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1.
A Patch-Clamp Study of Ion Channels in Protoplasts Prepared from the Marine Alga Valonia utricularis
The giant marine alga Valonia utricularis is a classical model system for studying the electrophysiology and water relations of plant cells by using microelectrode
and pressure probe techniques. The recent finding that protoplasts can be prepared from the giant ``mother cells' (Wang,
J., Sukhorukov, V.L., Djuzenova, C.S., Zimmermann, U., Müller, T., Fuhr, G., 1997, Protoplasma
196:123–134) allowed the use of the patch-clamp technique to examine ion channel activity in the plasmalemma of this species.
Outside-out and cell-attached experiments displayed three different types of voltage-gated Cl− channels (VAC1, VAC2, VAC3, Valonia Anion Channel 1,2,3), one voltage-gated K+ channel (VKC1, Valonia K
+
Channel 1) as well as stretch-activated channels. In symmetrical 150 mm Cl− media, VAC1 was most frequently observed and had a single channel conductance of 36 ± 7 pS (n= 4) in the outside-out and 33 ± 5 pS (n= 10) in the cell-attached configuration. The reversal potential of the corresponding current-voltage curves was within 0
± 4 mV (n= 4, outside-out) and 9 ± 7 mV (n= 10, cell-attached) close to the Nernst potential of Cl− and shifted towards more negative values when cell-attached experiments were performed in asymmetrical 50:150 mm Cl− media (bath/pipette; E
Cl−
−20 ± 7 mV (n= 4); Nernst potential −28 mV). Consistent with a selectivity for Cl−, VAC1 was inhibited by 100 μM DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid). VAC1 was activated by a hyperpolarization of the patch. Boltzmann
fits of the channel activity under symmetrical 150 mm Cl− conditions yielded a midpoint potential of −12 ± 5 mV (n= 4, outside-out) and −3 ± 6 mV (n= 9, cell-attached) and corresponding apparent minimum gating charges of 15 ± 3 (n= 4) and 18 ± 5 (n= 9). The midpoint potential shifted to more negative values in the presence of a Cl− gradient.
VAC2 was activated by voltages more negative than E
Cl−
and was always observed together with VAC1, but less frequently. It showed a ``flickering' gating. The single channel conductance
was 99 ± 10 pS (n= 6). VAC3 was activated by membrane depolarization and frequently exhibited several subconductance states. The single channel
conductance of the main conductance state was 36 ± 5 pS (n= 5). VKC1 was also activated by positive clamped voltages. Up to three conductance states occurred whereby the main conductance
state had a single channel conductance of 124 ± 27 pS (n= 6).
In the light of the above results it seems to be likely that VAC1 contributes mainly to the Cl− conductance of the plasmalemma of the turgescent ``mother cells' and that this channel (as well as VAC2) can operate in
the physiological membrane potential range. The physiological significance of VAC3 and VKC1 is unknown, but may be related
(as the stretch-activated channels) to processes involved in turgor regulation.
Received: 24 June 1999/Revised: 2 September 1999 相似文献
2.
We have characterized a Ca2+-dependent Cl− current (ClCa) in cultured Sertoli cells from immature rat testis by using the whole cell recording patch-clamp technique. Cells dialyzed
with pipette solutions containing 3 mm adenoside-triphosphate (ATP) and 1 μm free Ca2+, exhibited outward currents which were inhibited by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and anthracene-9-carboxylic
acid (9-AC) but insensitive to tetraethylammonium (TEA). Dialysis of cells with pipette solutions containing less than 1 nm free Ca2+ strongly reduced the currents indicating that they were Ca2+ dependent. With cells dialyzed with Cs+ glutamate-rich pipette solutions containing 0.2 mm EGTA, 10 μm ionomycin induced outward currents having properties of Ca2+-activated Cl− currents.
With ATP-free pipette solution, the magnitude of currents was not modified suggesting the direct control by Ca2+. By contrast, addition of 0.1 mm cAMP in the pipette solution or the superfusion of cells by a permeant analogue of cAMP strongly reduced the currents. These
results may suggest that ClCa is inhibited by cAMP-dependent protein kinase.
Finally, our results do not agree with the model of primary fluid secretion by exocrine cells, but are in agreement with a
hyperpolarizing effect of cAMP in primary culture of Sertoli cells and the release of a low Cl− and bicarbonate-rich primary fluid by these cells.
