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1.
CFTR, A Regulator of Channels 总被引:1,自引:0,他引:1
2.
Blockers of CFTR with well-characterized kinetics and mechanism of action will be useful as probes of pore structure. We
have studied the mechanism of block of CFTR by the arylaminobenzoates NPPB and DPC. Block of macroscopic currents by NPPB
and DPC exhibited similar voltage-dependence, suggestive of an overlapping binding region. Kinetic analysis of single-channel
currents in the presence of NPPB indicate drug-induced closed time constants averaging 2.2 msec at −100 mV. The affinity for
NPPB calculated from single-channel block, K
D
= 35 μm, exceeds that for other arylaminobenzoates studied thus far. These drugs do not affect the rate of activation of wild-type
(WT) channels expressed in oocytes, consistent with a simple mechanism of block by pore occlusion, and appear to have a single
binding site in the pore. Block by NPPB and DPC were affected by pore-domain mutations in different ways. In contrast to its
effects on block by DPC, mutation T1134F-CFTR decreased the affinity and reduced the voltage-dependence for block by NPPB.
We also show that the alteration of macroscopic block by NPPB and DPC upon changes in bath pH is due to both direct effects
(i.e., alteration of voltage-dependence) and indirect effects (alteration of cytoplasmic drug loading). These results indicate
that both NPPB and DPC block CFTR by entering the pore from the cytoplasmic side and that the structural requirements for
binding are not the same, although the binding regions within the pore are similar. The two drugs may be useful as probes
for overlapping regions in the pore.
Received: 14 October 1999/Revised: 18 January 2000 相似文献
3.
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
4.
CFTR Activation Raises Extracellular pH of NIH/3T3 Mouse Fibroblasts and C127 Epithelial Cells 总被引:2,自引:0,他引:2
Cystic Fibrosis (CF) is caused by mutations in the gene for CFTR, a cAMP-activated anion channel found in apical membranes
of wet epithelia. Since CFTR is permeable to HCO−
3 and changes in extracellular fluid composition may contribute to CF lung disease, we investigated possible differences in
extracellular pH (pHo) between CFTR-expressing and control cell lines. The Cytosensor™ Microphysiometer was used to study
forskolin-stimulated extracellular acidification rates in CFTR-expressing and control mouse mammary epithelial (C127) and
fibroblast (NIH/3T3) cell lines. Forskolin, which activates CFTR via raised cAMP, caused decreased extracellular acidification of CFTR-expressing NIH/3T3 and C127 cells by 15–35%. By contrast, forskolin caused increased extracellular acidification of control cells by 10–20%. Ionomycin, which may activate CFTR via PKC, also elicited this decreased
extracellular acidification signal only in cells expressing CFTR. In control experiments, dideoxyforskolin had no effect on
the acidification rates and osmotic stimuli were shown to equally stimulate all cell lines. These results suggest a role for
CFTR in controlling pHo and complement recent evidence that HCO−
3 dependent epithelial secretion may be reduced in amount and altered in composition in CF.
Received: 20 June 2000/Revised: 13 November 2000 相似文献
5.
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated, ATP-dependent chloride channel which
may have additional functions. Recent reports that CFTR mediates substantial electrodiffusion of ATP from epithelial cells
have led to the proposal that CFTR regulates other ion channels through an autocrine mechanism involving ATP. The aim of this
study was to determine the ATP conductance of wild-type CFTR channels stably expressed in Chinese hamster ovary cells using
patch clamp techniques. In the cell-attached configuration with 100 mm Mg · ATP or Tris · ATP solution in the pipette and 140 mm NaCl in the bath, exposing cells to forskolin caused the activation of a low-conductance channel having kinetics resembling
those of CFTR. Single channel currents were negative at the resting membrane potential (V
m
), consistent with net diffusion of Cl from the cell into the pipette. The transitions decreased in amplitude, but did not
reverse direction, as V
m
was clamped at increasingly positive potentials to enhance the driving force for inward ATP flow (>+80 mV). In excised patches,
single channel currents did not reverse under essentially biionic conditions (Clin/ATPout or ATPin/Clout), although PKA-activated currents were clearly visible in the same patches at voltages where they would be carried by chloride
ions. Moreover, with NaCl solution in the bath and a mixture of ATP and Cl in the pipette, the single channel I/V curve reversed at the predicted equilibrium potential for chloride. CFTR channel currents disappeared when patches were exposed
to symmetrical ATP solutions and were restored by reexposure to Cl solution. Finally, in the whole-cell configuration with
NaCl in the bath and 100 mm MgATP or TrisATP in the pipette, cAMP-stimulated cells had time-independent, outwardly rectifying currents consistent with
CFTR selectivity for external Cl over internal ATP. Whole-cell currents reversed near V
m
=−55 mV under these conditions, however the whole cell resistance measured at −100 mV was comparable to that of the gigaohm
seal between the plasma membrane and glass pipette (7 GΩ). We conclude that CFTR does not mediate detectable electrodiffusion
of ATP.
