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1.
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
2.
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
3.
T. Ishikawa 《The Journal of membrane biology》1996,153(2):147-159
A Ca2+-activated Cl− conductance in rat submandibular acinar cells was identified and characterized using whole-cell patch-clamp technique. When
the cells were dialyzed with Cs-glutamate-rich pipette solutions containing 2 mm ATP and 1 μm free Ca2+ and bathed in N-methyl-d-glucamine chloride (NMDG-Cl) or Choline-Cl-rich solutions, they mainly exhibited slowly activating currents. Dialysis of
the cells with pipette solutions containing 300 nm or less than 1 nm free Ca2+ strongly reduced the Cl− currents, indicating the currents were Ca2+-dependent. Relaxation analysis of the ``on' currents of slowly activating currents suggested that the channels were voltage-dependent.
The anion permeability sequence of the Cl− channels was: NO−
3 (2.00) > I− (1.85) ≥ Br− (1.69) > Cl− (1.00) > bicarbonate (0.77) ≥ acetate (0.70) > propionate (0.41) ≫ glutamate (0.09). When the ATP concentration in the pipette
solutions was increased from 0 to 10 mm, the Ca2+-dependency of the Cl− current amplitude shifted to lower free Ca2+ concentrations by about two orders of magnitude. Cells dialyzed with a pipette solution (pCa = 6) containing ATP-γS (2 mm) exhibited currents of similar magnitude to those observed with the solution containing ATP (2 mm). The addition of the calmodulin inhibitors trifluoperazine (100 μm) or calmidazolium (25 μm) to the bath solution and the inclusion of KN-62 (1 μm), a specific inhibitor of calmodulin kinase, or staurosporin (10 nm), an inhibitor of protein kinase C to the pipette solution had little, if any, effect on the Ca2+-activated Cl− currents. This suggests that Ca2+/Calmodulin or calmodulin kinase II and protein kinase C are not involved in Ca2+-activated Cl− currents. The outward Cl− currents at +69 mV were inhibited by NPPB (100 μm), IAA-94 (100 μm), DIDS (0.03–1 mm), 9-AC (300 μm and 1 mm) and DPC (1 mm), whereas the inward currents at −101 mV were not. These results demonstrate the presence of a bicarbonate- and weak acid-permeable
Cl− conductance controlled by cytosolic Ca2+ and ATP levels in rat submandibular acinar cells.
Received: 9 January 1996/Revised: 20 May 1996 相似文献
4.
Lens Major Intrinsic Protein (MIP) is a member of a family of membrane transport proteins including the Aquaporins and bacterial
glycerol transporters. When expressed in Xenopus oocytes, MIP increased both glycerol permeability and the activity of glycerol kinase. Glycerol permeability (p
Gly
) was 2.3 ± 0.23 × 10−6 cm sec−1 with MIP vs. 0.92 ± 0.086 × 10−6 cm sec−1 in control oocytes. The p
Gly
of MIP was independent of concentration from 5 × 10−5 to 5 × 10−2
m, had a low temperature dependence, and was inhibited approximately 90%, 80% and 50% by 1.0 mm Hg++, 0.2 mm DIDS (diisothiocyanodisulfonic stilbene), and 0.1 mm Cu++, respectively. MIP-enhanced glycerol phosphorylation, resulting in increased incorporation of glycerol into lipids. This
could arise from an increase in the total activity of glycerol kinase, or from an increase in its affinity for glycerol. Based
on methods we present to distinguish these mechanisms, MIP increased the maximum rate of phosphorylation by glycerol kinase
(0.12 ± 0.03 vs. 0.06 ± 0.01 pmol min−1 cell−1) without changing the binding of glycerol to the kinase (K
M
∼ 10 μm).
Received: 23 May 1997/Revised: 4 August 1997 相似文献
5.
Rate and equilibrium measurements of ryanodine binding to terminal cysternae fractions of heavy sarcoplasmic reticulum vesicles
demonstrate that its activation by high concentrations of monovalent salts is based on neither elevated osmolarity nor ionic
strength. The effect of the ions specifically depends on their chemical nature following the Hofmeister ion series for cations
(Li+ < NH+
4 < K−∼ Cs+≤ Na+) and anions (gluconate− < Cl− < NO3
−∼ ClO4
−∼ SCN−) respectively, indicating that both are involved in the formation of the salt-protein complex that can react with ryanodine.
