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1.
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
2.
Despite biochemical evidence for the existence of high-affinity phenylalkylamine receptors in higher plants, their effects
on channel activity have only been demonstrated at relatively high concentrations. We have performed a quantitative single-channel
analysis of the changes induced by extracellular verapamil in the rca channel [a wheat root plasma membrane Ca2+-selective channel (Pi?eros & Tester, 1995. Planta
195:478–488)]. Concentrations as low as 0.5 μm verapamil induced a blockade of the inward current, with no evident reduction of the single-channel current amplitude. Blockade
by verapamil was concentration and voltage dependent. Preliminary analysis suggested the blockade was due to a reduction in
the maximum open state probability rather than a change in V0.5. Further analysis of the association and dissociation rate constants revealed a binding site located 56 to 59% down the voltage
drop from the extracellular face of the channel, with a K
d
(0) of 24 to 26 μm. This results in a K
d
at −100 mV of 2 μm. Methoxyverapamil had qualitatively the same effects. This intra-pore binding site can be accessed directly from the extracellular
side of the rca channel, but apparently not from the cytosolic side.
Received: 15 August 1996/Revised: 23 December 1996 相似文献
3.
Plasma membrane anion channels are thought to play important roles in osmoregulation and signal transduction in higher plant
cells. Knowledge of their pharmacology and regulation is of importance to unravel their physiological functions. In this study,
we explore the pharmacological properties and the nucleotide regulation of the voltage-dependent anion channel of Arabidopsis hypocotyls. The pharmacological profile of this channel is characterized by a low sensitivity to most anion channel blockers.
It is inhibited by niflumic acid with an IC50 of 80 μm, but poorly sensitive to IAA-94 and NPPB and insensitive to 9-AC and DIDS. Nucleotides alter the amplitude, the kinetics
and the voltage-dependence of the channel. The main effect of nucleotides is a shift of the voltage-dependent gate of the
channel toward depolarized potentials leading to a strong reduction of the current amplitude. This regulation does not require
ATP hydrolysis as nonhydrolyzable ATP analogues—AMPPNP and ATPγS—also regulate the anion current. This suggests that a nucleotide
binding site is involved in the regulation. The study of the properties of this putative nucleotide binding site reveals that
(i) ATP regulates the channel with an EC50 of 0.7 mm, (ii) adenyl nucleotides modulate the channel with the following order of effectiveness: ATP > ADP ≫ AMP, and (iii) thiophosphate
nucleotide analogues are the most potent agonists with EC50 in the range of 80 μm. The hypothesis that this regulation may couple the electrical properties of the membrane with the metabolic status of the
cell is discussed.
Received: 26 December 1996/Revised: 21 March 1997 相似文献
4.
Harper AA Catacuzzeno L Trequattrini C Petris A Franciolini F 《The Journal of membrane biology》2001,179(2):103-111
The effects of verapamil on the large conductance Ca-activated K (BK) channel from rat aortic smooth muscle cells were examined
at the single channel level. Micromolar concentrations of verapamil produced a reversible flickering block of the BK channel
activity. Kinetic analysis showed that verapamil decreased markedly the time constants of the open states, without any significant
change in the time constants of the closed states. The appearance of an additional closed state — specifically, a nonconducting,
open-blocked state — was also observed, whose time constant would reflect the mean residence time of verapamil on the channel.
These observations are indicative of a state-dependent, open-channel block mechanism. Dedicated kinetic (group) analysis confirmed
the state-dependent block exerted by verapamil. D600 (gallopamil), the methoxy derivative of verapamil, was also tested and
found to exert a similar type of block, but with a higher affinity than verapamil. The permanently charged and membrane impermeant
verapamil analogue D890 was used to address other important features of verapamil block, such as the sidedness of action and
the location of the binding site on the channel protein. D890 induced a flickering block of BK channels similar to that observed
with verapamil only when applied to the internal side of the membrane, indicating that D890 binds to a site accessible from
the cytoplasmic side. Finally, the voltage dependence of D890 block was assessed. The experimental data fitted with a Langmuir
equation incorporating the Woodhull model for charged blockers confirms that the D890-binding site is accessed from the internal
mouth of the BK channel, and locates it approximately 40% of the membrane voltage drop along the permeation pathway.
