共查询到20条相似文献,搜索用时 31 毫秒
1.
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
2.
O. Strauß K. Steinhausen S. Mergler F. Stumpff M. Wiederholt 《The Journal of membrane biology》1999,169(3):141-153
This combined study of patch-clamp and intracellular Ca2+ ([Ca2+]
i
) measurement was undertaken in order to identify signaling pathways that lead to activation of Ca2+-dependent Cl− channels in cultured rat retinal pigment epithelial (RPE) cells. Intracellular application of InsP3 (10 μm) led to an increase in [Ca2+]
i
and activation of Cl− currents. In contrast, intracellular application of Ca2+ (10 μm) only induced transient activation of Cl− currents. After full activation by InsP3, currents were insensitive to removal of extracellular Ca2+ and to the blocker of I
CRAC, La3+ (10 μm), despite the fact that both maneuvers led to a decline in [Ca2+]
i
. The InsP3-induced rise in Cl− conductance could be prevented either by thapsigargin-induced (1 μm) depletion of intracellular Ca2+ stores or by removal of Ca2+ prior to the experiment. The effect of InsP3 could be mimicked by intracellular application of the Ca2+-chelator BAPTA (10 mm). Block of PKC (chelerythrine, 1 μm) had no effect. Inhibition of Ca2+/calmodulin kinase (KN-63, KN-92; 5 μm) reduced Cl−-conductance in 50% of the cells investigated without affecting [Ca2+]
i
. Inhibition of protein tyrosine kinase (50 μm tyrphostin 51, 5 μm genistein, 5 μm lavendustin) reduced an increase in [Ca2+]
i
and Cl− conductance. In summary, elevation of [Ca]
i
by InsP3 leads to activation of Cl− channels involving cytosolic Ca2+ stores and Ca2+ influx from extracellular space. Tyrosine kinases are essential for the Ca2+-independent maintenance of this conductance.
Received: 15 October 1998/Revised: 3 March 1999 相似文献
3.
Properties of large conductance Ca2+-activated K+ channels were studied in the soma of motoneurones visually identified in thin slices of neonatal rat spinal cord. The channels
had a conductance of 82 ± 5 pS in external Ringer solution (5.6 mm K+
o
//155 mm K+
i
) and 231 ± 4 pS in external high-K
o
solution (155 mm K+
o
//155 mm K+
i
). The channels were activated by depolarization and by an increase in internal Ca2+ concentration. Potentials of half-maximum channel activation (E50) were −13, −34, −64 and −85 mV in the presence of 10−6, 10−5, 10−4 and 10−3
m internal Ca2+, respectively. Using an internal solution containing 10−4
m Ca2+, averaged KCa currents showed fast activation within 2–3 msec after a voltage step to +50 mV. Averaged KCa currents did not inactivate during 400 msec voltage pulses. External TEA reduced the apparent single-channel amplitude with
a 50% blocking concentration (IC50) of 0.17 ± 0.02 mm. KCa channels were completely suppressed by externally applied 100 mm charybdotoxin. It is concluded that KCa channels activated by Ca2+ entry during the action potential play an important role in the excitability of motoneurones.
Received: 7 November 1996/Revised: 29 October 1997 相似文献
4.
Mechanically Activated Currents in Chick Heart Cells 总被引:7,自引:0,他引:7
As predicted from stretch-induced changes of rate and rhythm in the heart, acutely isolated embryonic chick heart cells exhibit
whole-cell mechanosensitive currents. These currents were evoked by pressing on cells with a fire polished micropipette and
measured through a perforated patch using a second pipette. The currents were carried by Na+ and K+ but not Cl−, and were independent of external Ca2+. The currents had linear I/V curves reversing at −16 mV and were completely blocked by Gd3+≥ 30 μm and Grammostola spatulata venom at a dilution of 1:1000. Approximately 20% of cells showed time dependent inactivation. In contrast to direct mechanical
stimulation, hypotonic volume stress produced an increase in conductance for anions rather than cations—the two stimuli are
not equivalent. The cells had two types of stretch-activated ion channels (SACs): a 21 pS nonspecific cation-selective reversing
at −2 mV and a 90 pS K+ selective reversing at −70 mV in normal saline. The activity of SACs was strongly correlated with the presence of whole-cell
currents. Both the whole-cell currents and SACs were blocked by Gd3+ and by Grammostola spatulata spider venom. Mechanical stimulation of spontaneously active cells increased the beating rate and this effect was blocked
by Gd3+. We conclude that physiologically active mechanosensitive currents arise from stretch activated ion channels.