Activation by rising salt concentrations exhibits saturation kinetics with different dissociation constants (25–11 m) and different degrees of cooperativity (n= 1.5–4.0) for the respective salts. Maximal second order binding rates between 40,000 and 80,000 (m
−1· sec−1) were obtained for chlorides and nitrates of 1a group alkali ions with the exception of lithium supporting only rates of
maximally 10,000 (M−1· sec−1). The nitrogen bases, NH+
4 and Tris+, in combination with chloride or nitrate, behave divergently. High maximal binding rates were achieved only with NH4NO3. The dissociation constants for the ryanodine–protein complexes obtained by measurements at equilibrium proved to depend
differently on salt concentration, yet, converging to 1–3 nm for the applied salts at saturating concentrations. The salts do not affect dissociation of the ryanodine protein complex
proving that the effect of salts on the protein's affinity for ryanodine is determined by their effect on the on-rate of ryanodine
binding. ATP and its analogues modify salt action resulting in elevated maximal binding rates and reduction or abolition of
binding cooperativity. Linear relations have been obtained by comparing the rates of ryanodine binding at different salt concentrations
with the rates or the initial amplitudes (15 sec) of salt induced calcium release from actively loaded heavy vesicles indicating
that the various salts promote specifically and concentration dependently channel opening and its reaction with ryanodine.
Received: 9 February 1998/Revised: 24 April 1998 相似文献
6.
Cell-volume changes induced by terbutaline (a specific β2-agonist) were studied morphometrically in rat fetal distal lung epithelium (FDLE) cells. Cell-volume changes qualitatively
differed with the concentration of terbutaline. Terbutaline of 10−10–10−8
m induced transient cell swelling. Terbutaline of 10−7
m induced transient cell swelling followed by slow cell shrinkage. Terbutaline of 10−6–10−5
m induced rapid cell shrinkage followed by slow cell shrinkage. Terbutaline of 10−3
m induced transient cell shrinkage; then cell volume oscillated during stimulation. Benzamil of 10−6
m suppressed the cell swelling induced by 10−10–10−8
m terbutaline and quinine of 10−3
m inhibited the cell shrinkage induced by 10−6–10−5
m terbutaline. These results suggest that cell swelling would be induced by NaCl influx and the cell shrinkage is by KCl efflux.
Dibutyryl cyclic AMP (DBcAMP) also induced similar cell-volume changes over a wide range of concentrations (10−9–10−3
m): a low concentration induced transient cell swelling; a high concentration, rapid and slow cell shrinkage. Forskolin (10−4
m), like terbutaline (10−5
m), induced rapid cell shrinkage followed by slow cell shrinkage, and this decrease in the cell volume was enhanced by the
presence of benzamil. On the other hand, cell shrinkage was induced by ionomycin (even low concentration; 3 × 10−10
m ionomycin), and after that cell volume remained at a plateau level. Removal of extracellular Ca2+ abolished the cell swelling caused by terbutaline of 10−10–10−8
m. With removal of extracellular Ca2+, the initial, rapid cell shrinkage induced by 10−5
m terbutaline became transient, but we still detected slow cell shrinkage similar to that in the presence of extracellular
Ca2+. Overall, at low concentrations (10−10–10−8
m), terbutaline induced benzamil-sensitive cell swelling in FDLE cells, which was cAMP- and Ca2+-dependent; high concentrations (≥−6) induced quinine-sensitive rapid cell shrinkage, which was Ca2+-dependent; high concentrations (≥−7) induced slow cell shrinkage, which was cAMP-dependent. These findings suggest that terbutaline regulates cell volume in
FDLE cells by cytosolic cAMP and Ca2+ through activation of Na+ and K+ channels.
Received: 13 March 1995/Revised: 17 January 1996 相似文献
7.
