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1.
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
2.
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
3.
The dependence of currents through the cyclic nucleotide-gated (CNG) channels of mammalian olfactory receptor neurons (ORNs)
on the concentration of NaCl was studied in excised inside-out patches from their dendritic knobs using the patch-clamp technique.
With a saturating concentration (100 μm) of adenosine 3′, 5′-cyclic monophosphate (cAMP), the changes in the reversal potential of macroscopic currents were studied
at NaCl concentrations from 25 to 300 mm. In symmetrical NaCl solutions without the addition of divalent cations, the current-voltage relations were almost linear,
reversing close to 0 mV. When the external NaCl concentration was maintained at 150 mm and the internal concentrations were varied, the reversal potentials of the cAMP-activated currents closely followed the
Na+ equilibrium potential indicating that P
Cl/P
Na≈ 0. However, at low external NaCl concentrations (≤100 mm) there was some significant chloride permeability. Our results further indicated that Na+ currents through these channels: (i) did not obey the independence principle; (ii) showed saturation kinetics with K
ms in the range of 100–150 mm and (iii) displayed a lack of voltage dependence of conductance in asymmetric solutions that suggested that ion-binding sites
were situated midway along the channel. Together, these characteristics indicate that the permeation properties of the olfactory
CNG channels are significantly different from those of photoreceptor CNG channels.
Received: 7 November 1996/Revised: 24 March 1997 相似文献
4.
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
5.
A large-conductance (or maxi-) chloride channel was identified in bovine pigmented ciliary epithelial (PCE) cells using inside-out
excised patch clamp recording. The channel had a mean conductance of 293 pS when excised patches were bathed in symmetrical
130 mm NaCl although the conductance decreased to 209 pS when the solution bathing the cytoplasmic face of the patch contained only
33 mm NaCl. The channel was highly selective for chloride, with a P
Cl/P
Na= 24. A flickery, reversible block was produced by the diuretic stilbene 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic
acid (SITS), while 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) produced a permanent block. The channel was rarely
active in cell-attached patches and usually required several minutes of polarization before activity could be detected in
excised patches, a process known as metagenesis. Once activated, the channel was voltage-dependent and was mainly open within
the voltage range −30 to +30 mV closing when the membrane was polarized to larger values. GTPγS (100 μm) activated the channel with a latency of 170 sec when applied to the cytoplasmic face of patches. This activation was not
reversible upon return to control solution within the duration of the experiment. We assess the available evidence and suggest
a role for this channel in volume regulation.
Received: 24 June 1996/Revised: 18 February 1997 相似文献
6.
A K+ channel with a main conductance of 29 pS was recorded after the incorporation of coronary artery membrane vesicles into lipid
bilayers. This channel was identified as an ATP-sensitive K+ channel (KATP) because its activity was diminished by the internal application of 50–250 μm ATP-Na2. Moreover, it was opened when 10–50 μm pinacidil was externally applied. Single-channel records revealed the existence of several (sub)conductance states. At 0
mV and with a 5/250 KCl gradient, the main conductance of the KATP channel was 29 pS. The other (sub)conductance states were less frequent and had discrete values of 12, 17 and 22 pS. Pinacidil
stabilized the channel open state primarily in the 29 pS conductance level; whereas ATP inhibited all the conductance levels.
In general, KATP channels were characterized by brief openings followed by long closings (open probability, P
o
≈ 0.02); only occasionally (3 out of 12 experiments) did the KATP channels have a high open probability (P
o
≥ 0.7). Channel activity could be increased or rescued by adding 2.5–10 mm UDP-TRIS and 0.5–2 mm MgCl2 to the internal side of the channel.
Received: 7 November 1995/Revised: 10 June 1996 相似文献
7.
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
8.