Received: 8 April 1996/Revised: 8 August 1996 相似文献
5.
Guyot A Dupré-Aucouturier S Ojeda C Rougier O Bilbaut A 《The Journal of membrane biology》2000,173(2):149-163
Voltage-activated Ca2+ currents, in zona fasciculata cells isolated from calf adrenal gland, were characterized using perforated patch-clamp recording.
In control solution (Ca2+: 2.5 mm) a transient inward current was followed, in 40% of the cells, by a sustained one. In 20 mm Ba2+, 61% of the cells displayed an inward current, which consisted of transient and sustained components. The other cells produced
either a sustained or a transient inward current. These different patterns were dependent upon time in culture. Current-voltage
relationships show that both the transient and sustained components activated, peaked and reversed at similar potentials:
−40, 0 and +60 mV, respectively. The two components, fully inactivated at −10 mV, were separated by double-pulse protocols
from different holding potentials where the transient component could be inactivated or reactivated. The decaying phase of
the sustained component was fitted by a double exponential (time constants: 1.9 and 20 sec at +10 mV); that of the transient
component was fitted by a single exponential (time constant: 19 msec at +10 mV). Steady-state activation and inactivation
curves of the two components were superimposed. Their half activation and inactivation potentials were similar, about −15
and −34 mV, respectively. The sustained component was larger in Ba2+ than in Sr2+ and Ca2+. Ni2+ (20 μm) selectively blocked the transient component while Cd2+ (10 μm) selectively blocked the sustained one. (±)Bay K 8644 (0.5 μm) increased the sustained component and nitrendipine (0.5–1 μm) blocked it selectively. The sustained component was inhibited by calciseptine (1 μm). Both components were unaffected by ω-conotoxin GVIA and MVIIC (0.5 μm). These results show that two distinct populations of Ca2+ channels coexist in this cell type. Although the voltage dependence of their activation and inactivation are comparable,
these two components of the inward current are similar to T- and L-type currents described in other cells.
Received: 12 July 1999/Revised: 5 October 1999 相似文献
6.
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
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.
The superficial (tonic) abdominal flexor muscles of Atya lanipes do not generate Ca2+ action potentials when depolarized and have no detectable inward Ca2+ current. These fibers, however, are strictly dependent on Ca2+ influx for contraction, suggesting that they depend on Ca2+-induced Ca2+ release for contractile activation. The nature of the communication between Ca2+ channels in the sarcolemmal/tubular membrane and Ca2+ release channels in the sarcoplasmic reticulum in this crustacean muscle was investigated. The effects of dihydropyridines
on tension generation and the passive electrical response were examined in current-clamped fibers: Bay K 8644 enhanced tension
about 100% but did not alter the passive electrical response; nifedipine inhibited tension by about 70%. Sr2+ and Ba2+ action potentials could be elicited in Ca2+-free solutions. The spikes generated by these divalent cations were abolished by nifedipine. As the Sr2+ or Ba2+ concentrations were increased, the amplitudes of the action potentials and their maximum rate of rise, V
max
, increased and tended towards saturation. Three-microelectrode voltage-clamp experiments showed that even at high (138 mm) extracellular Ca2+ concentration the channels were silent, i.e., no inward Ca2+ current was detected. In Ca2+-free solutions, inward currents carried by 138 mm Sr2+ or Ba2+ were observed. The currents activated at voltages above −40 mV and peaked at about 0 mV. This voltage-activation profile
and the sensitivity of the channels to dihydropyridines indicate that they resemble L-type Ca2+ channels. Peak inward current density values were low, ca.−33 μA/cm2 for Sr2+ and −14 μA/cm2 for Ba2+, suggesting that Ca2+ channels are present at a very low density. It is concluded that Ca2+-induced Ca2+ release in this crustacean muscle operates with an unusually high gain: Ca2+ influx through the silent Ca2+ channels is too low to generate a macroscopic inward current, but increases sufficiently the local concentration of Ca2+ in the immediate vicinity of the sarcoplasmic reticulum Ca2+ release channels to trigger the highly amplified release of Ca2+ required for tension generation.
Received: 5 April 1999/Revised: 15 September 1999 相似文献
9.
Cells from ten human meningiomas were electrophysiologically characterized in both living tissue slices and primary cultures.