Received: 30 November 1998/Revised: 2 March 1999 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.
J.D. Kibble S.L. Greenwood L.H. Clarson C.P. Sibley 《The Journal of membrane biology》1996,151(2):131-138
Whole-cell patch clamp experiments were performed on cultured human cytotrophoblast cells incubated for 24–48 hr after their
isolation from term placentas. Cl−-selective currents were examined using K+-free solutions. Under nonstimulated conditions, most cells initially expressed only small background leak currents. However,
inclusion of 0.2 mm GTPγS in the electrode solution caused activation of an outwardly rectifying conductance which showed marked time-dependent
activation at depolarized potentials above +20 mV. Stimulation of this conductance by GTPγS was found to be Ca2+-dependent since GTPγS failed to activate currents when included in a Ca2+-free electrode solution. In addition, similar currents could be activated by increasing the [Ca2+] of the pipette solution to 500 nm. The Ca2+-activated conductance was judged to be Cl−-selective, since reversal potentials were predicted by Nernst equilibrium potentials for Cl−. This conductance could also be reversibly inhibited by addition of the anion channel blocker DIDS to the bath solution at
a dose of 100 μm. Preliminary experiments indicated the presence of a second whole-cell anion conductance in human cytotrophoblast cells,
which may be activated by cell swelling. Possible roles for the Ca2+-activated Cl− conductance in human placental trophoblast are discussed.
Received: 9 November 1995/Revised: 18 January 1996 相似文献
5.
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
6.
An amiloride-sensitive, Ca2+-activated nonselective cation (NSC) channel in the apical membrane of fetal rat alveolar epithelium plays an important role
in stimulation of Na+ transport by a beta adrenergic agonist (beta agonist). We studied whether Ca2+ has an essential role in the stimulation of the NSC channel by beta agonists. In cell-attached patches formed on the epithelium,
terbutaline, a beta agonist, increased the open probability (P
o
) of the NSC channel to 0.62 ± 0.07 from 0.03 ± 0.01 (mean ±se; n= 8) 30 min after application of terbutaline in a solution containing 1 mm Ca2+. The P
o
of the terbutaline-stimulated NSC channel was diminished in the absence of extracellular Ca2+ to 0.26 ± 0.05 (n= 8). The cytosolic Ca2+ concentration ([Ca2+]
c
) in the presence and absence of extracellular Ca2+ was, respectively, 100 ± 6 and 20 ± 2 nm (n= 7) 30 min after application of terbutaline. The cytosolic Cl− concentration ([Cl−]
c
) in the presence and absence of extracellular Ca2+ was, respectively, 20 ± 1 and 40 ± 2 mm (n= 7) 30 min after application of terbutaline. The diminution of [Ca2+]
c
from 100 to 20 nm itself had no significant effects on the P
o
if the [Cl−]
c
was reduced to 20 mm; the P
o
was 0.58 ± 0.10 at 100 nm [Ca2+]
c
and 0.55 ± 0.09 at 20 nm [Ca2+]
c
(n= 8) with 20 mm [Cl−]
c
in inside-out patches. On the other hand, the P
o
(0.28 ± 0.10) at 20 nm [Ca2+]
c
with 40 mm [Cl−]
c
was significantly lower than that (0.58 ± 0.10; P < 0.01; n= 8) at 100 nm [Ca2+]
c
with 20 mm [Cl−]
c
, suggesting that reduction of [Cl−]
c
is an important factor stimulating the NSC channel. These observations indicate that the extracellular Ca2+ plays an important role in the stimulatory action of beta agonist on the NSC channel via reduction of [Cl−]
c
.
Received: 11 August 2000/Revised: 4 December 2000 相似文献
7.