Received: 8 November 1995/Revised: 23 January 1996 相似文献
6.
In cystic fibrosis airway epithelia, mutation of the CFTR protein causes a reduced response of Cl− secretion to secretagogues acting via cAMP. Using a Ca2+ imaging system, the hypothesis that CFTR activation may permit ATP release and regulate [Ca2+]
i
via a receptor-mediated mechanism, is tested in this study. Application of external nucleotides produced a significant increase
in [Ca2+]
i
in normal (16HBE14o− cell line and primary lung culture) and in cystic fibrosis (CFTE29o− cell line) human airway epithelia. The potency order of nucleotides on [Ca2+]
i
variation was UTP ≫ ATP > UDP > ADP > AMP > adenosine in both cell types. The nucleotide [Ca2+]
i
response could be mimicked by activation of CFTR with forskolin (20 μm) in a temperature-dependent manner. In 16HBE14o− cells, the forskolin-induced [Ca2+]
i
response increased with increasing temperature. In CFTE29o− cells, forskolin had no effect on [Ca2+]
i
at body temperature-forskolin-induced [Ca2+]
i
response in CF cells could only be observed at low experimental temperature (14°C) or when cells were cultured at 26°C instead
of 37°C. Pretreatment with CFTR channel blockers glibenclamide (100 μm) and DPC (100 μm), with hexokinase (0.5 U/mg), and with the purinoceptor antagonist suramin (100 μm), inhibited the forskolin [Ca2+]
i
response. Together, these results demonstrate that once activated, CFTR regulates [Ca2+]
i
by mediating nucleotide release and activating cell surface purinoceptors in normal and CF human airway epithelia.
Received: 3 April 2000/Revised: 30 June 2000 相似文献
7.
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
8.
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
9.
Twin-electrode voltage-clamp techniques were used to study the effect of calcium and calcium channel blockers on the transient
outward current in isolated F76 and D1 neurones of Helix aspersa subesophageal ganglia in vitro (soma only preparation with no cell processes). On lowering extracellular Ca2+ concentration from 10 to 2 mm or removing extracellular calcium from the bathing medium, the threshold for this current shifted in a negative direction
by 11.5 and 20 mV, respectively. On the other hand, increasing the extracellular Ca2+ concentration from 10 to 20 and to 40 mm shifted the steady-state inactivation curves in positive directions on the voltage axis by 7 and 15 mV, respectively. Upon
application of calcium channel blockers, Co2+, La3+, Ni2+ and Cd2+, transient potassium current amplitude was reduced in a voltage-dependent manner, being more effective at voltages close
to the threshold. The current was elicited even at a holding potential of −34 mV. The specific calcium channel blockers, amiloride
and nifedipine did not shift the activation and steady-state inactivation curves and did not reduce the transient outward
current amplitude. It was concluded that the transient outward current is not dependent on intracellular Ca2+ but that it is modulated by Ca2+ and di- and trivalent ions extracellularly. The effects of these ions are very unlikely to be due to a surface charge effect
because the addition of La3+ (200 μm) completely reverses the shift in a hyperpolarizing direction when the extracellular Ca2+ concentration was reduced from 10 to 1 mm and additionally shifts the kinetics further still in a depolarizing direction. The responses seen here are consistent with
a specific effect of di- and trivalent ions on the transient outward current channels leading to a modification of gating.
Received: 30 March 1999/Revised: 5 October 1999 相似文献
10.