In vivo studies with leaf cells of aquatic plant species such as Elodea nuttallii revealed the proton permeability and conductance of the plasma membrane to be strongly pH dependent. The question was posed
if similar pH dependent permeability changes also occur in isolated plasma membrane vesicles. Here we report the use of acridine
orange to quantify passive proton fluxes. Right-side out vesicles were exposed to pH jumps. From the decay of the applied
ΔpH the proton fluxes and proton permeability coefficients (PH+) were calculated. As in the intact Elodea plasma membrane, the proton permeability of the vesicle membrane is pH sensitive, an effect of internal pH as well as external
pH on PH+ was observed. Under near symmetric conditions, i.e., zero electrical potential and zero ΔpH, PH+ increased from 65 × 10−8 at pH 8.5 to 10−1 m/sec at pH 11 and the conductance from 13 × 10−6 to 30 × 10−4 S/m2. At a constant pH
i
of 8 and a pH
o
going from 8.5 to 11, PH+ increased more than tenfold from 2 to 26 × 10−6 m/sec. The calculated values of PH+ were several orders of magnitude lower than those obtained from studies on intact leaves. Apparently, in plasma membrane
purified vesicles the transport system responsible for the observed high proton permeability in vivo is either (partly) inactive
or lost during the procedure of vesicle preparation. The residue proton permeability is in agreement with values found for
liposome or planar lipid bilayer membranes, suggesting that it reflects an intrinsic permeability of the phospholipid bilayer
to protons. Possible implications of these findings for transport studies on similar vesicle systems are discussed.
Received: 5 April 1995/Revised: 28 March 1996 相似文献
8.
We hypothesized that inhibition and activation of basolateral to luminal chloride transport mechanisms were associated with
respective decreases and increases in basolateral to luminal water fluxes. The luminal to basolateral (J
W
L→B
) and basolateral to luminal (J
W
B→L
) water fluxes across ovine tracheal epithelia were measured simultaneously. The mean J
W
L→B
(6.5 μl/min/cm2) was larger than J
W
B→L
(6.1 μl/min/cm2). Furosemide reduced J
W
B→L
from 6.0 to 5.6 μl/min/cm2. Diphenylamine-2-carboxylate (DPC) reduced J
W
B→L
from 7.9 to 7.3 μl/min/cm2 and reduced the membrane potential difference by 38%. Furosemide together with DPC decreased J
W
L→B
by 30% and J
W
B→L
by 15%. Norepinephrine increased J
W
B→L
from 4.9 to 6.0 μl/min/cm2. Neuropeptide Y in the presence of norepinephrine decreased J
W
L→B
(6.4 to 5.2 μl/min/cm2) and returned J
W
B→L
to its baseline value. Vasopressin increased J
W
B→L
from 4.1 to 5.1 μl/min/cm2. Endothelin-1 induced a simultaneous increase in J
W
B→L
(7.0 to 7.7 μl/min/cm2) and decrease in J
W
L→B
(7.4 to 6.4 μl/min/cm2); and decreased the membrane resistance. These data indicate that in tracheal epithelia under homeostatic conditions J
W
B→L
has a ∼15% actively coupled component. Consistent with our hypothesis, inhibition and receptor-induced stimulation of chloride
effluxes were associated with decreases and increases in J
W
B→L
, respectively. However, as inhibition of transcellular chloride transport always decreased J
W
L→B
more than J
W
B→L
, reducing transepithelial chloride transport did not result in less water being transported into the airway lumen.
Received: 12 October 1999/Revised: 14 March 2000 相似文献
9.
Aqueous exposure of critical residues in the selectivity region of voltage gated Na+ channels was studied by cysteine-scanning mutagenesis at three positions in each of the SS2 segments of domains III (D3)
and IV (D4) of the human heart Na+ channel. Ionic currents were modified by charged cysteine-specific methanethiosulfonate (MTS) reagents, (2-aminoethyl)methanethiosulfonate
(MTSEA+) and (2-sulfonatoethyl)methanethiosulfonate (MTSES−) in all six of the Cys-substituted channels, including Trp → Cys substitutions at homologous positions in D3 and D4 that
were predicted in secondary structure models to have buried side chains. Furthermore, in the absence of MTS modification,
each of the Cys mutants showed a reduction in tetrodotoxin (TTX) block by a factor >102. Cysteine substitution without MTS modification abolished the alkali metal ion selectivity in K1418C (D3), but not in A1720C
(the corresponding position in D4) suggesting that the lysine but not the alanine side chains contribute to selectivity even
though both were exposed. Neither position responded to MTSES− suggesting that these residues occupy either a size- or charge-restricted region of the pore. By contrast, MTSES− markedly increased, and MTSEA+ markedly decreased conductance of D1713C (D4) suggesting that the acidic side chain of Asp1713 acts electrostatically in an unrestricted region. These results suggest that Lys1418 lies in a restricted region favorable to cations, whereas Asp1713 is at a more peripheral location in the Na+ channel pore.