These experiments were conducted to determine the membrane K+ currents and channels in human urinary bladder (HTB-9) carcinoma cells in vitro. K+ currents and channel activity were assessed by the whole-cell voltage clamp and by either inside-out or outside-out patch
clamp recordings. Cell depolarization resulted in activation of a Ca2+-dependent outward K+ current, 0.57 ± 0.13 nS/pF at −70 mV holding potential and 3.10 ± 0.15 nS/pF at 30 mV holding potential. Corresponding patch
clamp measurements demonstrated a Ca2+-activated, voltage-dependent K+ channel (KCa) of 214 ± 3.0 pS. Scorpion venom peptides, charybdotoxin (ChTx) and iberiotoxin (IbTx), inhibited both the activated current
and the KCa activity. In addition, on-cell patch recordings demonstrated an inwardly rectifying K+ channel, 21 ± 1 pS at positive transmembrane potential (V
m
) and 145 ± 13 pS at negative V
m
. Glibenclamide (50 μm), Ba2+ (1 mm) and quinine (100 μm) each inhibited the corresponding nonactivated, basal whole-cell current. Moreover, glibenclamide inhibited K+ channels in inside/out patches in a dose-dependent manner, and the IC50= 46 μm. The identity of this K+ channel with an ATP-sensitive K+ channel (KATP) was confirmed by its inhibition with ATP (2 mm) and by its activation with diazoxide (100 μm). We conclude that plasma membranes of HTB-9 cells contain the KCa and a lower conductance K+ channel with properties consistent with a sulfonylurea receptor-linked KATP.
Received: 12 June 1997/Revised: 21 October 1997 相似文献
8.
Sitsapesan R 《The Journal of membrane biology》1999,168(2):159-168
Tonoplast K+ channels of Chara corallina are well characterized but only a few reports mention anion channels, which are likely to play an important role in the tonoplast
action potential and osmoregulation of this plant. For experiments internodal cells were isolated. Cytoplasmic droplets were
formed in an iso-osmotic bath solution according to a modified procedure. Ion channels with conductances of 48 pS and 170
pS were detected by the patch-clamp technique. In the absence of K+ in the bath solution the 170 pS channel was not observed at negative pipette potential values. When Cl− on either the vacuolar side or the cytoplasmic side was partly replaced with F−, the reversal potential of the 48 pS channel shifted conform to the Cl− equilibrium potential with similar behavior in droplet-attached and excised patch mode. These results showed that the 48
pS channel was a Cl− channel. In droplet-attached mode the channel rectified outward current flow, and the slope conductance was smaller. When
Chara droplets were formed in a bath solution containing low (10−8
m) Ca2+, then no Cl− channels could be detected either in droplet-attached or in inside-out patch mode. Channel activity was restored if Ca2+ was applied to the cytoplasmic side of inside-out patches. Rectification properties in the inside-out patch configuration
could be controlled by the holding pipette potential. Holding potential values negative or positive to the calculated reversal
potential for Cl− ions induced opposite rectification properties. Our results show Ca2+-activated Cl− channels in the tonoplast of Chara with holding potential dependent rectification.
Received: 30 March 1999/Revised: 10 August 1999 相似文献
9.
Selective Inhibition of Glucose-Stimulated β-Cell Activity by an Anion Channel Inhibitor 总被引:2,自引:0,他引:2
4,4′-dithiocyanatostilbene-2,2′-disulfonic acid (DIDS), an inhibitor of the volume-sensitive anion channel, was used to investigate
the role of this channel in the stimulation of rat pancreatic β-cells by glucose and by tolbutamide. Glucose-stimulated electrical
activity in β-cells was markedly and reversibly inhibited by DIDS. The increase in cytosolic [Ca2+] and stimulated insulin release evoked by glucose were also inhibited by DIDS. In contrast to its inhibitory effect on glucose-induced
β-cell activity, DIDS had no effect on electrical activity, the rise in [Ca2+]
i
or insulin release induced by tolbutamide.
DIDS failed to increase β-cell input conductance, an index of whole-cell K
ATP
channel activity, or the rate of efflux of 86Rb+ from perifused islets, a measure of net K+ permeability. Furthermore, DIDS had no effect on intracellular pH or on regulatory volume increase following exposure of
cells to hypertonic solutions, indicating that the effects of DIDS were not the result of inhibition of Cl− transport systems. It is suggested that the DIDS-induced repolarization is caused by inactivation of the volume-sensitive
anion channel. The stimulation of β-cell electrical and secretory activity by glucose, but not tolbutamide, may therefore
involve activation of the anion channel.
Received: 30 November 1999/Revised: 23 June 2000 相似文献
10.
Using the whole-cell configuration of the patch-clamp technique, we studied the conditions necessary for the activation of
Cl−-currents in retinal pigment epithelial (RPE) cells from rats with retinal dystrophy (RCS) and nondystrophic control rats.