In whole cells, depolarization to voltages higher than +80 mV evoked a large K+ outward current, which could be blocked by iberiotoxin (100 nm) and TEA (half blocking concentration IC50= 5.3 mm). Raising the internal Ca2+ from 10 nm to 2 mm shifted the voltage of half-maximum activation (V
1/2) of the K+ current from +106 to +4 mV. Respective inside-out patch recordings showed a voltage- and Ca2+-activated (BK
Ca
) K+ channel with a conductance of 296 pS (130 mm K+ at both sides of the patch). V
1/2 of single-channel currents was +6, −12, −46, and −68 mV in the presence of 1, 10, 100, and 1000 μm Ca2+, respectively, at the internal face of the patch. In cell-attached patches the open probability (P
o
) of BK
Ca
channels was nearly zero at potentials below +80 mV, matching the activation threshold for whole-cell K+ currents with 10 nm Ca2+ in the pipette. Application of 20 μm cytochalasin D increased P
o
of BK
Ca
channels in cell-attached patches within minutes. These data suggest that the activation of BK
Ca
channels in meningioma cells does not only depend on voltage and internal Ca2+ but is also controlled by the cytoskeleton.
Received 18 June 1999/Revised: 18 January 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.
The present study demonstrates that B-type Ca2+ channels observed in rat ventricular myocytes markedly reacted to agents known to affect the ion-motive plasma membrane Ca2+-ATPase (PMCA) pump. Chlorpromazine (CPZ)-activated B-type Ca2+ channels were completely blocked by internal application of PMCA pump inhibitors, namely La3+ (100 μm), eosin (10 μm) and AIF3 (100 μm). Calmodulin (50 U/ml), the main endogenous positive regulator of PMCA, was unable to activate but significantly reduced
CPZ-activated B-type channel activity. In the same manner, ATP (1 and 4 mm), the main energizing substrate of PMCA, was able to reversibly and significantly reduce this activity in a dose-dependent
manner. Interestingly, anti-PMCA antibody 5F10, but not anti-Na/K ATPase antibody (used as a negative control) induced a marked
Ba2+-conducting channel activity that shared the same characteristics with that of CPZ-activated B-type channels. 5F10-Activated
channels were mostly selective towards Ba2+, mainly had three observed conductance levels (23, 47 and 85 pS), were observed with a frequency of about 1 out of 5 membrane
patches and were completely blocked by 10 μm eosin. These results suggest that B-type Ca2+ channels are some form of the PMCA pump.
Received: 24 July 2000/Revised: 5 October 2000 相似文献
12.
K. Yoshimura 《The Journal of membrane biology》1998,166(2):149-155
Mechanosensitive channels appear ubiquitous but they have not been well characterized in cells directly responding to mechanical
stimuli. Here, we identified tension-sensitive channel currents on the cell body of Chlamydomonas, a protist that shows a marked behavioral response to mechanical stimulation. When a negative pressure was applied to the
cell body with a patch clamp electrode, single-ion-channel currents of 2.4 pA in amplitude were observed. The currents were
inhibited by 10 μm gadolinium, a general blocker of mechanosensitive channels. The currents were most likely due to Ca2+ influxes because the current was absent in Ca2+-free solutions and the reversal potential was 98 mV positive to the resting potential. The distribution of channel-open times
conformed to a single exponential component and that of closed times to two exponential components. This mechanosensitive
channel was similar to the one found in the flagella in the following respects: both channels were inhibited by Gd3+ at 10 μm but not at 1 μm; both passed Ca2+ and Ba2+; their kinetic parameters for channel opening were similar. These observations raise the possibility that identical mechanosensitive
channels may function both in the behavioral control through the mechanoreception by the flagella and in the regulation of
cellular physiology in response to mechanical perturbation on the cell body.
Received: 13 May 1998/Revised: 2 September 1998 相似文献
13.
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
14.