Received: 11 April 2000/Revised: 17 October 2000 相似文献
5.
Current-voltage relationships of a cation channel in the tonoplast of Beta vulgaris, as recorded in solutions with different activities of Ca2+ and K+ (from Johannes & Sanders 1995, J. Membrane Biol. 146:211–224), have been reevaluated for Ca2+/K+ selectivity. Since conversion of reversal voltages to permeability ratios by constant field equations is expected to fail
because different ions do not move independently through a channel, the data have been analyzed with kinetic channel models
instead. Since recent structural information on K+ channels show one short and predominant constriction, selectivity models with only one binding site are assumed here to reflect
this region kinetically. The rigid-pore model with a main binding site between two energy barriers (nine free parameters)
had intrinsic problems to describe the observed current-saturation at large (negative) voltages. The alternative, dynamic-pore
model uses a selectivity filter in which the binding site alternates its orientation (empty, or occupied by either Ca2+ or K+) between the cytoplasmic side and the luminal side within a fraction of the electrical distance and in a rate-limiting fashion.
Fits with this model describe the data well. The fits yield about a 10% electrical distance of the selectivity filter, located
about 5% more cytoplasmic than the electrical center. For K+ translocation, reorientation of the unoccupied binding site (with a preference of about 6:5 to face the lumenal side) is
rate limiting. For Ca2+, the results show high affinity to the binding site and low translocation rates (<1% of the K+ translocation rate). With the fitted model Ca2+ entry through the open channel has been calculated for physiological conditions. The model predicts a unitary open channel
current of about 100 fA which is insensitive to cytoplasmic Ca2+ concentrations (between 0.1 and 1 μm) and which shows little sensitivity to the voltage across the tonoplast.
Received: 19 February 1997/Revised: 19 May 1997 相似文献
6.
Mechanosensitive (MS) ion channels, with 560 pS conductance, opened transiently by rapid application of suction pulses to
patches of E. coli protoplast membrane. The adaptation phase of the response was voltage-independent. Application of strong suction pulses,
which were sufficient to cause saturation of the MS current, did not abolish the adaptation. Multiple-pulse experimental protocols
revealed that once MS channels had fully adapted, they could be reactivated by a second suction pulse of similar amplitude,
providing the time between pulses was long enough and suction had been released between pulses. Limited proteolysis (0.2 mg/ml
pronase applied to the cytoplasmic side of the membrane patch) reduced the number of open channels without affecting the adaptation.
Exposing patches to higher levels of pronase (1 mg/ml) removed responsiveness of the channel to suction and abolished adaptation
consistent with disruption of the tension transmission mechanism responsible for activating the MS channel. Based on these
data we discuss a mechanism for mechanosensitivity mediated by a cytoplasmic domain of the MS channel molecule or associated
protein.
Received: 29 January 1998/Revised: 16 April 1998 相似文献
7.
The rate-limiting step for the maternofetal exchange of low molecular-weight solutes in humans is constituted by transport
across a single epithelial layer (syncytiotrophoblast) of the placenta. Other than the well-established presence of a large-conductance,
multisubstate Cl− channel, the ionic channels occurring in this syncytial tissue are, for the most part, unknown. We have found that fusion
of apical plasma membrane-enriched vesicle fractions with planar lipid bilayers leads, mainly (96% of 353 reconstitutions),
to the reconstitution of nonselective cation channels. Here we describe the properties of this novel placental conductance
at the single-channel level. The channel has a large (>200 pS) and variable conductance, is cation selective (P
Cl
/P
K
≅ 0.024), is reversibly inhibited (presumably blocked) by submillimolar La3+, has very unstable kinetics, and displays a large number (>10) of current sublevels with a ``promiscuous' connectivity pattern.