R. Moreau A.M. Hurst J-Y. Lapointe D. Lajeunesse 《The Journal of membrane biology》1996,150(2):175-184
Patch clamp experiments were performed on two human osteosarcoma cell lines (MG-63 and SaOS-2 cells) that show an osteoblasticlike
phenotype to identify and characterize the specific K channels present in these cells. In case of MG-63 cells, in the cell-attached
patch configuration (CAP) no channel activity was observed in 2 mm Ca Ringer (control condition) at resting potential. In contrast, a maxi-K channel was observed in previously silent CAP upon
addition of 50 nm parathyroid hormone (PTH), 5 nm prostaglandin E2 (PGE2) or 0.1 mm dibutyryl cAMP + 1 μm forskolin to the bath solution. However, maxi-K channels were present in excised patches from both stimulated and nonstimulated
cells in 50% of total patches tested. A similar K channel was also observed in SaOS-2 cells. Characterization of this maxi-K
channel showed that in symmetrical solutions (140 mm K) the channel has a conductance of 246 ± 4.5 pS (n = 7 patches) and, when Na was added to the bath solution, the permeability ratio (PK/PNa) was 10 and 11 for MG-63 and SaOS-2 cells respectively. In excised patches from MG-63 cells, the channel open probability
(P
o
) is both voltage- (channel opening with depolarization) and Ca-dependent; the presence of Ca shifts the P
o
vs. voltage curve toward negative membrane potential. Direct modulation of this maxi-K channel via protein kinase A (PKA) is very
unlikely since in excised patches the activity of this channel is not sensitive to the addition of 1 mm ATP + 20 U/ml catalytic subunit of PKA. We next evaluated the possibility that PGE2 or PTH stimulated the channel through a rise in intracellular calcium. First, calcium uptake (45Ca++) by MG-63 cells was stimulated in the presence of PTH and PGE2, an effect inhibited by Nitrendipine (10 μm). Second, whereas PGE2 stimulated the calcium-activated maxi-K channel in 2 mm Ca Ringer in 60% of patches studied, in Ca-free Ringer bath solution, PGE2 did not open any channels (n = 10 patches) nor did cAMP + forskolin (n = 3 patches), although K channels were present under the patch upon excision. In addition, in the presence of 2 mm Ca Ringer and 10 μm Nitrendipine in CAP configuration, PGE2 (n = 5 patches) and cAMP + forskolin (n = 2 patches) failed to open K channels present under the patch. As channel activation by phosphorylation with the catalytic
subunit of PKA was not observed, and Nitrendipine addition to the bath or the absence of calcium prevented the opening of
this channel, it is concluded that activation of this channel by PTH, PGE2 or dibutyryl cAMP + forskolin is due to an increase in intracellular calcium concentration via Ca influx.
Received: 17 September 1995/Revised: 7 December 1995 相似文献
9.
A.E. Alekseev L.A. Gomez L.A. Aleksandrova P.A. Brady A. Terzic 《The Journal of membrane biology》1997,157(2):203-214
Opening of ATP-sensitive K+ (KATP) channels by the uncoupler of oxidative phosphorylation, 2,4 dinitrophenol (DNP), has been assumed to be secondary to metabolic
inhibition and reduced intracellular ATP levels. Herein, we present data which show that DNP (200 μm) can induce opening of cardiac KATP channels, under whole-cell and inside-out conditions, despite millimolar concentrations of ATP (1–2.5 mm). DNP-induced currents had a single channel conductance (71 pS), inward rectification, reversal potential, and intraburst
kinetic properties (open time constant, τopen: 4.8 msec; fast closed time constant, τclosed(f): 0.33 msec) characteristic of KATP channels suggesting that DNP did not affect the pore region of the channel, but may have altered the functional coupling
of the ATP-dependent channel gating. A DNP analogue, with the pH-titrable hydroxyl replaced by a methyl group, could not open
KATP channels. The pH-dependence of the effect of DNP on channel opening under whole-cell, cell-attached, and inside-out conditions
suggested that transfer of protonated DNP across the sarcolemma is essential for activation of KATP channels in the presence of ATP. We conclude that the use of DNP for metabolic stress-induced KATP channel opening should be reevaluated.
Received: 10 September 1996/Revised: 27 December 1996 相似文献
10.
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
11.
D.P. Lotshaw 《The Journal of membrane biology》1997,156(3):261-277
Nystatin perforated-patch clamp and single-channel recording methods were used to characterize macroscopic and single-channel
K+ currents and the effects of angiotensin II (AngII) in cultured rat adrenal glomerulosa cells. Two basic patterns of macroscopic
current-voltage relationships were observed: type 1 exhibited a rapidly activating, noninactivating, voltage-dependent outward
current and type 2 exhibited an inactivating voltage-dependent outward current attributed to charybdotoxin sensitive Ca++-dependent K+ channels. Most cells exhibited the type 1 pattern and experiments focused on this cell type. Cell-attached and inside-out
patches were dominated by a single K+ channel class which exhibited an outward conductance of 12 pS (20 mm K+ pipette in cell-attached and inside-out configurations, 145 mm K+
in), a mean open time of 2 msec, and a weakly voltage-dependent low open probability that increased with depolarization. Channel
open probability was reversibly inhibited by bath stimulation with AngII. At the macroscopic level, type 1 cell macroscopic
K+ currents appeared comprised of two components: a weakly voltage-dependent current controlling the resting membrane potential
(−85 mV) which appeared mediated by the 12 pS K+ channel and a rapidly activating, noninactivating voltage-dependent current activated above −50 mV. The presence of the second
voltage-dependent K+ channel class was suggested by the effects of AngII, the blocking effects of quinidine and Cs+, and the properties of the weakly voltage-dependent K+ channel described. The K+ selectivity of the macroscopic current was demonstrated by the dependence of current reversal potentials on the K+ equilibrium potential and by the effects of K+ channel blockers, Cs+ and quinidine. AngII (10 pm to 1 nm) reversibly inhibited macroscopic K+ currents and this effect was blocked by the AT1 receptor antagonist losartin.