In RPE cells from both rat strains, intracellular application of 10 μm inositol-1,4,5-triphosphate (IP3) via the patch pipette led to a sustained activation of voltage-dependent Cl− currents, blockable by 1 mm 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS). IP3 activated Cl− currents in the presence of a high concentration of the calcium chelator BAPTA (10 mm) in the pipette solution, but failed to do so when extracellular calcium was removed. Intracellular application of 10−5
m Ca2+ via the patch pipette also led to a transient activation of Cl− currents. When the cells were preincubated in a bath solution containing thapsigargin (1 μm) for 5 min before breaking into the whole-cell configuration, IP3 failed to activate voltage-dependent currents. Thus, IP3
led to release of Ca2+ from cytosolic calcium stores. This in turn activated an influx of extracellular calcium into the submembranal space by a
mechanism as yet unknown, leading to an activation of calcium-dependent chloride currents. In RPE cells from RCS rats, which
show an increased membrane conductance for calcium compared to normal rats, we observed an accelerated speed of Cl−-current activation induced by IP3 which could be reduced by nifedipine (1 μm). Thus, the increased membrane conductance to calcium in RPE cells from RCS rats changes the response of the cell to the
second messenger IP3.
Received: 17 July 1995/Revised: 31 January 1996 相似文献
11.
S.L. Wladkowski W. Lin M. McPheeters S.C. Kinnamon S. Mierson 《The Journal of membrane biology》1998,164(1):91-101
We used Ussing chamber measurements and whole-cell recordings to characterize a chloride conductance in rat lingual epithelium.
Niflumic acid (NFA) and flufenamic acid (FFA), nonsteroidal anti-inflammatory aromatic compounds known to inhibit Cl− conductances in other tissues, reduced transepithelial short-circuit current (I
sc
) in the intact dorsal anterior rat tongue epithelium when added from the serosal side, and reduced whole-cell currents in
rat fungiform taste cells. In both Ussing chamber and patch-clamp experiments, the effect of NFA was mimicked by replacement
of bath Cl− with methanesulfonate or gluconate. In low Cl− bath solution, the effect of NFA on whole-cell current was reduced. Replacement of bath Ca2+ with Ba2+ reduced the whole-cell Cl− current. We conclude that a Ca2+-activated Cl− conductance is likely present in the basolateral membrane of the rat lingual epithelium, and is present in the taste receptor
cells from fungiform papillae. Further experiments will be required to identify the role of this conductance in taste transduction.
Received: 8 September 1997/Revised: 27 March 1998 相似文献
12.
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
13.
It has previously been shown that osmotic cell shrinkage activates a nonselective cation (NSC) channel in M-1 mouse cortical
collecting duct cells [54] and in a variety of other cell types [20]. In the present study we further characterized the shrinkage-activated
NSC channel in M-1 cells and its mechanism of activation using whole-cell current recordings. Osmotic cell shrinkage induced
by addition of 100 mm sucrose to the bath solution caused a 20-fold increase in whole-cell inward currents from −10.8 ± 1.5 pA to −211 ± 10.2 pA
(n= 103). A similar response was observed when cell shrinkage was elicited using a hypo-osmotic pipette solution. This indicates
that cell shrinkage and not extracellular osmolarity per se is the signal for current activation. Cation substitution experiments revealed that the activated channels discriminate poorly
between monovalent cations with a selectivity sequence NH4 (1.2) ≥ Na+ (1) ≈ K+ (0.9) ≈ Li+ (0.9). In contrast there was no measurable permeability for Ca2+ or Ba2+ and the cation-to-anion permeability ratio was about 14. The DPC-derivatives flufenamic acid, 4-methyl-DPC and DCDPC were
the most effective blockers followed by LOE 908, while amiloride and bumetanide were ineffective. The putative channel activator
maitotoxin had no effect. Current activation was dependent upon the presence of intracellular ATP and Mg2+ and was inhibited by staurosporine (1 μm) and calphostin C (1 μm). Moreover, cytochalasin D (10 μm) and taxol (2 μm) reduced the current response to cell shrinkage. These findings suggest that the activation mechanism of the shrinkage-activated
NSC channel involves protein kinase mediated phosphorylation steps and cytoskeletal elements.
Received: 3 May 2000/Revised: 6 July 2000 相似文献
14.
15.
We observed intermediate conductance channels in approximately 20% of successful patch-clamp seals made on collecting tubules
dissected from Ambystoma adapted to 50 mm potassium. These channels were rarely observed in collecting tubules taken from animals which were maintained in tap water.