Received: 8 May 1996/Revised: 15 August 1996 相似文献
10.
To study vacuolar chloride (Cl−) transport in the halophilic plant Mesembryanthemum crystallinum L., Cl− uptake into isolated tonoplast vesicles was measured using the Cl−-sensitive fluorescent dye lucigenin (N,N′-dimethyl-9,9′-bisacridinium dinitrate). Lucigenin was used at excitation and emission wavelengths of 433 nm and 506 nm,
respectively, and showed a high sensitivity towards Cl−, with a Stern-Volmer constant of 173 m
−1 in standard assay buffer. While lucigenin fluorescence was strongly quenched by all halides, it was only weakly quenched,
if at all, by other anions. However, the fluorescence intensity and Cl−-sensitivity of lucigenin was shown to be strongly affected by alkaline pH and was dependent on the conjugate base used as
the buffering ion. Chloride transport into tonoplast vesicles of M. crystallinum loaded with 10 mm lucigenin showed saturation-type kinetics with an apparent K
m
of 17.2 mm and a V
max
of 4.8 mm min−1. Vacuolar Cl− transport was not affected by sulfate, malate, or nitrate. In the presence of 250 μm
p-chloromercuribenzene sulfonate, a known anion-transport inhibitor, vacuolar Cl− transport was actually significantly increased by 24%. To determine absolute fluxes of Cl− using this method, the average surface to volume ratio of the tonoplast vesicles was measured by electron microscopy to be
1.13 × 107 m−1. After correcting for a 4.4-fold lower apparent Stern-Volmer constant for intravesicular lucigenin, a maximum rate of Cl− transport of 31 nmol m−2 sec−1 was calculated, in good agreement with values obtained for the plant vacuolar membrane using other techniques.
Received: 18 February 2000/Revised: 30 June 2000 相似文献
11.
Mo L Hellmich HL Fong P Wood T Embesi J Wills NK 《The Journal of membrane biology》1999,168(3):253-264
Loss of function mutations of the renal chloride channel, ClC-5, have been implicated in Dent's disease, a genetic disorder
characterized by low weight proteinuria, hypercalciuria, nephrolithasis and, in some cases, eventual renal failure. Recently,
our laboratory used an RT-PCR/RACE cloning strategy to isolate an amphibian cDNA from the renal epithelial cell line A6 that
had high homology to human ClC-5. We now report a full-length native ClC-5 clone (xClC-5, containing 5′ and 3′ untranslated
regions) isolated by screening a cDNA library from A6 cells that was successfully expressed in Xenopus oocytes. In addition, we compared the properties of xClC-5 and hClC-5 using isogenic constructs of xClC-5 and hClC-5 consisting
of the open reading frame subcloned into an optimized Xenopus expression vector. Expression of the full-length ``native'
xClC-5 clone resulted in large, strongly rectifying, outward currents that were not significantly affected by the chloride
channel blockers DIDS, DPC, and 9AC. The anion conductivity sequence was NO−
3 > Cl−= I− > HCO−
3 >> glutamate for xClC-5 and NO−
3 > Cl− > HCO−
3 > I− >> glutamate for hClC-5. Reduction of the extracellular pH (pH
o
) from 7.5 to 5.7 inhibited outward ClC-5 currents by 27 ± 9% for xClC-5 and 39 ± 7% for hClC-5. The results indicate that
amphibian and mammalian ClC-5 have highly similar functional properties. Unlike hClC-5 and most other ClC channels, expression
of xClC-5 in oocytes does not require the removal of its untranslated 5′ and 3′ regions. Acidic solutions inhibited both amphibian
and human ClC-5 currents, opposite to the stimulatory effects of low external pH on other ClC channels, suggesting a possibly
distinct regulatory mechanism for ClC-5 channels.
Received: 28 August 1998/Revised: 13 January 1999 相似文献
12.
