PKC on the plasma membrane. A similar increase
in volume sensitivity of the Cl− channel was also induced by the expression of the ATP hydrolysis-deficient PGP mutant, K433M. We conclude that P-glycoprotein
does not represent the volume-sensitive Cl− channel but that its expression modulates volume sensitivity of the Cl− channel in a manner independent of its ATPase activity or of the protein kinase C activity.
Received: 25 September 1996/Revised: 12 December 1996 相似文献
10.
Nuclear magnetic resonance (NMR) microimaging and proton relaxation times were used to monitor differences between the hydration
state of the nucleus and cytoplasm in the Rana pipiens oocyte. Individual isolated ovarian oocytes were imaged in a drop of Ringer's solution with an in-plane resolution of 80
μm. Proton spin echo images of oocytes arrested in prophase I indicated a marked difference in contrast between nucleoplasm
and cytoplasm with additional intensity gradations between the yolk platelet-rich region of the cytoplasm and regions with
little yolk. Neither shortening τe (spin echo time) to 9 msec (from 18 msec) nor lengthening τr (spin recovery time) to 2 sec (from 0.5 sec) reduced the observed contrast between nucleus and cytoplasm. Water proton T1 (spin-lattice) relaxation times of oocyte suspensions indicated three water compartments that corresponded to extracellular
medium (T1= 3.0 sec), cytoplasm (T1= 0.8 sec) and nucleoplasm (T1= 1.6 sec). The 1.6 sec compartment disappeared at the time of nuclear breakdown. Measurements of plasma and nuclear membrane
potentials with KCl-filled glass microelectrodes demonstrated that the prophase I oocyte nucleus was about 25 mV inside positive
relative to the extracellular medium. A model for the prophase-arrested oocyte is proposed in which a high concentration of
large impermeant ions together with small counter ions set up a Donnan-type equilibrium that results in an increased distribution
of water within the nucleus in comparison with the cytosol. This study indicates: (i) a slow exchange between two or more
intracellular water compartments on the NMR time-scale, (ii) an increased rotational correlation time for water molecules
in both the cytoplasmic and nuclear compartments compared to bulk water, and (iii) a higher water content (per unit dry mass)
of the nucleus compared to the cytoplasm, and (iv) the existence of a large (about 75 mV positive) electropotential difference
between the nuclear and cytoplasmic compartments.
Received: 18 January 1996/Revised: 29 April 1996 相似文献
11.
Removal of extracellular divalent cations activated a Cl− channel in the plasma membrane of Xenopus laevis oocytes. This so-called Ca2+-inactivated Cl− channel (CaIC) was present in every oocyte and was investigated using two-electrode whole-cell voltage clamp and single-channel
patch-clamp techniques. Beside other Cl− channel inhibitors, anthracene-9-carboxylic acid (9-AC) and 3′azido-3′deoxythymidine (AZT), a nucleoside analogue commonly
used as an antiviral drug, blocked at least partly the CalC-mediated currents.
Using the Cl−-sensitive dye 6-methoxy-N-(sulfopropyl)quinolinium (SPQ) we could visualize the transport of Cl− from the oocyte cytoplasm to the surrounding medium after activation of the CaIC by Ca2+ removal. In the absence of external Cl− and Ca2+, the emission intensity of SPQ declined continuously, indicating a quenching of fluorescence by the efflux of Cl− in the millimolar range. In the presence of external Ca2+, no emission changes could be observed during the same time period. Chelating external Ca2+ in absence of Cl− immediately activated Ca2+-inactivated Cl− channels leading to subsequent emission decrease of SPQ.
Investigations on the selectivity of the CaIC revealed only poor discrimination between different anions. With single-channel
measurements, we found an anion selectivity sequence I− > Br− > Cl−≫ gluconate as it is also typical for maxi Cl− channels.
Contrary to the majority of all other transport systems of the Xenopus oocyte, which show reduced activity due to membrane depolarization or endocytotic removal of the transport protein from the
plasma membrane during oocyte maturation, the CaIC remained active in maturated oocytes. Single-channel measurements on maturated
oocytes, also known as eggs, showed the presence of Ca2+-inactivated Cl− channels. However, this egg CaIC revealed an altered sensitivity to external Ca2+ concentrations.