Melanoma cells are transformed melanocytes of neural crest origin. K+ channel blockers have been reported to inhibit melanoma cell proliferation. We used whole-cell recording to characterize
ion channels in four different human melanoma cell lines (C8161, C832C, C8146, and SK28). Protocols were used to identify
voltage-gated (KV), Ca2+-activated (KCa), and inwardly rectifying (KIR) K+ channels; swelling-sensitive Cl− channels (Clswell); voltage-gated Ca2+ channels (CaV) and Ca2+ channels activated by depletion of intracellular Ca2+ stores (CRAC); and voltage-gated Na+ channels (NaV). The presence of Ca2+ channels activated by intracellular store depletion was further tested using thapsigargin to elicit a rise in [Ca2+]
i
. The expression of K+ channels varied widely between different cell lines and was also influenced by culture conditions. KIR channels were found in all cell lines, but with varying abundance. Whole-cell conductance levels for KIR differed between C8161 (100 pS/pF) and SK28 (360 pS/pF). KCa channels in C8161 cells were blocked by 10 nm apamin, but were unaffected by charybdotoxin (CTX). KCa channels in C8146 and SK28 cells were sensitive to CTX (K
d
= 4 nm), but were unaffected by apamin. KV channels, found only in C8146 cells, activated at ∼−20 mV and showed use dependence. All melanoma lines tested expressed
CRAC channels and a novel Clswell channel. Clswell current developed at 30 pS/sec when the cells were bathed in 80% Ringer solution, and was strongly outwardly rectifying (4:1
in symmetrical Cl−). We conclude that different melanoma cell lines express a diversity of ion channel types.
Received: 2 April 1996/Revised: 22 August 1996 相似文献
15.
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
16.
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
17.
Nonselective Cation and BK Channels in Apical Membrane of Outer Sulcus Epithelial Cells 总被引:5,自引:0,他引:5
The outer sulcus epithelium was recently shown to absorb cations from the lumen of the gerbil cochlea. Patch clamp recordings
of excised apical membrane were made to investigate ion channels that participate in this reabsorptive flux. Three types of
channel were observed: (i) a nonselective cation (NSC) channel, (ii) a BK (large conductance, maxi K or K
Ca
) channel and (iii) a small K+ channel which could not be fully characterized. The NSC channel found in excised insideout patch recordings displayed a linear
current-voltage (I-V) relationship (27 pS) and was equally conductive for Na+ and K+, but not permeable to Cl− or N-methyl-d-glucamine. Channel activity required the presence of Ca2+ at the cytosolic face, but was detected at Ca2+ concentrations as low as 10−7
m (open probability (P
o
) = 0.11 ± 0.03, n= 8). Gadolinium decreased P
o
of the NSC channel from both the external and cytosolic side (IC50∼ 0.6 μm). NSC currents were decreased by amiloride (10 μm− 1 mm) and flufenamic acid (0.1 mm). The BK channel was also frequently (38%) observed in excised patches. In symmetrical 150 mm KCl conditions, the I-V relationship was linear with a conductance of 268 pS. The Goldman-Hodgkin-Katz equation for current carried solely by K+ could be fitted to the I-V relationship in asymmetrical K+ and Na+ solutions. The channel was impermeable to Cl− and N-methyl-d-glucamine. P
o
of the BK channel increased with depolarization of the membrane potential and with increasing cytosolic Ca2+. TEA (20 mm), charybdotoxin (100 nm) and Ba2+ (1 mm) but not amiloride (1 mm) reduced P
o
from the extracellular side. In contrast, external flufenamic acid (100 μm) increased P
o
and this effect was inhibited by charybdotoxin (100 nm). Flufenamic acid inhibited the inward short-circuit current measured by the vibrating probe and caused a transient outward
current. We conclude that the NSC channel is Ca2+ activated, voltage-insensitive and involved in both constitutive K+ and Na+ reabsorption from endolymph while the BK channel might participate in the K+ pathway under stimulated conditions that produce an elevated intracellular Ca2+ or depolarized membrane potential.
Received: 14 October 1999/Revised: 10 December 1999 相似文献
18.
We studied the characteristics of the basal and antidiuretic hormone (arginine vasotocin, AVT)-activated whole cell currents
of an aldosterone-treated distal nephron cell line (A6) at two different cytosolic Ca2+ concentrations ([Ca2+]
c
, 2 and 30 nm). A6 cells were cultured on a permeable support filter for 10 ∼ 14 days in media with supplemental aldosterone (1 μm). At 30 nm [Ca2+]
c
, basal conductances mainly consisted of Cl− conductances, which were sensitive to 5-nitro-2-(3-phenylpropylamino)-benzoate. Reduction of [Ca2+]
c
to 2 nm abolished the basal Cl− conductance. AVT evoked Cl− conductances at 2 as well as 30 nm [Ca2+]
c
. In addition to Cl− conductances, AVT induced benzamil-insensitive nonselective cation (NSC) conductances. This action on NSC conductances was
observed at 30 nm [Ca2+]
c
but not at 2 nm [Ca2+]
c
. Thus, cytosolic Ca2+ regulates NSC and Cl− conductances in a distal nephron cell line (A6) in response to AVT. Keeping [Ca2+]
c
at an adequate level seems likely to be an important requirement for AVT regulation of ion conductances in aldosterone-treated
A6 cells.