Gating Kinetics of E. coli Poly-3-Hydroxybutyrate/Polyphosphate Channels in Planar Bilayer Membranes
Nonproteinaceous calcium channel complexes from Escherichia coli, composed of poly-(R)-3-hydroxybutyrate (PHB) and inorganic polyphosphate (polyP), exhibit two distinct gating modes (modes
1 and 2) in planar lipid bilayers. Here we report the kinetic characterization of the channel in mode 2, a mode characterized
by two well-defined conductance levels, a fully open state (87 ± 3 pS), and a major subconductance state (56 ± 2 pS). Other
subconductance states and full closures are rare (<0.5% of total time). Several kinetic properties of the channel showed asymmetric
voltage-dependence indicating an asymmetry in the channel structure. Accordingly, single channels responded to potential change
in one of two mirror-image patterns, postulated to arise from opposite orientations of the asymmetrical channel complex in
the bilayer. The fraction of time spent in each conductance level was strongly voltage-sensitive. For channels reported in
this study, presumably all oriented in the same direction, residence time in the fully open state increased as clamping potentials
became more positive whereas residence time in the major subconductance state increased at more negative potentials. Analysis
of open time distributions revealed existence of two kinetically distinct states for each level. The shorter time constants
for both conductance states exhibited weak voltage-sensitivity; however, the longer time constants were strongly voltage-sensitive.
A kinetic scheme, consistent with the complex voltage dependence of the channel, is proposed.
Received: 1 February 1999/Revised: 2 April 1999 相似文献
11.
In attached patches on the plasma membrane of nonexcited Chara corallina cells, randomly activating, transient Cl− currents with variable amplitudes were recorded. The peak amplitudes of these currents could be grouped into distinct populations
with approximately equidistant mean peak currents. Generally, the mean current of the smallest population measured about half
of the distance between the means of subsequent populations. Currents of the smallest population occurred most frequently
at all voltages; the frequency of observations decreased with increasing amplitudes of the currents. At all voltages transient
currents from different populations were similar in duration with the exception of the smallest currents, which lasted only
0.6 times as long as larger currents. Furthermore, transient currents were most frequent at positive voltages, but once initiated
at a positive conditioning pulse they were also observed during subsequent pulses to negative voltages. The results are consistent
with the idea that Chara contains Ca2+ stores in the vicinity of the plasma membrane, which are indirectly filled from the external medium. Upon quantal Ca2+ discharge from adjacent stores, a process independent of membrane voltage, the concentration of Ca2+ in the cytoplasm increases transiently. Depending on the number of discharging stores, distinct numbers of Ca2+-stimulated Cl− channels activate, giving rise to the macroscopic excitatory Cl− current in these cells.
Received: 27 October 1997/Revised: 26 February 1998 相似文献
12.
The objective of the present study was to clarify the mechanism by which the sulfonylurea drug, glibenclamide, inhibits single CFTR channels in excised patches from Xenopus oocytes. Glibenclamide blocks the open pore of the channel via binding at multiple sites with varying kinetics. In the absence of glibenclamide, open-channel bursts exhibited a flickery intraburst closed state (C1); this is due to block of the pore by the pH buffer, TES. Application of 25 microM glibenclamide to the cytoplasmic solution resulted in the appearance of two drug-induced intraburst closed states (C2, C3) of widely different duration, which differed in pH-dependence. The kinetics of interaction with the C3 state, but not the C2 state, were strongly voltage-dependent. The durations of both the C2 and C3 states were concentration-dependent, indicating a non-linear reaction scheme. Application of drug also increased the burst duration, which is consistent with an open-channel blocking mechanism. A kinetic model is proposed. These results indicate that glibenclamide interacts with open CFTR channels in a complex manner, involving interactions with multiple binding sites in the channel pore. 相似文献
13.
Certain COOH-terminus mutants of connexin32 (Cx32) were previously shown to form channels with unusual transjuctional voltage
(V
j
) sensitivity when tested heterotypically in oocytes against Cx32 wild type. Junctional conductance (G
j
) slowly increased by severalfold or decreases to nearly zero with V
j
positive or negative, respectively, at mutant side, and V
j
positive at mutant side reversed CO2-induced uncoupling. This suggested that the CO2-sensitive gate might be a V
j
-sensitive slow gate. Based on previous data for calmodulin (CaM) involvement in gap junction function, we have hypothesized
that the slow gate could be a CaM-like pore plugging molecule (cork gating model). This study describes a similar behavior
in heterotypic channels between Cx32 and each of four new Cx32 mutants modified in cytoplasmic-loop and/or COOH-terminus residues.