The occurrence of both ``staircaselike' and ``all-or-nothing' transitions between the minimum and maximum current levels
was intriguing, particularly considering the large number of conductance levels spanned at a time during the concerted current
steps. Single-channel data simulated according to a multistate linear reaction scheme, with rate constants that can vary spontaneously
in time, reproduce many aspects of the recorded subconductance behavior. The channel's sensitivity to lanthanides is reminiscent
of stretch-sensitive channels which, in turn, suggests a physiological role for this ion channel as a mechanotransducer during
syncytiotrophoblast-volume regulation.
Received: 30 August 1999/Revised: 12 November 1999 相似文献
8.
N.B. Brovtsyna D.B. Tikhonov O.B. Gorbunova V.E. Gmiro S.E. Serduk N.Y. Lukomskaya L.G. Magazanik B.S. Zhorov 《The Journal of membrane biology》1996,152(1):77-87
Structure-activity relationships of 56 pentamethylenbis-ammonium compounds, the blockers of the neuronal nicotinic acetylcholine
receptor (nAChR) ion channel, have been studied to estimate the cross-sectional dimensions of the channel pore. The cat superior
cervical sympathetic ganglion in situ and isolated guinea pig ileum were used to evaluate the potency of the compounds to block ganglionic transmission. Minimum-energy
conformations of each compound were calculated by the molecular mechanics method. A topographic model of the binding site
of the blockers was proposed. It incorporates two narrowings, a large and a small one. The small narrowing is located between
the large one and the cytoplasmic end of the pore. The cross-sectional dimensions of the large and small narrowings estimated
from the dimensions of the blockers are 6.1 × 8.3 ? and 5.5 × 6.4 ?, respectively, the distance between the narrowings along
the pore being approximately 7 ?. Most potent blockers would occlude the pore via binding to the channel at the levels of
both narrowings. Less potent blockers are either too large or too small to bind to both narrowings simultaneously: large blockers
would occlude the pore at the level of large narrowing, while small blockers would pass the large narrowing and occlude the
pore at the level of small narrowing only. A comparison of the topographic model with a molecular five-helix bundle model
of nAChR pore predicts Serine and Threonine rings to be the most probable candidates for the large and small narrowings, respectively.
Received: 6 September 1995/Revised: 12 March 1996 相似文献
9.
The four-state simple carrier model (SCM) is employed to describe ligand translocation by diverse passive membrane transporters.
However, its application to systems like facilitative sugar transporters (GLUTs) is controversial: unidirectional fluxes under
zero-trans and equilibrium-exchange experimental conditions fit a SCM, but flux data from infinite-cis and infinite-trans
experiments appear not to fit the same SCM. More complex kinetic models have been proposed to explain this ``anomalous' behavior
of GLUTs, but none of them accounts for all the experimental findings. We propose an alternative model in which GLUTs are
channels subject to conformational transitions, and further assume that the results from zero-trans and equilibrium-exchange
experiments as well as trans-effects corresponds to a single-occupancy channel regime, whereas the results from the infinite-cis
and infinite-trans experiments correspond to a regime including higher channel occupancies. We test the plausibility of this
hypothesis by studying a kinetic model of a two-site channel with two conformational states. In each state, the channel can
bind the ligand from only one of the compartments. Under single-occupancy, for conditions corresponding to zero-trans and
equilibrium-exchange experiments, the model behaves as a SCM capable of exhibiting trans-stimulations. For a regime including
higher degrees of occupancy and infinite-cis and infinite-trans conditions, the same channel model can exhibit a behavior
qualitatively similar to a SCM, albeit with kinetic parameters different from those for the single-occupancy regime. Numerical
results obtained with our model are consistent with available experimental data on facilitative glucose transport across erythrocyte
membranes. Hence, if GLUTs are multiconformational channels, their particular kinetic properties can result from transitions
between single and double channel occupancies.
Received: 12 April 1995/Revised: 28 August 1995 相似文献
10.
Exact algorithms for the kinetic analysis of multichannel patch-clamp records require hours to days for a single record.