Received: 6 May 1996/Revised: 28 June 1996 相似文献
19.
Inhibition of large-conductance calcium-activated potassium channel by 2-methoxyestradiol in cultured vascular endothelial (HUV-EC-C) cells 总被引:4,自引:0,他引:4
2-Methoxyestradiol, an endogenous metabolite of 17β-estradiol, is known to have antitumor and antiangiogenic actions. The
effects of 2-methoxyestradiol on ionic currents were investigated in an endothelial cell line (HUV-EC-C) originally derived
from human umbilical vein. In the whole-cell patch-clamp configuration, 2-methoxyestradiol (0.3–30 μm) reversibly suppressed the amplitude of K+ outward currents. The IC
50 value of the 2-methoxyestradiol-induced decrease in outward current was 3 μm. Evans blue (30 μm) or niflumic acid (30 μm), but not diazoxide (30 μm), reversed the 2-methoxyestradiol-induced decrease in outward current. In the inside-out configuration, application of 2-methoxyestradiol
(3 μm) to the bath did not modify the single-channel conductance of large-conductance Ca2+-activated K+ (BKCa) channels; however, it did suppress the channel activity. 2-Methoxyestradiol (3 μm) produced a shift in the activation curve of BKCa channels to more positive potentials. Kinetic studies showed that the 2-methoxyestradiol-induced inhibition of BKCa channels is primarily mediated by a decrease in the number of long-lived openings. 2-Methoxyestradiol-induced inhibition
of the channel activity was potentiated by membrane stretch. In contrast, neither 17β-estradiol (10 μm) nor estriol (10 μm) affected BKCa channel activity, whereas 2-hydroxyestradiol (10 μm) slightly suppressed it. Under current-clamp condition, 2-methoxyestradiol (10 μm) caused membrane depolarization and Evans blue (30 μm) reversed 2-methoxyestradiol-induced depolarization. The present study provides evidence that 2-methoxyestradiol can suppress
the activity of BKCa channels in endothelial cells. These effects of 2-methoxyestradiol on ionic currents may contribute to its effects on functional
activity of endothelial cells.
Received: 27 November 2000/Revised: 13 April 2001 相似文献
20.
The effects of Ni2+ were evaluated on slowly-decaying, high-voltage-activated (HVA) Ca2+ currents expressed by pyramidal neurons acutely dissociated from guinea-pig piriform cortex. Whole-cell, patch-clamp recordings
were performed with Ba2+ as the charge carrier. Ni2+ blocked HVA Ba2+ currents (I
Bas) with an EC50 of approximately 60 μm. Additionally, after application of nonsaturating Ni2+ concentrations, residual currents activated with substantially slower kinetics than both total and Ni2+-sensitive I
Bas. None of the pharmacological components of slowly decaying, HVA currents activated with kinetics significantly different
from that of total currents, indicating that the effect of Ni2+ on I
Bas kinetics cannot be attributed to the preferential inhibition of a fast-activating component. The effect of Ni2+ on I
Ba amplitude was voltage-independent over the potential range normally explored in our experiments (−60 to +20 mV), hence the
Ni2+-dependent decrease of I
Ba activation rate is not due to a voltage- and time-dependent relief from block. Moreover, Ni2+ significantly reduced I
Ba deactivation speed upon repolarization, which also is not compatible with a depolarization-dependent unblocking mechanism.
The dependence on Ni2+ concentration of the I
Ba activation-rate reduction was remarkably different from that found for I
Ba block, with an EC50 of ∼20 μm and a Hill coefficient of ∼1.73 vs.∼1.10. These results demonstrate that Ni2+, besides inhibiting the I
Bas under study probably by exerting a blocking action on the pore of the underlying Ca2+ channels, also interferes with Ca2+-channel gating kinetics, and strongly suggest that the two effects depend on Ni2+ occupancy of binding sites at least partly distinct.
Received: 13 July 2000/Revised: 9 November 2000 相似文献