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1.
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
2.
Glucagon is known to increase intracellular cAMP levels and enhance glucose-induced electrical activity and insulin secretion
in pancreatic β-cell perfused with Krebs-Ringer bicarbonate solution. The present experiments were aimed at evaluation of
the hypothesis that changes in β-cells ATP-sensitive K+ (K(ATP)) channel activity are involved in the glucagon-induced enhancement of electrical activity. Channel activity was recorded
using the cell-attached configuration of the patch-clamp technique. Addition of glucagon (2.9 × 10−7
m) in the presence of 11.1 mm glucose caused closure of K(ATP) channels followed by an increase in the frequency of biphasic current transients (action currents) due to action potential
generation in the cell. Three calmodulin-antagonists (W-7, chlorpromazine, and trifluoperazine) restored with similar efficacy
K(ATP) channel activity in cells being exposed to glucagon. At 2.8 mm glucose, glucagon did not affect K(ATP) channel activity until Ca2+ was released from Nitr-5 by flash photolysis, at which point channel activity was transiently suppressed. Similar effects
were seen when db-cAMP was used instead of glucagon.These results support the view that glucagon and other cAMP-generating
agonists enhance glucose-induced β-cell electrical activity through a Ca2+/calmodulin dependent-closure of K(ATP) channels.
Received: 26 May 1998/Revised: 18 September 1998 相似文献
3.
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
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.
Michinori Ichikawa Masashi Urayama Gen Matsumoto 《The Journal of membrane biology》1991,120(3):211-222
Summary The effects of calmodulin (CaM) antagonists (W-7, W-5, trifluoperazine, chlorpromazine, quinacrine, diazepam, propericyazine and carmidazolium) on the sodium and potassium channels were studied on the intracellularly perfused and voltage-clamped giant axon of the squid. It was found that the drugs are more potent blockers of the sodium current than of the potassium current. The drugs also reduce the sodium gating current. The blockage of the sodium and gating current can be explained by assuming that the drugs interact with the sodium gating subunit in one of its closed states. The site of action is probably the intracellular surface of the axolemma where presumably a Ca2+-calmodulin complex can be formed. 相似文献
6.
Characterization of the Stretch-activated Chloride Channel in Isolated Human Atrial Myocytes 总被引:2,自引:0,他引:2
Macroscopic and unitary currents through stretch-activated Cl− channels were examined in isolated human atrial myocytes using whole-cell, excised outside-out and inside-out configurations
of the patch-clamp technique. When K+ and Ca2+ conductances were blocked and the intracellular Ca2+ concentration ([Ca2+]
i
) was reduced, application of positive pressure via the pipette activated membrane currents under whole-cell voltage-clamp
conditions. The reversal potential of the current shifted by 60 mV per 10-fold change in the external Cl− concentration, indicating that the current was Cl− selective. The current was inhibited by bath application of 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and
9-anthracenecarboxylic acid (9-AC). β-Adrenergic stimulation failed to activate a Cl− current. In single channel recordings from outside-out patches, positive pressure in the pipette activated the unitary current
with half-maximal activation of 14.7 mm Hg at +40 mV. The current-voltage relationship of single channel activity obtained
in inside-out patches was linear in symmetrical Cl− solution with the averaged slope conductance of 8.6 ± 0.7 pS (mean ±sd, n= 10). The reversal potential shift of the channel by changing Cl− concentration was consistent with a Cl− selective channel. The open time distribution was best described by a single exponential function with mean open lifetime
of 80.4 ± 9.6 msec (n= 9), while at least two exponentials were required to fit the closed time distributions with a time constant for the fast
component of 11.5 ± 2.2 msec (n= 9) and that for the slow component of 170.2 ± 21.8 msec (n= 9). Major changes in the single channel activity in response to pressure were caused by changes in the interburst interval.
Single channel activity was inhibited by DIDS and 9-AC in a manner similar to whole-cell configuration. These results suggest
that membrane stretch induced by applying pressure via the pipette activated a Cl− current in human atrial myocytes. The current was sensitive to Cl− channel blockers and exhibited membrane voltage-independent bursting opening without sensitive to β-adrenergic stimulation.
Received: 21 October 1996/Revised: 17 December 1997 相似文献
7.