Previous measurements with CsF pipette solutions using whole-cell patch-clamp techniques in dissociated rat olfactory receptor
neurons (ORNs) indicated that the sodium currents had very negative inactivation characteristics with the implication that
the cell resting potential must also normally have a very negative value. This study supports the conclusions that such an
effect was real and not dependent on either the nature of the pipette anions or the recording situation previously used. For
all pipette solutions, sodium currents showed a threshold activation ≈−80 mV and half-maximal activation voltages ≈−55 with
half-inactivation potential ≤−100 mV, without being significantly affected by the replacement of F− by other pipette anions (H2PO−
4 and acetate−) or the addition of nucleotides and glutathione (which did cause a very slight positive shift). F−, followed by H2PO−
4 and to a much lesser extent by acetate−, was the most favorable pipette anion for obtaining good seals and whole-cell sodium currents in these extremely small ORNs.
These results implied that resting potentials, for viable responsive cells, should be more negative than about −90 mV, as
supported by the observation that action potentials could only be evoked from holding potentials more negative than −90 mV.
Received: 23 December 1999/Revised: 2 March 2000 相似文献
13.
Hirono C Nakamoto T Sugita M Iwasa Y Akagawa Y Shiba Y 《The Journal of membrane biology》2001,180(1):11-19
Forskolin-induced anion currents and depolarization were investigated to clarify the mechanism of HCO3
− secretion in the intralobular duct cells of rat parotid glands. Anion currents of the cells were measured at the equilibrium
potential of K+, using a gramicidin-perforated patch technique that negligibly affects intracellular anion concentration. The forskolin-induced
anion current was sustained and significantly (54%) suppressed by glibenclamide (200 μm), a blocker of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel. The anion current was markedly suppressed by addition of 1 mm methazolamide, a carbonic anhydrase inhibitor, and removal of external HCO3
−. Forskolin depolarized the cells in the current-clamp mode. Addition of methazolamide and removal of external HCO3
− significantly decreased the depolarizing level. These results suggest that activation of anion channels (mainly the CFTR
Cl− channel located in luminal membranes) and production of cytosolic HCO3
− induce the inward anion current and resulting depolarization. Inhibition of the Na+-K+-2Cl− cotransporter and the Cl−-HCO3
− exchanger had no significant effect on the current or depolarization, indicating that the uptake of Cl− via the Na+-K+-2Cl− cotransporter or the Cl−-HCO3
− exchanger is not involved in the responses. Taken together, we conclude that forskolin activates the outward movement (probably
secretion) of HCO3
− produced intracellularly, but not of Cl− due to lack of active Cl− transport in parotid duct cells, and that the gramicidin-perforated patch method is very useful to analyze anion transport.
Received: 17 June 2000/Revised: 14 November 2000 相似文献
14.
Chloride (Cl−) conductances were studied in primary cultures of the bright part of rabbit distal convoluted tubule (DCTb) by the whole
cell patch clamp technique. The bath solution (33°C) contained (in mm): 140 NaCl, 1 CaCl2, 10 N-2-hydroxy-ethylpiperazine-N′-2-ethanesulfonic acid (HEPES), pH 7.4 and the pipette solution 140 N-methyl-d-glucamine (NMDG)-Cl, 5 MgATP, 1 ethylene-glycol-bis(b-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 10 HEPES, pH 7.4. We identified a Cl− current activated by 10−5
m forskolin, 10−3
m 8-bromo adenosine 3′,5′-cyclic monophophosphate (8 Br-cAMP), 10−6
m phorbol 12-myristate 13-acetate (PMA), 10−3
m intracellular adenosine 3′,5′-cyclic monophophosphate (cAMP) and 10−7
m calcitonin. The current-voltage relationship was linear and the relative ion selectivity was Br− > Cl−≫ I− > glutamate. This current was inhibited by 10−3
m diphenylamine-2-carboxylate (DPC) and 10−4
m 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and was insensitive to 10−3
m 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). These characteristics are similar to those described for the cystic
fibrosis transmembrane conductance regulator (CFTR) Cl− conductance. In a few cases, forskolin and calcitonin induced an outwardly rectifying Cl− current blocked by DIDS. To determine the exact location of the Cl− conductance 6-methoxy-1-(3-sulfonatopropyl) quinolinium (SPQ) fluorescence experiments were carried out. Cultures seeded
on collagen-coated permeable filters were loaded overnight with 5 mm SPQ and the emitted fluorescence analyzed by laser-scan cytometry. Cl− removal from the apical solution induced a Cl− efflux which was stimulated by 10−5
m forskolin, 10−7 calcitonin and inhibited by 10−5
m NPPB. In 140 mm NaBr, forskolin stimulated an apical Br− influx through the Cl− pathway. Forskolin and calcitonin had no effect on the basolateral Cl− permeability. Thus in DCTb cultured cells, exposure to calcitonin activates a Cl− conductance in the apical membrane through a cAMP-dependent mechanism.