Thus, it may be reasonable to use a fast but less accurate method for the analysis of all data sets and to use the results
for a reanalysis of some selected records with more sophisticated approaches. For the first run, the tools of single-channel
analysis were used for the evaluation of the single-channel rate constants from multichannel dwell-time histograms. This could
be achieved by presenting an ensemble of single channels by a ``macrochannel' comprising all possible states of the ensemble
of channels. Equations for the calculations of the elements of the macrochannel transition matrix and for the steady-state
concentrations for individual states are given. Simulations of multichannel records with 1 to 8 channels with two closed and
one open states and with 2 channels with two open and two closed states were done in order to investigate under which conditions
the one-dimensional dwell-time analysis itself already provides reliable results. Distributions of the evaluated single-channel
rate constants show that a bias of the estimations of the single-channel rate constants of 10 to 20% has to be accepted. The
comparison of simulations with signal-to-noise ratios of SNR = 1 or SNR = 25 demonstrates that the major problem is not the
convergence of the fitting routine, but failures of the level detector algorithm which creates the dwell-times distributions
from noisy time series.
The macrochannel presentation allows the incorporation of constraints like channel interaction. The evaluation of simulated
4-channel records in which the rate-constant of opening increased by 20% per already open channel could reveal the interaction
factor.
Received: 9 June 1997/Revised: 28 April 1998 相似文献
11.
P.J. White 《The Journal of membrane biology》1998,164(3):275-281
The maxi cation channel from the plasma membrane of rye (Secale cereale L.) roots was studied following its incorporation into planar phosphatidylethanolamine bilayers. Current recordings were
made in the presence of 100-mm KCl containing quinine on both sides of the bilayer. Quinine produced voltage- and concentration-dependent blockade of the
channel, reducing its apparent unitary current and open probability. The voltage-dependence suggested that blockade was effected
from the cytoplasmic side by cationic quinine. Blockade was modelled using a kinetic scheme with two independent blocked states
termed B1 and B2 (B1⇆O⇄B2). Rate constants promoting fast kinetics (k
1
and k
−1
) were found to be several orders of magnitude greater than those promoting slow kinetics (k
2
and k
−2
). Analysis of the fast kinetics indicated that the rate constants for blockade of the open channel at the first site (k
1
) and its clearance (k
−1
) had voltage-dependencies (zδ
p
) of 0.41 and −0.71, respectively, and that the equilibrium dissociation constant for the binding site (K
d
(0)) was about 1 mm. Analysis of the slow kinetics indicated that the rate constants for blockade of the open channel at the second site (k
2
) and its clearance (k
−2
) had zδ
p
values of 0.12 and −1.27, respectively. The K
d
(0) value for the second binding site was about 10 mm.
Received: 20 January 1998/Revised: 1 May 1998 相似文献
12.
Plant growth requires a continuous supply of intracellular solutes in order to drive cell elongation. Ion fluxes through
the plasma membrane provide a substantial portion of the required solutes. Here, patch clamp techniques have been used to
investigate the electrical properties of the plasma membrane in protoplasts from the rapid growing tip of maize coleoptiles.
Inward currents have been measured in the whole cell configuration from protoplasts of the outer epidermis and from the cortex.
These currents are essentially mediated by K+ channels with a unitary conductance of about 12 pS. The activity of these channels was stimulated by negative membrane voltage
and inhibited by extracellular Ca2+ and/or tetraethylammonium-CI (TEA). The kinetics of voltage- and Ca2+-gating of these channels have been determined experimentally in some detail (steady-state and relaxation kinetics). Various
models have been tested for their ability to describe these experimental data in straightforward terms of mass action. As
a first approach, the most appropriate model turned out to consist of an active state which can equilibrate with two inactive
states via independent first order reactions: a fast inactivation/activation by Ca2+-binding and -release, respectively (rate constants >>103 sec−1) and a slower inactivation/activation by positive/negative voltage, respectively (voltage-dependent rate constants in the
range of 103 sec−1).