In the first part of this study, photofrin II sensitized membrane modifications of OK-cells were investigated at the level
of macroscopic membrane currents. In this second part, the inside-out configuration of the patch-clamp technique is applied
to analyze the phenomena at the microscopic level. It is shown that the characteristic single channel fluctuations of the
electric current disappear after the start of illumination of membrane patches in the presence of photofrin II. This holds
for all three types of ion channels investigated: the large-conductance Ca2+-dependent K+ channel (maxi-KCa), a K+ channel of small conductance (sK), and a stretch-activated nonselective cation channel (SA-cat). Part of the experiments
show a transient activation of the channels (indicated by an increase of the probability in the open-channel state) before
the channels are converted into a closed nonconductive state. Inactivation of all three channel types proceeds by a continuous
reduction of their open probability, while the single channel conductance values are not affected. The process of photodynamically
induced channel inactivation is followed by a pronounced increase of the leak conductance of the plasma membrane. The latter
process — after light-induced initiation — is found to continue in the dark. The ionic pathways underlying the leak conductance
also allow permeation of Ca2+ ions. The resulting Ca2+-flux may contribute to the photodynamically induced increase of the intracellular Ca2+ concentration observed in various cell lines.
Received: 26 May 1998/Revised: 8 September 1998 相似文献
8.
Ion Channel Permeable for Divalent and Monovalent Cations in Native Spinach Thylakoid Membranes 总被引:6,自引:0,他引:6
A cation-selective channel was characterized in isolated patches from osmotically swollen thylakoids of spinach (Spinacea oleracea). This channel was permeable for K+ as well as for Mg2+ and Ca2+ but not for Cl−. When K+ was the main permeant ion (symmetrical 105 mm KCl) the conductance of the channel was about 60 pS. The single channel conductance for different cations followed a sequence
K+ > Mg2+≥ Ca2+. The permeabilities determined by reversal potential measurements were comparable for K+, Ca2+, and Mg2+. The cation channel displayed bursting behavior. The total open probability of the channel increased at more positive membrane
potentials. Kinetic analysis demonstrated that voltage dependence of the total open probability was determined by the probability
of bursts formation while the probability to find the channel in open state within a burst of activity was hardly voltage-dependent.
The cation permeability of intact spinach thylakoids can be explained on the single channel level by the data presented here.
Received: 26 December 1995/Revised: 17 April 1996 相似文献
9.
In liver cells, cation-selective channels are permeable to Ca2+ and have been postulated to represent a pathway for receptor-mediated Ca2+ influx. This study examines the mechanisms involved in the regulation of these channels in a model liver cell line. Using
patch-clamp recording techniques, it is shown that channel open probability is a saturable function of cytosolic [Ca2+], with half-maximal opening at 660 nm. By contrast, channel opening is not affected by membrane voltage or cytosolic pH. In intact cells, reduction of cytosolic
[Cl−], a physiological response to Ca2+-mobilizing hormones and cell swelling, is also associated with an increase in channel opening. Finally, channel opening is
inhibited by intracellular ATP through a mechanism that does not involve ATP hydrolysis. These findings suggest that opening
of cation-selective channels is coupled to the metabolic state of the cell and provides a positive feedback mechanism for
regulation of receptor-mediated Na+ and Ca2+ influx.
Received: 8 October 1996 相似文献
10.
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
11.
Removal of extracellular divalent cations activated a Cl− channel in the plasma membrane of Xenopus laevis oocytes. This so-called Ca2+-inactivated Cl− channel (CaIC) was present in every oocyte and was investigated using two-electrode whole-cell voltage clamp and single-channel
patch-clamp techniques. Beside other Cl− channel inhibitors, anthracene-9-carboxylic acid (9-AC) and 3′azido-3′deoxythymidine (AZT), a nucleoside analogue commonly
used as an antiviral drug, blocked at least partly the CalC-mediated currents.
Using the Cl−-sensitive dye 6-methoxy-N-(sulfopropyl)quinolinium (SPQ) we could visualize the transport of Cl− from the oocyte cytoplasm to the surrounding medium after activation of the CaIC by Ca2+ removal. In the absence of external Cl− and Ca2+, the emission intensity of SPQ declined continuously, indicating a quenching of fluorescence by the efflux of Cl− in the millimolar range. In the presence of external Ca2+, no emission changes could be observed during the same time period. Chelating external Ca2+ in absence of Cl− immediately activated Ca2+-inactivated Cl− channels leading to subsequent emission decrease of SPQ.
Investigations on the selectivity of the CaIC revealed only poor discrimination between different anions. With single-channel
measurements, we found an anion selectivity sequence I− > Br− > Cl−≫ gluconate as it is also typical for maxi Cl− channels.
Contrary to the majority of all other transport systems of the Xenopus oocyte, which show reduced activity due to membrane depolarization or endocytotic removal of the transport protein from the
plasma membrane during oocyte maturation, the CaIC remained active in maturated oocytes. Single-channel measurements on maturated
oocytes, also known as eggs, showed the presence of Ca2+-inactivated Cl− channels. However, this egg CaIC revealed an altered sensitivity to external Ca2+ concentrations.