Received: 6 August 1996/Revised: 15 November 1996 相似文献
12.
P. Light Y. Shimoni S. Harbison W. Giles R.J. French 《The Journal of membrane biology》1998,162(3):217-223
The effects of thyroid status on the properties of ATP-sensitive potassium channels were investigated. Single-channel recordings
were made using excised inside-out membrane patches from enzymatically dissociated ventricular myocytes from hearts of control
and thyroidectomized rats and each group was studied with and without administration of thyroid hormone.
In patches excised from hypothyroid myocytes the IC50 for ATP inhibition of KATP channels was 110 μm. This value was 3-fold higher than the IC50 in control myocytes (43 μm). Treatment of hypothyroid rats to restore physiological levels of thyroid hormone (tri-iodothyronine, T3), resulted in a return to normal ATP-sensitivity (IC50= 46 μm). In patches from animals rendered hyperthyroid, the IC50 for ATP was 50 μm and this value was not significantly different from the control. There was no difference in the cooperativity of ATP-binding
(Hill coefficient, nH) among control (nH= 2.2), hypothyroid (nH= 2.1), T3-treated (nH= 2.0) and hyperthyroid groups (nH= 2.4). The unitary conductance was unchanged and there was no apparent change in intraburst kinetics between examples of
single KATP channels from control and hypothyroid rats. Action potentials recorded in myocytes from hypothyroid rats were significantly
shortened by 50 μm levcromakalim, a KATP channel opener (P < 0.001) but unchanged in control myocytes.
We conclude that hypothyroidism significantly decreased the ATP-sensitivity of KATP channels, whereas the induction of hyperthyroid conditions did not alter the ATP-sensitivity of these channels. Thus, hypothyroidism
is likely to have important physiological consequences under circumstances in which KATP channels are activated, such as during ischemia.
Received: 1 July 1997/Revised: 24 December 1997 相似文献
13.
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
14.
N.A. Sunstrom L.S. Premkumar A. Premkumar G. Ewart G.B. Cox P.W. Gage 《The Journal of membrane biology》1996,150(2):127-132
The influenza B virus protein, NB, was expressed in Escherichia coli, either with a C-terminal polyhistidine tag or with NB fused to the C-terminus of glutathione S-transferase (GST), and purified
by affinity chromatography. NB produced ion channel activity when added to artificial lipid bilayers separating NaCl solutions
with unequal concentrations (150–500 mm
cis, 50 mm
trans). An antibody to a peptide mimicking the 25 residues at the C-terminal end of NB, and amantadine at high concentration (2–3
mm), both depressed ion channel activity. Ion channels had a variable conductance, the lowest conductance observed being approximately
10 picosiemens. At a pH of 5.5 to 6.5, currents reversed at positive potentials indicating that the channel was more permeable
to sodium than to chloride ions (PNa/PCl∼ 9). In asymmetrical NaCl solutions at a pH of 2.5, currents reversed closer to the chloride than to the sodium equilibrium
potential indicating that the channel had become more permeable to chloride than to sodium ions (PCl/PNa∼ 4). It was concluded that, at normal pHs, NB forms cation-selective channels.
Received: 6 March 1995/Revised: 17 November 1995 相似文献
15.
Other than the fact that the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel can be activated by cAMP dependent kinase (PKA), little is known about the signal transduction pathways regulating
CFTR. Since G-proteins play a principal role in signal transduction regulating several ion channels [4, 5, 9], we sought to
test whether G-proteins control CFTR Cl− conductance (CFTR G
Cl
) in the native sweat duct (SD). We permeabilized the basolateral membrane with α-toxin so as to manipulate cytosolic nucleotides.