The mutants are: ML/NN+3R/N, 3R/N, ML/NN and ML/EE; in these mutants, N or E replace M105 and L106, and N replace R215, R219
and R220. This study also reports that inhibition of CaM expression strongly reduces V
j
and CO2 sensitivities of two of the most effective mutants, suggesting a CaM role in slow and chemical gating.
Received: 19 April 2000/Revised: 11 August 2000 相似文献
14.
To determine whether the persistent nature of hepatitis C infection is related to the emergence of antigenic variants driven
by immune selection, we examined the sequence heterogeneity in a portion of the hepatitis C virus (HCV) nonstructural 3 (NS3)
gene of a patient infected over the course of more than 2 years. By PCR amplification, cloning, and sequencing, we observed
several variable and conserved regions in the NS3 segment of the HCV genome. All variable regions had higher ratios of nonsynonymous/synonymous
mutations and encompassed immunodominant epitopes, and their locations were not essential to maintain the known function of
HCV RNA helicase. In contrast, the regions that are critical for HCV RNA helicase activity were found to be conserved with
lower heterogeneity or lower ratios of nonsynonymous/synonymous mutations, and none except one of these regions was encoded
within immunodominant epitopes. Our results are consistent with immune selection of viral variants at the epitope and molecular
levels that may enable HCV to evade host defenses over time. Plotting the relatedness of sequence variants revealed a star
topology suggesting that a wild-type HCV sequence is maintained, unlike HIV.
Received: 2 November 2000 / Accepted: 1 October 2001 相似文献
15.
A number of peptide toxins derived from marine snails and various spiders have been shown to potently inhibit voltage-dependent
calcium channels. Here, we describe the effect of calcicludine, a 60 amino-acid peptide isolated from the venom of the green
mamba (Dendroaspis angusticeps), on transiently expressed high voltage-activated calcium channels. Upon application of calcicludine, L-type (α1
C
) calcium channels underwent a rapid, irreversible decrease in peak current amplitude with no change in current kinetics,
or any apparent voltage-dependence. However, even at saturating toxin concentrations, block was always incomplete with a maximum
inhibition of 58%, indicating either partial pore block, or an effect on channel gating. Block nonetheless was of high affinity
with an IC50 value of 88 nm. Three other types of high voltage activated channels tested (α1
A
, α1
B
, and α1
E
) exhibited a diametrically different response to calcicludine. First, the maximal inhibition observed was around 10%, furthermore,
the voltage-dependence of channel activation was shifted slightly towards more negative potentials. Thus, at relatively hyperpolarized
test potentials, calcicludine actually upregulated current activity of (N-type) α1
B
channels by as much as 50%. Finally, the use of several chimeric channels combining the major transmembrane domains of α1
C
and α1
E
revealed that calcicludine block of L-type calcium channels involves interactions with multiple structural domains. Overall,
calcicludine is a potent and selective inhibitor of neuronal L-type channels with a unique mode of action.
Received: 22 September 1999/Revised: 1 December 1999 相似文献
16.
17.
A plant hyperpolarization-activating K+ channel, KAT1, is highly selective for K+ over Na+ and is little affected by external Na+, which is crucial to take up K+ effectively in a Na+-containing environment. It has been shown that a mutation at the location (Thr256) preceding the selectivity signature sequence
dramatically enhanced the sensitivity of the KAT1 channel to external Na+. We report here electrophysiological experiments for the mechanism of action of external Na+ on KAT1 channels. The Thr256 residue was substituted with either glutamine (Q) or glutamate (E). The wild-type channel was
insensitive to external Na+. However, the activity of both mutant channels was significantly depressed by Na+ with apparent dissociation constants of 6.7 mm and 11.3 mm for T256Q and T256E, respectively. The instantaneous current-voltage relationships revealed distinct blocking mechanisms
for these mutants. For T256Q a typical voltage-dependent fast blocking was shown. On the other hand, the blocking for the
T256E mutant was voltage-independent at low Na+ concentrations and became voltage-dependent at higher concentrations. At extreme hyperpolarization the blocking was relieved
significantly. These data strongly suggest that the mutation at the end of the pore helix rearranged the selectivity filter
and allows Na+ to penetrate into the pore.
Received: 16 October 2000/Revised: 20 February 2001 相似文献
18.