With 10 mm K+ and 1 mm Ca2+ in the external solution, intact coleoptile cells have a membrane voltage (V) of −105 ± 7 mV. At this V, the density and open probability of the inward-rectifying channels is sufficient to mediate K+ uptake required for cell elongation. Extracellular TEA or Ca2+, which inhibit the K+ inward conductance, also inhibit elongation of auxin-depleted coleoptile segments in acidic solution. The comparable effects
of Ca2+ and TEA on both processes and the similar Ca2+ concentration required for half maximal inhibition of growth (4.3 mm Ca2+) and for conductance (1.2 mm Ca2+) suggest that K+ uptake through the inward rectifier provides essential amounts of solute for osmotic driven elongation of maize coleoptiles.
Received: 6 June 1995/Revised: 12 September 1995 相似文献
13.
The present study deals with photomodification of the electrical properties of the plasma membrane of an epithelial cell
line (opossum kidney (OK) cells). The effect of photofrin II (previously investigated) is compared with that of 5 other membrane-active
sensitizers: sulfonated Zn-phthalocyanine, merocyanine 540, rose bengal, methylene blue and protoporphyrin IX (an endogenous
sensitizer induced by addition of its biosynthetic precursor 5-aminolaevulinic acid). The study was performed in order to
investigate whether photomodification of the ion transport properties of the plasma membrane by membrane-active sensitizers
is a general and early event in cellular photosensitization. The changes in the electrical properties were monitored by application
of the whole-cell and the inside-out configuration of the patch-clamp technique.
Illumination in the presence of the compounds (apart from merocyanine 540) gave rise to similar changes of the electrical
properties of the membrane: depolarization of the membrane potential, inactivation of a large-conductance, Ca2+-dependent K+-channel (maxi-KCa), and a strong increase of the leak conductance of the membrane. This similarity indicates the general character of the functional
photomodifications by membrane-active sensitizers previously reported for photofrin II.
Received: 5 September 2000/Revised: 28 December 2000 相似文献
14.
The effects of the calmodulin antagonists W-7 and trifluoperazine have been measured on the Ca2+-activated potassium channel in the membrane surrounding protoplasmic drops expressed from internodal cells of charophyte
plants. The large-conductance (170 pS), voltage- and Ca2+-dependent gating, and prominent conductance substrate of this channel shows a strong kinetic resemblance to those of the
Maxi-K channel from animal cells.
This is the first study of the action of calmodulin antagonists which measures their effects on the most populated substates
as well as the closed and main open states of Maxi-K channels. The substate analysis provides new evidence for different modes
of action of- and different bindings sites for these calmodulin antagonists.
Neither antagonist produces the simple closure of the channel reported previously as its effect on the Maxi-K channel, though
both do induce flicker-block, reducing the mean current to near zero at high concentrations following an inverted Michaelis-Menten
curve.
W-7 reduces residence time in the fully open state, thus raising, in the same proportions, the probabilities of finding the
channel in the closed state or a pre-existing substate. Its binding to the channel is voltage- and calcium-dependent.
In contrast, trifluoperazine reduces residence in the open state and promotes an apparently new substate which overlaps the
closed state at −50 mV but is distinguishable from it at voltages more negative than −100 mV. This substate may represent
times that trifluoperazine is bound to the channel.
Both antagonists have effects clearly distinguishable from that of withdrawing calcium from the channel, which does not affect
open state residence time but increases closed state residence time. Thus neither antagonist reverses the activating effect
of Ca2−. This is good kinetic evidence against the view that the channel is activated by Ca2+-calmodulin and that the effect of a calmodulin antagonist is to reverse this process by making Ca2−-calmodulin less available.