All these data confirm and extend our previous observations on the CaIC and give clear evidence that this channel is peculiar
among all Cl− channels described up to now.
Received: 16 May 1996/Revised: 4 September 1996 相似文献
12.
In cystic fibrosis, the mutation of the CFTR protein causes reduced transepithelial Cl− secretion. As recently proposed, beside its role of Cl− channel, CFTR may regulate the activity of other channels such as a Ca2+-activated Cl− channel. Using a calcium imaging system, we show, in adenovirus-CFTR infected Chinese Hamster Ovary (CHO) cell monolayers,
that CFTR can act as a regulator of intracellular [Ca2+]
i
([Ca2+]
i
), involving purino-receptors. Apical exposure to ATP or UTP produced an increase in ([Ca2+]
i
in noninfected CHO cell monolayers (CHO-WT), in CHO monolayers infected with an adenovirus-CFTR (CHO-CFTR) or infected with
an adenovirus-LacZ (CHO-LacZ). The transient [Ca2+]
i
increase produced by ATP or UTP could be mimicked by activation of CFTR with forskolin (20 μm) in CHO-CFTR confluent monolayers. However, forskolin had no significant effect on [Ca2+]
i
in noninfected CHO-WT or in CHO-LacZ cells. Pretreatment with purino-receptor antagonists such as suramin (100 μm) or reactive blue-2. (100 μm), and with hexokinase (0.28 U/mg) inhibited the [Ca2+]
i
response to forskolin in CHO-CFTR infected cells. Taken together, our experiments provide evidence for purino-receptor activation
by ATP released from the cell and regulation of [Ca2+]
i
by CFTR in CHO epithelial cell membranes.
Received: 5 April 1999/Revised: 28 June 1999 相似文献
13.
The calcium-dependent modulation of the affinity of the cyclic nucleotide-gated (CNG) channels for adenosine 3′,5′-cyclic
monophosphate (cAMP) was studied in enzymatically dissociated rat olfactory receptor neurons, by recording macroscopic cAMP-activated
currents from inside-out patches excised from their dendritic knobs. Upon intracellular addition of 0.2 mm Ca2+ (0.2 Ca) the concentration of cAMP required for the activation of half-maximal current (EC50) was reversibly increased from 3 μm to about 30 μm. This Ca2+-induced affinity shift was insensitive to the calmodulin antagonist, mastoparan, was abolished irreversibly by a 2-min exposure
to 3 mm Mg2++ 2 mm EGTA (Mg + EGTA), and was not restored by the application of calmodulin (CAM). Addition of CAM plus 0.2 mm Ca2+ (0.2 Ca + CAM), further reversibly shifted the cAMP affinity from 30 μm to about 200 μm. This affinity shift was not affected by Mg + EGTA exposure, but was reversed by mastoparan. Thus, the former Ca2+-only effect must be mediated by an unknown endogenous factor, distinct from CAM. Removal of this factor also increased the
affinity of the channel for CAM. The affinity shift induced by Ca2+-only was maintained in the presence of the nonhydrolyzable cAMP analogue, 8-bromo-cAMP and the phosphatase inhibitor, microcystin-LR,
ruling out modulation by phosphodiesterases or phosphatases. Our results indicate that the olfactory CNG channels are modulated
by an as yet unidentified factor distinct from CAM.
Received: 26 December 1995/Revised: 14 March 1996 相似文献
14.
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
15.
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
16.
The gating and conduction properties of a channel activated by intracellular Na+ were studied by recording unitary currents in inside-out patches excised from lobster olfactory receptor neurons. Channel
openings to a single conductance level of 104 pS occurred in bursts. The open probability of the channel increased with increasing
concentrations of Na+. At 210 mm Na+, membrane depolarization increased the open probability e-fold per 36.6 mV. The distribution of channel open times could
be fit by a single exponential with a time constant of 4.09 msec at −60 mV and 90 mm Na+. The open time constant was not affected by the concentration of Na+, but was increased by membrane depolarization. At 180 mm Na+ and −60 mV, the distribution of channel closed times could be fit by the sum of four exponentials with time constants of
0.20, 1.46, 8.92 and 69.9 msec, respectively. The three longer time constants decreased, while the shortest time constant
did not vary with the concentration of Na+. Membrane depolarization decreased all four closed time constants. Burst duration was unaffected by the concentration of
Na+, but was increased by membrane depolarization. Permeability for monovalent cations relative to that of Na+ (P
X
/P
Na
), calculated from the reversal potential, was: Li+ (1.11) > Na+ (1.0) > K+ (0.54) > Rb+ (0.36) > Cs+ (0.20). Extracellular divalent cations (10 mm) blocked the inward Na+ current at −60 mV according to the following sequence: Mn2+ > Ca2+ > Sr2+ > Mg2+ > Ba2+. Relative permeabilities for divalent cations (P
Y
/P
Na
) were Ca2+ (39.0) > Mg2+ (34.1) > Mn2+ (15.5) > Ba2+ (13.8) > Na+ (1.0). Both the reversal potential and the conductance determined in divalent cation-free mixtures of Na+ and Cs+ or Li+ were monotonic functions of the mole fraction, suggesting that the channel is a single-ion pore that behaves as a multi-ion
pore when the current is carried exclusively by divalent cations. The properties of the channel are consistent with the channel
playing a role in odor activation of these primary receptor neurons.