All these data confirm and extend our previous observations on the CaIC and give clear evidence that this channel is peculiar
among all Cl− channels described up to now.
Received: 16 May 1996/Revised: 4 September 1996 相似文献
12.
Muscarinic m3 receptor-mediated changes in cytosolic Ca2+ concentration ([Ca2+]l) occur by activation of Ca2+ release channels present in the endoplasmic reticulum membrane and Ca2+ entry pathways across the plasma membrane. In this report we demonstrate the coupling of m3 muscarinic receptors to the activation
of a voltage-insensitive, cation-selective channel of low conductance (3.2 ± 0.6 pS; 25 mm Ca2+ as charge carrier) in a fibroblast cell line expressing m3 muscarinic receptor clone (A9m3 cells). Carbachol (CCh)-induced
activation of the cation-selective channel occurred both in whole cell and excised membrane patches (CCh on the external side),
suggesting that the underlying mechanism involves receptor-channel coupling independent of intracellular messengers. In excised
inside-out membrane patches from nonstimulated A9m3 cells GTP (10 μm) and GDP (10 μm) activated cation-selective channels with conductances of approximately 4.3 and 3.3 pS, (25 mm Ca2+ as charge carrier) respectively. In contrast, ATP (10 μm), UTP (10 μm) or CTP (10 μm) failed to activate the channel. Taken together, these results suggest that carbachol and guanine nucleotides regulate the
activation of a cation channel that conducts calcium.
Received: 14 November 1996/Revised: 4 April 1997 相似文献
13.
M.J.C. Bijvelds Z.I. Kolar S.E. Wendelaar Bonga G. Flik 《The Journal of membrane biology》1996,154(3):217-225
In tilapia (Oreochromis mossambicus) intestine, Mg2+ transport across the epithelium involves a transcellular, Na+- and Na+/K+-ATPase dependent pathway. In our search for the Mg2+ extrusion mechanism of the basolateral compartment of the enterocyte, we could exclude Na+/Mg2+ antiport or ATP-driven transport. Evidence is provided, however, that Mg2+ movement across the membrane is coupled to anion transport. In basolateral plasma membrane vesicles, an inwardly directed
Cl− gradient stimulated Mg2+ uptake (as followed with the radionuclide 27Mg) twofold. As Cl−-stimulated uptake was inhibited by the detergent saponin and by the ionophore A23187, Mg2+ may be accumulated intravesicularly above chemical equilibrium. Valinomycin did not affect uptake, suggesting that electroneutral
symport activity occurred. The involvement of anion coupled transport was further indicated by the inhibition of Mg2+ uptake by the stilbene derivative, 4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid. Kinetic analyses of the Cl−-stimulated Mg2+ uptake yielded a K
m
(Mg2+) of 6.08 ± 1.29 mmol · l−1 and a K
m
(Cl−) of 26.5 ± 6.5 mmol · l−1, compatible with transport activity at intracellular Mg2+- and Cl−-levels. We propose that Mg2+ absorption in the tilapia intestine involves an electrically neutral anion symport mechanism.
Received: 19 January 1996/Revised: 1 August 1996 相似文献
14.
The putative role of lysophospholipids in activation and regulation of the volume-sensitive taurine efflux was investigated
in HeLa cells using tracer technique. Lysophosphatidylcholine (LPC, 10 μm) with oleic acid increased taurine efflux during hypotonic and isotonic conditions. Substituting palmitic or stearic acid
for oleic acid enhanced taurine release during isotonic conditions, whereas ethanolamine, serine or inositol containing lysophospholipids
were ineffective. High concentrations of LPC (25 μm) induced Ca2+ influx, loss of adenosine nucleotides, taurine and the Ca2+-sensitive probe Fura-2, and thus reflected a general breakdown of the membrane permeability barrier. Low concentrations of
LPC (5–10 μm) solely induced taurine efflux. The LPC-induced taurine release was unaffected by anion channel blockers (DIDS, MK196) and
the 5-lipoxygenase inhibitor ETH 615-139, which all blocked the volume sensitive taurine efflux. Furthermore, LPC-induced
taurine release was reduced by antioxidants (NDGA, vitamin E) and the protein tyrosine kinase inhibitor genistein. The swelling-induced
taurine efflux was in the absence of LPC unaffected by vitamin E, blocked by genistein, and increased by H2O2 and the protein tyrosine phosphatase inhibitor vanadate. It is suggested that low concentrations of LPC permeabilizes the
plasma membrane in a Ca2+-independent process that involves generation of reactive oxygen species and tyrosine phosphorylation, and that LPC is not
a second messenger in activation of the volume sensitive taurine efflux in HeLa cells.