Potassium-adaptation either leads to an increase in the number of channels present or activates quiescent channels.
In cell-attached patches the conductance averaged 30.3 ± 2.4 (9) pS. Since replacement of the chloride in the patch pipette
with gluconate did not change the conductance, the channel carries cations, not anions. Notably, channel activity was observed
at both positive and negative pipette voltages. When the pipette was voltage clamped at 0 mV or positive voltages, the current
was directed inward, consistent with the movement of sodium into the cell. The pipette voltage at which the polarity of the
current reversed (movement of potassium into the pipette) was −29.6 ± 6.5(9) mV.
Open probability at 0 mV pipette voltage was 0.08 ± 0.03 and was unaffected when the apical membrane was exposed to either
2 × 10−6 or 2 × 10−5
m of amiloride. Exposure of the basolateral surface of the tubule to a saline containing 15 mm potassium caused a significant increase (P less than 0.001) in the open probability of these channels to 0.139 ± 0.002 without affecting the conductance of the apical
channel.
These data illustrate the presence of an intermediate conductance, poorly selective, amiloride-insensitive cation channel
in native vertebrate collecting tubule. We postulate that, at least in amphibia, this channel may be used to secrete potassium.
Received: 14 January 2000/Revised: 16 June 2000 相似文献
16.
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
17.
A. Chorvatova A. Guyot C. Ojeda O. Rougier A. Bilbaut 《The Journal of membrane biology》1998,162(1):39-50
The effects of angiotensin II (100 nm) on the electrical membrane properties of zona fasciculata cells isolated from calf adrenal gland were studied using the
whole cell patch recording method. In current-clamp condition, angiotension II induced a biphasic membrane response which
began by a transient hyperpolarization followed by a depolarization more positive than the control resting potential. These
effects were abolished by Losartan (10−5
m), an antagonist of angiotensin receptors of type 1. The angiotensin II-induced transient hyperpolarization was characterized
in voltage-clamp condition from a holding potential of −10 mV. Using either the perforated or the standard recording method,
a transient outward current accompanied by an increase of the membrane conductance was observed in response to the hormonal
stimulation. This outward current consisted of an initial fast peak followed by an oscillating or a slowly decaying plateau
current. In Cl−-free solution, the outward current reversed at −78.5 mV, a value close to E
K. It was blocked by external TEA (20 mm) and by apamin (50 nm). In K+-free solution, the transient outward current, sensitive to Cl− channel blocker DPC (400 μm), reversed at −52 mV, a more positive potential than E
Cl. Its magnitude changed in the same direction as the driving force for Cl−. The hormone-induced transient outward current was never observed when EGTA (5 mm) was added to the pipette solution. The plateau current was suppressed in nominally Ca2+-free solution (47% of cells) and was reversibly blocked by Cd2+ (300 μm) but not by nisoldipine (0.5–1 μm) which inhibited voltage-gated Ca2+ currents identified in this cell type. The present experiments show that the transient hyperpolarization induced by angiotensin
II is due to Ca2+-dependent K+ and Cl− currents. These two membrane currents are co-activated in response to an internal increase of [Ca2+]
i
originating from intra- and extracellular stores.
Received: 29 May 1997/Revised: 4 November 1997 相似文献
18.
The Ca2+-activated maxi K+ channel was found in the apical membrane of everted rabbit connecting tubule (CNT) with a patch-clamp technique. The mean
number of open channels (NP
o
) was markedly increased from 0.007 ± 0.004 to 0.189 ± 0.039 (n= 7) by stretching the patch membrane in a cell-attached configuration. This activation was suggested to be coupled with the
stretch-activation of Ca2+-permeable cation channels, because the maxi K+ channel was not stretch-activated in both the cell-attached configuration using Ca2+-free pipette and in the inside-out one in the presence of 10 mm EGTA in the cytoplasmic side. The maxi K+ channel was completely blocked by extracellular 1 μm charybdotoxin (CTX), but was not by cytoplasmic 33 μm arachidonic acid (AA). On the other hand, the low-conductance K+ channel, which was also found in the same membrane, was completely inhibited by 11 μm AA, but not by 1 μm CTX. The apical K+ conductance in the CNT was estimated by the deflection of transepithelial voltage (ΔV
t
) when luminal K+ concentration was increased from 5 to 15 mEq. When the tubule was perfused with hydraulic pressure of 0.5 KPa, the ΔV
t
was only −0.7 ± 0.4 mV. However, an increase in luminal fluid flow by increasing perfusion pressure to 1.5 KPa markedly enhanced
ΔV
t
to −9.4 ± 0.9 mV. Luminal application of 1 μm CTX reduced the ΔV
t
to −1.3 ± 0.6 mV significantly in 6 tubules, whereas no significant change of ΔV
t
was recorded by applying 33 μm AA into the lumen of 5 tubules (ΔV
t
=−7.2 ± 0.5 mV in control vs.ΔV
t
=−6.7 ± 0.6 mV in AA). These results suggest that the Ca2+-activated maxi K+ channel is responsible for flow-dependent K+ secretion by coupling with the stretch-activated Ca2+-permeable cation channel in the rabbit CNT.