Received: 26 August 1996/Revised: 7 October 1996 相似文献
15.
cDNA encoding the full-length hKv1.3 lymphocyte channel and a C-terminal truncated (Δ459-523) form that lacks the putative
PKA Ser468 phosphorylation site were stably transfected in human embryonic kidney (HEK) 293 cells. Immunostaining of the transfected
cells revealed a distribution at the plasma membrane that was uniform in the case of the full-length channel whereas clustering
was observed in the case of the truncated channel. Some staining within the cell cytoplasm was found in both instances, suggesting
an active process of biosynthesis. Analyses of the K+ current by the patch-clamp technique in the whole cell configuration showed that depolarizing steps to 40 mV from a holding
potential (HP) of −80 mV elicited an outward current of 2 to 10 nA. The current threshold was positive to −40 mV and the current
amplitude increased in a voltage-dependent manner. The parameters of activation were −5.7 and −9.9 mV (slope factor) and −35
mV (half activation, V
0.5) in the case of the full-length and truncated channels, respectively. The characteristics of the inactivation were 14.2 and
24.6 mV (slope factor) and −17.3 and −39.0 mV (V
0.5) for the full-length and truncated channels, respectively. The activation time constant of the full-length channel for potentials
ranging from −30 to 40 mV decreased from 18 to 12 msec whereas the inactivation time constant decreased from 6600 msec at
−30 mV to 1800 msec at 40 mV. The unit current amplitude measured in cells bathing in 140 mm KCl was 1.3 ± 0.1 pA at 40 mV, the unit conductance, 34.5 pS and the zero current voltage, 0 mV. Both forms of the channels
were inhibited by TEA, 4-AP, Ni2+ and charybdotoxin. In contrast to the native (Jurkat) lymphocyte Kv1.3 channel that is fully inhibited by PKA and PKC, the
addition of TPA resulted in 34.6 ± 7.3% and 38.7 ± 9.4% inhibition of the full-length and the truncated channels, respectively.
8-BrcAMP induced a 39.4 ± 5.4% inhibition of the full-length channel but had no effect (8.6 ± 8.3%) on the truncated channel.
Cell dialysis with alkaline phosphatase had no effects, suggesting that the decreased sensitivity of the transfected channels
to PKA and PKC was not due to an already phosphorylated channel. Patch extract experiments suggested that the hKv1.3 channel
was partially sensitive to PKA and PKC. Cotransfecting the Kvβ1.2 subunit resulted in a decrease in the value of the time
constant of inactivation of the full-length channel but did not modify its sensitivity to PKA and PKC. The cotransfected Kvβ2
subunit had no effects. Our results indicate that the hKv1.3 lymphocyte channel retains its electrophysiological characteristics
when transfected in the Kvβ-negative HEK 293 cell line but its sensitivity to modulation by PKA and PKC is significantly reduced.
Received: 18 June 1997/Revised: 7 October 1997 相似文献
16.
J. I. Kourie 《The Journal of membrane biology》1997,157(1):39-51
Chloride channels in the sarcoplasmic reticulum (SR) are thought to play an essential role in excitation-contraction (E-C)
coupling by balancing charge movement during calcium release and uptake. In this study the nucleotide-sensitivity of Cl− channels in the SR from rabbit skeletal muscle was investigated using the lipid bilayer technique. Two distinct ATP-sensitive
Cl− channels that differ in their conductance and kinetic properties and in the mechanism of ATP-induced channel inhibition were
observed. The first, a nonfrequent 150 pS channel was inhibited by trans (luminal) ATP, and the second, a common 75 pS small chloride (SCl) channel was inhibited by cis (cytoplasmic) ATP. In the case of the SCl channel the ATP-induced reversible decline in the values of current (maximal current
amplitude, I
max and integral current, I′) and kinetic parameters (frequency of opening F
O
, probability of the channel being open P
O
, mean open T
O
and closed T
c
times) show a nonspecific block of the voltage- and Ca2+-dependent SCl channel. ATP was a more potent blocker from the cytoplasmic side than from the luminal side of the channel.
The SCl channel block was not due to Ca2+ chelation by ATP, nor to phosphorylation of the channel protein. The inhibitory action of ATP was mimicked by the nonhydrolyzable
analogue adenylylimidodiphosphate (AMP-PNP) in the absence of Mg2+. The inhibitory potency of the adenine nucleotides was charge dependent in the following order ATP4− > ADP3− > > > AMP2−. The data suggest that ATP-induced effects are mediated via an open channel block mechanism. Modulation of the SCl channel
by [ATP]
cis
and [Ca2+]
cis
indicates that (i) this channel senses the bioenergetic state of the muscle fiber and (ii) it is linked to the ATP-dependent
cycling of the Ca2+ between the SR and the sarcoplasm.