Received: 5 July 1995/Revised: 21 December 1995 相似文献
15.
The chloride conductance of inner medullary collecting duct cells (mIMCD-3 cell line) has been investigated using the whole
cell configuration of the patch clamp technique. Seventy-seven percent of cells were chloride selective when measured with
a NaCl-rich bathing solution and a TEACl-rich pipette solution. Seventy-five percent of chloride-selective cells (90/144)
had whole cell currents which exhibited an outwardly-rectifying (OR) current-voltage (I/V) relationship, while the remaining cells exhibited a linear (L) I/V relationship. The properties of the OR and L chloride currents were distinct. OR currents (mean current densities at ±60
mV of 66 ± 5 pA/pF and 44 ± 3 pA/pF), were time- and voltage-independent with an anion selectivity (from calculated permeability
ratios) of SCN− (2.3), NO−
3 (1.8), ClO−
4 (1.7), Br− (1.7), I− (1.6), Cl− (1.0), HCO−
3 (0.5), gluconate− (0.2). Bath additions of NPPB, flufenamate, glibenclamide (all 100 μm) and DIDS (500 μm) produced varying degrees of block of OR currents with NPPB being the most potent (IC50 of approximately 50 μm) while DIDS was the least effective. Linear chloride currents had similar current densities to the OR chloride currents and
were also time- and voltage-independent. The anion selectivity sequence was SCN− (2.5), NO−
3 (1.9), Br− (1.4), I− (1.1), Cl− (1.0), ClO−
4 (0.5), HCO−
3 (0.5), gluconate− (0.3). In contrast to the OR conductance, glibenclamide was the most potent and DIDS the least potent blocker of L currents.
An IC50 of >100 μm was observed for NPPB block. Neither OR of L chloride currents were affected by acutely or chronically increased intracellular
cAMP and were not affected when intracellular Ca2+ levels were increased or decreased. The molecular identity and physiological role of OR and linear currents in mIMCD-3 cells
are discussed.
Received: 13 June 1995/Revised: 15 September 1995 相似文献
16.
Davis BA Hogan EM Cooper GJ Bashi E Zhao J Boron WF 《The Journal of membrane biology》2001,183(1):25-32
Previous squid-axon studies identified a novel K/HCO3 cotransporter that is insensitive to disulfonic stilbene derivatives. This cotransporter presumably responds to intracellular
alkali loads by moving K+ and HCO−
3 out of the cell, tending to lower intracellular pH (pHi). With an inwardly directed K/HCO3 gradient, the cotransporter mediates a net uptake of alkali (i.e., K+ and HCO−
3 influx). Here we test the hypothesis that intracellular quaternary ammonium ions (QA+) inhibit the inwardly directed cotransporter by interacting at the intracellular K+ site. We computed the equivalent HCO−
3 influx (J
HCO3) mediated by the cotransporter from the rate of pHi increase, as measured with pH-sensitive microelectrodes. We dialyzed axons to pHi 8.0, using a dialysis fluid (DF) free of K+, Na+ and Cl−. Our standard artificial seawater (ASW) also lacked Na+, K+ and Cl−. After halting dialysis, we introduced an ASW containing 437 mm K+ and 0.5% CO2/12 mm HCO−
3, which (i) caused membrane potential to become transiently very positive, and (ii) caused a rapid pHi decrease, due to CO2 influx, followed by a slower plateau-phase pHi increase, due to inward cotransport of K+ and HCO−
3. With no QA+ in the DF, J
HCO3 was ∼58 pmole cm−2 sec−1. With 400 mm tetraethylammonium (TEA+) in the DF, J
HCO3 was virtually zero. The apparent K
i
for intracellular TEA+ was ∼78 mm, more than two orders of magnitude greater than that obtained by others for inhibition of K+ channels. Introducing 100 mm inhibitor into the DF reduced J
HCO3 to ∼20 pmole cm−2 sec−1 for tetramethylammonium (TMA+), ∼24 for TEA+, ∼10 for tetrapropylammonium (TPA+), and virtually zero for tetrabutylammonium (TBA+). The apparent K
i
value for TBA+ is ∼0.86 mm. The most potent inhibitor was phenyl-propyltetraethylammonium (PPTEA+), with an apparent K
i
of ∼91 μm. Thus, trans-side quaternary ammonium ions inhibit K/HCO3 influx in the potency sequence PPTEA+ > TBA+ > TPA+ > TEA+≅ TMA+. The identification of inhibitors of the K/HCO3 cotransporter, for which no inhibitors previously existed, will facilitate the study of this transporter.