Received: 17 December 1999/Revised: 13 April 2000 相似文献
15.
The hyperpolarization of the electrical plasma membrane potential difference has been identified as an early response of
plant cells to various signals including fungal elicitors. The hyperpolarization-activated influx of Ca2+ into tomato cells was examined by the application of conventional patch clamp techniques. In both whole cell and single-channel
recordings, clamped membrane voltages more negative than −120 mV resulted in time- and voltage-dependent current activation.
Single-channel currents saturated with increasing activities of Ca2+ and Ba2+ from 3 to 26 mm and the single channel conductance increased from 4 pS to 11 pS in the presence of 20 mm Ca2+ or Ba2+, respectively. These channels were 20–25 and 10–13 times more permeable to Ca2+ than to K+ and to Cl−, respectively. Channel currents were strongly inhibited by 10 μm lanthanum and 50% inhibited by 100 μm nifedipine. This evidence suggests that hyperpolarization-activated Ca2+-permeable channels provide a mechanism for the influx of Ca2+ into tomato cells.
Received: 13 February 1996/Revised: 12 August 1996 相似文献
16.
A voltage-activated Ca++ channel has been identified in the apical membranes of cultured rabbit proximal tubule cells using the patch-clamp technique.
With 105 mm CaCl2 solution in the pipette and 180 NaAsp in the bath, the channel had a conductance of 10.4 ± 1.0 pS (n= 8) in on-cell patches, and 9.8 ± 1.1 pS (n= 8) in inside-out patches. In both on-cell and inside-out patches, the channel is active by membrane depolarization. For
this channel, the permeation to Ba++ and Ca++ is highly selective over Na+ and K+ (PCa(Ba):PNa(K) >200:1). The sensitivity to dihydropyridines is similar to that for L-type channels where the channel was blocked by nifedipine
(10 μm), and activated by Bay K 8644 (5 μm). When activated by Bay K 8644, the channel showed subconductance levels. Treatment with forskolin (12.5 μm), phorbol ester (1 μm), or stretching (40 cm water) did not activate this channel. These results indicate that this Ca++ channel is mostly regulated by membrane voltage, and appears to be an epithelial class of L-type Ca++ channel. As such, it may participate in calcium reabsorption during periods of enhanced sodium reabsorption, or calcium signaling
in volume regulation, where membrane depolarization occurs for prolonged periods.
Received: 1 April 1996/Revised: 5 August 1996 相似文献
17.
The proximal tubule Na+-HCO−
3 cotransporter is located in the basolateral plasma membrane and moves Na+, HCO−
3, and net negative charge together out of the cell. The presence of charge transport implies that at least two HCO−
3 anions are transported for each Na+ cation. The actual ratio is of physiological interest because it determines direction of net transport at a given membrane
potential. To determine this ratio, a thermodynamic approach was employed that depends on measuring charge flux through the
cotransporter under defined ion and electrical gradients across the basolateral plasma membrane. Cells from an immortalized
rat proximal tubule line were grown as confluent monolayer on porous substrate and their luminal plasma membrane was permeabilized
with amphotericin B. The electrical properties of these monolayers were measured in a Ussing chamber, and ion flux through
the cotransporter was achieved by applying Na+ or HCO−
3 concentration gradients across the basolateral plasma membrane. Charge flux through the cotransporter was identified as difference
current due to the reversible inhibitor dinitro-stilbene disulfonate. The cotransporter activity was Cl− independent; its conductance ranged between 0.12 and 0.23 mS/cm2 and was voltage independent between −60 and +40 mV. Reversal potentials obtained from current-voltage relations in the presence
of Na+ gradients were fitted to the thermodynamic equivalent of the Nernst equation for coupled ion transport. The fit yielded a
cotransport ratio of 3HCO−
3:1Na+.