Received: 4 September 1996/Revised: 6 December 1996 相似文献
17.
The aim of this study is to evaluate directly, using a reduced experimental system, the nature of interactions between voltage-gated
potassium channels and the resting membrane potential. Xenopus oocytes were injected with various concentrations of cRNA coding for a delayed-rectifier potassium channel Shaker-IR. The effects of the density and kinetics of the expressed channels on resting membrane potential is explored in isolated
(``inside-out') patches. The channel density is given in terms of maximal conductance (G
max), measured from the maximal slope of the I-V curve under voltage clamp conditions. The capacitance of the experimental setup is approximately 1 pF. At high channel densities
(G
max > 10 pA/mV) the mean membrane potential is stabilized at approximately −60 mV. This resting membrane potential is more than
35 mV positive to the reversal potential for potassium ions under the same experimental conditions. Analyses of voltage clamp
experiments indicate that at high channel densities the mean membrane potential is determined by the rates of channel activation
and deactivation, but is not affected by the rates involved in the process of slow (C-type) inactivation. In contrast, at
lower channel densities membrane potential is very unstable, and its mean value and amplitude of fluctuations are strongly
affected by the process of slow (C-type) inactivation.
Received: 21 March 1996/Revised: 6 August 1996 相似文献
18.
Ion channel activity in cell-attached patch recordings shows channel behavior under more physiological conditions than whole-cell
and excised patch measurements. Yet the analysis of cell-attached patch measurements is complicated by the fact that the system
is ill defined with respect to the intracellular ion activities and the electrical potential actually experienced by the membrane
patch. Therefore, of the several patch-clamp configurations, the information that is obtained from cell-attached patch measurements
is the most ambiguous. The present study aims to achieve a better understanding of cell-attached patch measurements. Here
we describe a method to calculate the intracellular ion concentration and membrane potential prevailing during cell-attached
patch recording. The first step is an analysis of the importance of the input resistance of the intact cell on the cell-attached
patch measurement. The second step, and actual calculation, is based on comparison of the single channel conductance and reversal
potential in the cell-attached patch and excised patch configurations. The method is demonstrated with measurements of membrane
potential and cytosolic K+ concentrations in Vicia faba guard cells. The approach described here provides an attractive alternative to the measurement of cytosolic ion concentrations
with fluorescent probes or microelectrodes.
Received: 3 April 1998/Revised: 6 August 1998 相似文献
19.
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
20.
The possible correlation between P-glycoprotein (PGP) and volume-sensitive Cl− channel was examined in a pair of cell lines: a subline of the human epidermoid KB cell (KB-3-1) and the corresponding MDR1-transfected cell line (KB-G2). Western blot analysis and indirect immunofluorescence studies indicated that KB-G2, but not
KB-3-1, exhibits the PGP expression. Patch-clamp whole-cell recordings showed that osmotic swelling activates Cl− currents not only in PGP-expressing but also in PGP-lacking cells. The amplitude of the maximal current was indistinguishable
between both cells. Activation of protein kinase C (PKC) or loading with a PKC inhibitor failed to affect the swelling-induced
activation of the Cl− currents in both cells. The relation between whole-cell Cl− currents and cell size measured simultaneously showed that volume sensitivity of the Cl− channel was augmented by the PGP expression irrespective of the activity of PKC on the plasma membrane. A similar increase
in volume sensitivity of the Cl− channel was also induced by the expression of the ATP hydrolysis-deficient PGP mutant, K433M. We conclude that P-glycoprotein
does not represent the volume-sensitive Cl− channel but that its expression modulates volume sensitivity of the Cl− channel in a manner independent of its ATPase activity or of the protein kinase C activity.
Received: 25 September 1996/Revised: 12 December 1996 相似文献