Hyperpolarization-activated K channels (K
H
channels) in the plasmalemma of guard cells operate at apoplastic pH range of 5 to over 7. Using patch clamp in a whole-cell
mode, we characterized the effect of varying the external pH between 4.4–8.1 on the activity of the K
H
channels in isolated guard cell protoplasts from Vicia faba leaves.
Acidification from pH 5.5 to 4.4 increased the macroscopic conductance of the K
H
channels by 30–150% while alkalinization from pH 5.5 to 8.1 decreased it only by roughly 15%. The voltage-independent maximum cell conductance, increased by ∼60% between pH 8.1 and 4.4 with an apparent pK
a
of 5.3, most likely owing to the increased availability of channels. Voltage-dependent gating was affected only between pH 5.5 and 4.4. Acidification in this range shifted the voltage-dependent open probability by over 10 mV. We interpret this shift as an increase of the electrical field sensed by the gating subunits
caused by the protonation of external negative surface charges. Within the framework of a surface charge model the mean spacing
of these charges was ∼30 ? and their apparent dissociation constant was 10−4.6. The overall voltage sensitivity of gating was not altered by pH changes. In a subgroup of protoplasts analyzed within the
framework of a Closed-Closed-Open model, the effect of protons on gating was limited to shifting of the voltage-dependence
of all four transition rate constants.
Received: 26 April 1996/Revised: 29 June 1996 相似文献
19.
A mathematical model of the HCO−
3-secreting pancreatic ductal epithelium was developed using network thermodynamics. With a minimal set of assumptions, the
model accurately reproduced the experimentally measured membrane potentials, voltage divider ratio, transepithelial resistance
and short-circuit current of nonstimulated ducts that were microperfused and bathed with a CO2/HCO−
3-free, HEPES-buffered solution, and also the intracellular pH of duct cells bathed in a CO2/HCO−
3-buffered solution. The model also accurately simulated: (i) the effect of step changes in basolateral K+ concentration, and the effect of K+ channel blockers on basolateral membrane potential; (ii) the intracellular acidification caused by a Na+-free extracellular solution and the effect of amiloride on this acidification; and (iii) the intracellular alkalinization
caused by a Cl−-free extracellular solution and the effect of DIDS on this alkalinization. In addition, the model predicted that the luminal
Cl− conductance plays a key role in controlling both the HCO−
3 secretory rate and intracellular pH during HCO−
3 secretion. We believe that the model will be helpful in the analysis of experimental data and improve our understanding of
HCO−
3-transporting mechanisms in pancreatic duct cells.
Received: 18 October 1995/Revised: 5 July 1996 相似文献
20.
We have characterized the conduction and blocking properties of a chloride channel from rough endoplasmic reticulum membranes
of rat hepatocytes after incorporation into a planar lipid bilayer. Our experiments revealed the existence of a channel with
a mean conductance of 164 ± 5 pS in symmetrical 200 mm KCl solutions. We determined that the channel was ten times more permeable for Cl− than for K+, calculated from the reversal potential using the Goldman-Hodgkin-Katz equation. The channel was voltage dependent, with
an open probability value ranging from 0.9 at −20 mV to 0.4 at +60 mV. In addition to its fully open state, the channel could
also enter a flickering state, which appeared to involve rapid transitions to zero current level. Our results showed a decrease
of the channel mean open time combined with an increase of the channel mean closed time at positive potentials. An analysis
of the dwell time distributions for the open and closed intervals led to the conclusion that the observed fluctuation pattern
was compatible with a kinetic scheme containing a single open state and a minimum of three closed states. The permeability
sequence for test halides determined from reversal potentials was Br− > Cl− > I−≈ F−. The voltage dependence of the open probability was modified by the presence of halides in trans with a sequence reflecting the permeability sequence, suggesting that permeant anions such as Br− and Cl− have access to an internal site capable of controlling channel gating. Adding NPPB to the cis chamber inhibited the channel activity by increasing fast flickering and generating long silent periods, whereas channel
activity was not affected by 50 μm DNDS in trans. The channel was reversibly inhibited by adding phosphate to the trans chamber. The inhibitory effect of phosphate was voltage-dependent and could be reversed by addition of Cl−. Our results suggest that channel block involves the interaction of HPO2−
4 with a site located at 70% of the membrane span.
Received: 10 January 1997/Revised: 29 May 1997 相似文献