We activated G-proteins and monitored CFTR G
Cl
activity as described earlier [20, 23, 25]. We now show that activating G-proteins with GTP-γ-S (100 μm) also activates CFTR G
Cl
in the presence of 5 mm ATP alone (without exogenous cAMP). GTP-γ-S increased CFTR G
Cl
by 44 ± 20 mS/cm2 (mean ±se; n= 7). GDP (10 mm) inhibited G-protein activation of CFTR G
Cl
even in the presence of GTP-γ-S. The heterotrimeric G-protein activator (AlF4
−) in the cytoplasmic bath activated CFTR G
Cl
(increased by 51.5 ± 9.4 mS/cm2 in the presence of 5 mm ATP without cAMP, n= 6), the magnitude of which was similar to that induced by GTP-γ-S. Employing immunocytochemical-labeling techniques, we
localized Gαs, Gαi, Gαq, and Gβ at the apical membranes of the sweat duct. Further, we showed that the mutant CFTR G
Cl
in ducts from cystic fibrosis (CF) subjects could be partially activated by G-proteins. The magnitude of mutant CFTR G
Cl
activation by G-proteins was smaller as compared to non-CF ducts but comparable to that induced by cAMP in CF ducts. We conclude
that heterotrimeric G-proteins are present in the apical membrane of the native human sweat duct which may help regulate salt
absorption by controlling CFTR G
Cl
activity.
Received: 9 June 2000/Revised: 5 October 2000 相似文献
16.
F. Lemtiri-Chlieh 《The Journal of membrane biology》1996,153(2):105-116
One of the main effects of abscisic acid (ABA) is to induce net loss of potassium salts from guard cells enabling the stomata
to close. K+ is released from the vacuole into the cytosol and then to the extracellular space. The effects of increasing cytosolic K+ on the voltage- and time-dependence of the outwardly rectifying K+-current (I
K,out) in guard cell protoplasts (GCP) was examined in the whole-cell configuration of the patch-clamp technique. The same quantitative
analysis was performed in the presence of ABA at different internal K+ concentrations ([K+]
i
). Varying [K+]
i
in the patch pipette from 100 to 270 mm increased the magnitude of I
K,out in a nonlinear manner and caused a negative shift in the midpoint (V
0.5) of its steady-state activation curve. External addition of ABA (10–20 μm) also increased the magnitude of I
K,out at all [K+]
i
, but caused a shift in V
0.5 of the steady-state activation curve only in those GCP loaded with 150 mm internal K+ or less. Indeed, V
0.5 did not shift upon addition of ABA when the [K+]
i
was above 150 mm and up to 270 mm, i.e., the shift in V
0.5 caused by ABA depended on the [K+]
i
. Both increase in [K+]
i
and external addition of ABA, decreased (by ≈ 20%) the activation time constant (τ
n
) of I
K,out. The small decrease in τ
n
, in both cases, was found to be independent of the membrane voltage. The results indicate that ABA mimics the effect of increasing
cytoplasmic K+, and suggest that ABA may increase I
K,out and alter V
0.5 of its steady-state activation curve via an enhancement in cytosolic K+. This report describes for the first time the effects of [K+]
i
on the voltage- and time-dependence of I
K,out in guard cells. It also provides an explanation for the quantitative (total membrane current) and qualitative (current kinetics)
differences found between intact guard cells and their protoplasts.
Received: 1 December 1995/Revised: 8 May 1996 相似文献
17.
GABAA channels were activated by GABA in outside-out patches from rat cultured hippocampal neurons. They were blocked by bicuculline
and potentiated by diazepam. In 109 of 190 outside-out patches, no channels were active before exposure to GABA (silent patches).
The other 81 patches showed spontaneous channel activity. In patches containing spontaneous channel activity, rapid application
of GABA rapidly activated channels. In 93 of the silent patches, channels could be activated by GABA but only after a delay
that was sometimes as long as 10 minutes. The maximum channel conductance of the channels activated after a delay increased
with GABA concentration from less than 10 pS (0.5 μm GABA) to more than 100 pS (10 mm GABA). Fitting the data with a Hill-type equation gave an EC
50 value of 33 μm and a Hill coefficient of 0.6. The channels showed outward rectification and were chloride selective. In the presence of
1 μm diazepam, the GABA EC
50 decreased to 0.2 μm but the maximum conductance was unchanged. Diazepam decreased the average latency for channel opening. Bicuculline, a GABA
antagonist, caused a concentration-dependent decrease in channel conductance. In channels activated with 100 μm GABA the bicuculline IC
50 was 19 μm. The effect of GABA on channel conductance shows that the role of the ligand in GABAA receptor channel function is more complex than previously thought.