Received: 19 January 1996/Revised: 24 April 1996 相似文献
18.
Plasma membrane anion channels are thought to play important roles in osmoregulation and signal transduction in higher plant
cells. Knowledge of their pharmacology and regulation is of importance to unravel their physiological functions. In this study,
we explore the pharmacological properties and the nucleotide regulation of the voltage-dependent anion channel of Arabidopsis hypocotyls. The pharmacological profile of this channel is characterized by a low sensitivity to most anion channel blockers.
It is inhibited by niflumic acid with an IC50 of 80 μm, but poorly sensitive to IAA-94 and NPPB and insensitive to 9-AC and DIDS. Nucleotides alter the amplitude, the kinetics
and the voltage-dependence of the channel. The main effect of nucleotides is a shift of the voltage-dependent gate of the
channel toward depolarized potentials leading to a strong reduction of the current amplitude. This regulation does not require
ATP hydrolysis as nonhydrolyzable ATP analogues—AMPPNP and ATPγS—also regulate the anion current. This suggests that a nucleotide
binding site is involved in the regulation. The study of the properties of this putative nucleotide binding site reveals that
(i) ATP regulates the channel with an EC50 of 0.7 mm, (ii) adenyl nucleotides modulate the channel with the following order of effectiveness: ATP > ADP ≫ AMP, and (iii) thiophosphate
nucleotide analogues are the most potent agonists with EC50 in the range of 80 μm. The hypothesis that this regulation may couple the electrical properties of the membrane with the metabolic status of the
cell is discussed.
Received: 26 December 1996/Revised: 21 March 1997 相似文献
19.
Mouse myeloma cells were electropermeabilized by single square-wave electric pulses with amplitudes of up to ∼150 kV/cm and
durations of 10–100 nsec. The effects of the field intensity, pulse duration and medium conductivity on cell viability and
field-induced uptake of molecules were analyzed by quantitative flow cytometry using the membrane-impermeable fluorescent
dye propidium iodide as indicator molecule. Despite the extremely large field strengths, the majority of cells survived the
exposure to ultra-short field pulses. The electrically induced dye uptake increased markedly with decreasing conductivity
of the suspending medium. We assigned this phenomenon to the transient electrodeformation (stretching) force that assumes
its maximum value if cells are suspended in low-conductivity media, i.e., if the external conductivity σe is smaller than that of the cytosol σi. The stretching force vanishes when σe is equal to or larger than σi. Due to their capability of delivering extremely large electric fields, the pulse power systems used here appear to be a
promising tool for the electropermeabilization of very small cells and vesicles (including intracellular organelles, liposomes,
etc.).
Received: 15 May 2001/Revised: 20 July 2001 相似文献
20.
In attached patches on the plasma membrane of nonexcited Chara corallina cells, randomly activating, transient Cl− currents with variable amplitudes were recorded. The peak amplitudes of these currents could be grouped into distinct populations
with approximately equidistant mean peak currents. Generally, the mean current of the smallest population measured about half
of the distance between the means of subsequent populations. Currents of the smallest population occurred most frequently
at all voltages; the frequency of observations decreased with increasing amplitudes of the currents. At all voltages transient
currents from different populations were similar in duration with the exception of the smallest currents, which lasted only
0.6 times as long as larger currents. Furthermore, transient currents were most frequent at positive voltages, but once initiated
at a positive conditioning pulse they were also observed during subsequent pulses to negative voltages. The results are consistent
with the idea that Chara contains Ca2+ stores in the vicinity of the plasma membrane, which are indirectly filled from the external medium. Upon quantal Ca2+ discharge from adjacent stores, a process independent of membrane voltage, the concentration of Ca2+ in the cytoplasm increases transiently. Depending on the number of discharging stores, distinct numbers of Ca2+-stimulated Cl− channels activate, giving rise to the macroscopic excitatory Cl− current in these cells.
Received: 27 October 1997/Revised: 26 February 1998 相似文献