Received: 21 November 2000/Revised: 14 May 2001 相似文献
17.
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
18.
Using the whole-cell patch-clamp technique, we examined Cl−-selective currents manifested by strial marginal cells isolated from the inner ear of gerbils. A large Cl−-selective conductance of ∼18 nS/pF was found from nonswollen cells in isotonic buffer containing 150 mm Cl−. Under a quasi-symmetrical Cl− condition, the `instantaneous' current-voltage relation was close to linear, while the current-voltage relation obtained
at the end of command pulses of duration 400 msec showed weak outward rectification. The permeability sequence for anionic
currents was as SCN− > Br−≅ Cl− > F− > NO−
3≅ I− > gluconate−, corresponding to Eisenmann's sequence V. When whole-cell voltage clamped in isotonic bathing solutions, the cells exhibited
volume changes that were accounted for by the Cl− currents driven by the imposed electrochemical potential gradients. The volume change was elicited by lowered extracellular
Cl− concentration, anion substitution and altered holding potentials. The Cl− conductance varied in parallel with cell volume when challenged by bath anisotonicity. The whole-cell Cl− current was only partially blocked by both 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB, 0.5 mm) and diphenylamine-2-carboxylic acid (DPC, 1.0 mm), but 4-acetamido-4′-isothiocyanato-stilbene-2,2′-disulfonic acid (SITS, 0.5 mm) was without effect. The properties of the present whole-cell Cl− current resembled those of the single Cl− channel previously found in the basolateral membrane of the marginal cell (Takeuchi et al., Hearing Res.
83:89–100, 1995), suggesting that the volume-correlated Cl− conductance could be ascribed predominantly to the basolateral membrane. This Cl− conductance may function not only in cell volume regulation but also for the transport of Cl− and the setting of membrane potential in marginal cells under physiological conditions.
Received: 15 August 1995/Revised: 3 November 1995 相似文献
19.
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
20.
GABA-activated Cl− current was expressed in Xenopus oocytes after injecting cRNA that had been transcribed in vitro from complementary DNA (cDNA) coding for a single GABA ρi-subunit cloned from human retina. The expressed current was insensitive to 100 μm bicuculline, but was activated by the GABA analogue trans-4-aminocrontonic acid (TACA). Anion-selective permeability of the
expressed ρ1-subunit was determined by isotonically replacing the extracellular Cl− with different anions. The anion permeability was very similar to the native GABAA receptor/channel following a sequence of SCN− > I− > NO3
− > Br−≥ Cl−. Halogenated fatty acids, such as chlorotrifluoroethylene (CTFE) and perfluorinated oligomer acids inhibited the GABA-induced
current in oocytes expressing the human retinal GABA ρ1-subunit or rat brain GABAA receptor α1,β2,γ2 subunits. The inhibitory effect of halogenated fatty acids demonstrated a carbon chain length-dependent manner of: C10 > C8 > C6 > C4. Perfluorinated C8-oligomer acid (PFOA) was less effective at blocking this channel than the C8-CTFE oligomer acid. Radiolabeled GABA binding assay indicated that CTFE oligomer acids do not interfere at the GABA binding
site of the receptor. Furthermore, the C8-CTFE oligomer fatty acid did not compete with picrotoxin for binding sites within the pore of the channel. These studies
demonstrated that the heterologous expression system is useful for studying the molecular interaction between potential neurotoxic
agents and neuroreceptors. Our results provide detailed information that should contribute to our understanding of the structure
and function of retinal GABA receptors.
Received: 12 June 1995/Revised: 21 September 1995 相似文献