Received: 23 October 2000/Revised: 27 February 2001 相似文献
18.
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
19.
The gating cycle of CFTR (Cystic Fibrosis Transmembrane conductance Regulator) chloride channels requires ATP hydrolysis
and can be interrupted by exposure to the nonhydrolyzable nucleotide AMP-PNP. To further characterize nucleotide interactions
and channel gating, we have studied the effects of AMP-PNP, protein kinase C (PKC) phosphorylation, and temperature on gating
kinetics. The rate of channel locking increased from 1.05 × 10−3 sec−1 to 58.7 × 10−3 sec−1 when AMP-PNP concentration was raised from 0.5 to 5 mm in the presence of 1 mm MgATP and 180 nm protein kinase A catalytic subunit (PKA). Although rapid locking precluded estimation of P
o
or opening rate immediately after the addition of AMP-PNP to wild-type channels, analysis of locking rates in the presence
of high AMP-PNP concentrations revealed two components. The appearance of a distinct, slow component at high [AMP-PNP] is
evidence for AMP-PNP interactions at a second site, where competition with ATP would reduce P
o
and thereby delay locking. All channels exhibited locking when they were strongly phosphorylated by PKA, but not when exposed
to PKC alone. AMP-PNP increased P
o
at temperatures above 30°C but did not cause locking, evidence that the stabilizing interactions between domains, which have
been proposed to maintain CFTR in the open burst state, are relatively weak. The temperature dependence of normal CFTR gating
by ATP was strongly asymmetric, with the opening rate being much more temperature sensitive (Q
10= 9.6) than the closing rate (Q
10= 3.6). These results are consistent with a cyclic model for gating of phosphorylated CFTR.
Received: 28 August 1997/Revised: 4 February 1998 相似文献
20.
The structural determinants of mibefradil inhibition were analyzed using wild-type and inactivation-modified CaV1.2 (α1C) and CaV2.3 (α1E) channels. Mibefradil inhibition of peak Ba2+ currents was dose- and voltage-dependent. An increase of holding potentials from −80 to −100 mV significantly shifted dose-response
curves toward higher mibefradil concentrations, namely from a concentration of 108 ± 21 μm (n= 7) to 288 ± 17 μm (n= 3) for inhibition of half of the Cav1.2 currents (IC
50) and from IC
50= 8 ± 2 μm (n= 9) to 33 ± 7 μm (n= 4) for CaV2.3 currents. In the presence of mibefradil, CaV1.2 and CaV2.3 experienced significant use-dependent inhibition (0.1 to 1 Hz) and slower recovery from inactivation suggesting mibefradil
could promote transition(s) to an absorbing inactivated state. In order to investigate the relationship between inactivation
and drug sensitivity, mibefradil inhibition was studied in inactivation-altered CaV1.2 and CaV2.3 mutants. Mibefradil significantly delayed the onset of channel recovery from inactivation in CEEE (Repeat I + part of
the I–II linker from CaV1.2 in the CaV2.3 host channel), in EC(AID)EEE (part of the I–II linker from CaV1.2 in the CaV2.3 host channel) as well as in CaV1.2 E462R, and CaV2.3 R378E (point mutation in the β-subunit binding motif) channels. Mibefradil inhibited the faster inactivating chimera EC(IS1-6)EEE with an IC
50= 7 ± 1 μm (n= 3), whereas the slower inactivating chimeras EC(AID)EEE and CEEE were, respectively, inhibited with IC
50= 41 ± 5 μm (n= 4) and IC
50= 68 ± 9 μm (n= 5). Dose-response curves were superimposable for the faster EC(IS1-6)EEE and CaV2.3, whereas intermediate-inactivating channel kinetics (CEEE, CaV1.2 E462R, and CaV1.2 E462K) were inhibited by similar concentrations of mibefradil with IC
50≈ 55–75 μm. The slower CaV1.2 wild-type and CaV1.2 Q473K channels responded to higher doses of mibefradil with IC
50≈ 100–120 μm. Mibefradil was also found to significantly speed up the inactivation kinetics of slower channels (CaV1.2, CEEE) with little effect on the inactivation kinetics of faster-inactivating channels (CaV2.3). A open-channel block model for mibefradil interaction with high-voltage-activated Ca2+ channels is discussed and shown to qualitatively account for our observations. Hence, our data agree reasonably well with
a ``receptor guarded mechanism' where fast inactivation kinetics efficiently trap mibefradil into the channel.
Received: 14 March 2001/Revised: 25 June 2001 相似文献