Received: 21 August 1997/Revised: 20 March 1998 相似文献
19.
H. Chabot M.F. Vives A. Dagenais Cz. Grygorczyk Y. Berthiaume R. Grygorczyk 《The Journal of membrane biology》1999,169(3):175-188
Defective regulatory interactions between the cystic fibrosis conductance regulator (CFTR) and the epithelial sodium channel
(ENaC) have been implicated in the elevated Na+ transport rates across cystic fibrosis airway epithelium. It has recently been proposed that ENaC downregulation by CFTR
depends on the ability of CFTR to conduct Cl− into the cell and is negligible when Cl− flows out of the cell. To study the mechanisms of this downregulation we have measured amiloride-inhibitable Na+ current (I
amil
) in oocytes co-expressing rat ENaC and human wild-type CFTR. In oocytes voltage-clamped to −60 mV, stimulating CFTR with
1 mm IBMX reduced I
amil
by up to 80%, demonstrating that ENaC is inhibited when Cl− is conducted out of the cell. Decreasing the level of CFTR stimulation in a single oocyte, decreased both the degree of I
amil
downregulation and the CFTR-mediated plasma membrane Cl− conductance, suggesting a direct correlation. However, I
amil
downregulation was not affected when Cl− flux across oocyte membrane was minimized by holding the oocyte membrane potential near the Cl− reversal potential (67% ± 10% inhibition at −20 mV compared to 79% ± 4% at −60 mV) demonstrating that I
amil
downregulation was independent of the amount of current flow through CFTR. Studies with the Ca2+-sensitive photoprotein aequorin showed that Ca2+ is not involved in I
amil
downregulation by CFTR, although Ca2+ injection into the cytoplasm did inhibit I
amil
. These results demonstrate that downregulation of ENaC by CFTR depends on the degree of CFTR stimulation, but does not involve
Ca2+ and is independent of the direction and magnitude of Cl− transport across the plasma membrane.
Received: 15 December 1998/Revised: 5 March 1999 相似文献
20.
The pharmacological profile of a voltage-independent Ca2+-activated potassium channel of intermediate conductance (IK(Ca2+)) present in bovine aortic endothelial cells (BAEC) was investigated in a series of inside-out and outside-out patch-clamp
experiments. Channel inhibition was observed in response to external application of ChTX with a half inhibition concentration
of 3.3 ± 0.3 nm (n= 4). This channel was insensitive to IbTX, but channel block was detected following external application of MgTX and StK
leading to the rank order toxin potency ChTX > StK > MgTX >>IbTX. A reduction of the channel unitary current amplitude was
also measured in the presence of external TEA, with half reduction occurring at 23 ± 3 mm TEA (n= 3). The effect of TEA was voltage insensitive, an indication that TEA may bind to a site located on external side of the
pore region of this channel. Similarly, the addition of d-TC to the external medium caused a reduction of the channel unitary
current amplitude with half reduction at 4.4 ± 0.3 mm (n= 4). In contrast, application of d-TC to the bathing medium in inside-out experiments led to the appearance of long silent
periods, typical of a slow blocking process. Finally, the IK(Ca2+) in BAEC was found to be inhibited by NS1619, an activator of the Ca2+-activated potassium channel of large conductance (Maxi K(Ca2+)), with a half inhibition value of 11 ± 0.8 μm (n= 4). These results provide evidence for a pharmacological profile distinct from that reported for the Maxi K(Ca2+) channel, with some features attributed to the voltage-gated KV1.2 potassium channel.
Received: 6 November 1997/Revised: 19 February 1998 相似文献