Received: 17 September 1996/Revised: 15 November 1996 相似文献
17.
Sitsapesan R 《The Journal of membrane biology》1999,168(2):159-168
Tonoplast K+ channels of Chara corallina are well characterized but only a few reports mention anion channels, which are likely to play an important role in the tonoplast
action potential and osmoregulation of this plant. For experiments internodal cells were isolated. Cytoplasmic droplets were
formed in an iso-osmotic bath solution according to a modified procedure. Ion channels with conductances of 48 pS and 170
pS were detected by the patch-clamp technique. In the absence of K+ in the bath solution the 170 pS channel was not observed at negative pipette potential values. When Cl− on either the vacuolar side or the cytoplasmic side was partly replaced with F−, the reversal potential of the 48 pS channel shifted conform to the Cl− equilibrium potential with similar behavior in droplet-attached and excised patch mode. These results showed that the 48
pS channel was a Cl− channel. In droplet-attached mode the channel rectified outward current flow, and the slope conductance was smaller. When
Chara droplets were formed in a bath solution containing low (10−8
m) Ca2+, then no Cl− channels could be detected either in droplet-attached or in inside-out patch mode. Channel activity was restored if Ca2+ was applied to the cytoplasmic side of inside-out patches. Rectification properties in the inside-out patch configuration
could be controlled by the holding pipette potential. Holding potential values negative or positive to the calculated reversal
potential for Cl− ions induced opposite rectification properties. Our results show Ca2+-activated Cl− channels in the tonoplast of Chara with holding potential dependent rectification.
Received: 30 March 1999/Revised: 10 August 1999 相似文献
18.
Marunaka Y Niisato N O'Brodovich H Post M Tanswell AK 《The Journal of membrane biology》1999,168(1):91-101
The aim of the present study was to investigate the roles of Ca2+ and protein tyrosine kinase (PTK) in the insulin action on cell volume in fetal rat (20-day gestational age) type II pneumocytes.
Insulin (100 nm) increased cell volume in the presence of extracellular Ca2+ (1 mm), while cell shrinkage was induced by insulin in the absence of extracellular Ca2+ (<1 nm). This insulin action in a Ca2+-containing solution was completely blocked by co-application of bumetanide (50 μm, an inhibitor of Na+/K+/2Cl− cotransporter) and amiloride (10 μm, an inhibitor of epithelial Na+ channel), but not by the individual application of either bumetanide or amiloride. On the other hand, the insulin action
on cell volume in a Ca2+-free solution was completely blocked by quinine (1 mm, a blocker of Ca2+-activated K+ channel), but not by bumetanide and/or amiloride. These observations suggest that insulin activates an amiloride-sensitive
Na+ channel and a bumetanide-sensitive Na+/K+/2Cl− cotransporter in the presence of 1 mm extracellular Ca2+, that the stimulatory action of insulin on an amiloride-sensitive Na+ channel and a bumetanide-sensitive Na+/K+/2Cl− cotransporter requires Ca2+, and that in a Ca2+-free solution insulin activates a quinine-sensitive K+ channel but not in the presence of 1 mm Ca2+. The insulin action on cell volume in a Ca2+-free solution was almost completely blocked by treatment with BAPTA (10 μm) or thapsigargin (1 μM, an inhibitor of Ca2+-ATPase which depletes the intracellular Ca2+ pool). Further, lavendustin A (10 μm, an inhibitor of receptor type PTK) blocked the insulin action in a Ca2+-free solution. These observations suggest that the stimulatory action of insulin on a quinine-sensitive K+ channel is mediated through PTK activity in a cytosolic Ca2+-dependent manner. Lavendustin A, further, completely blocked the activity of the Na+/K+/2Cl− cotransporter in a Ca2+-free solution, but only partially blocked the activity of the Na+/K+/2Cl− cotransporter in the presence of 1 mm Ca2+. This observation suggests that the activity of the Na+/K+/2Cl− cotransporter is maintained through two different pathways; one is a PTK-dependent, Ca2+-independent pathway and the other is a PTK-independent, Ca2+-dependent pathway. Further, we observed that removal of extracellular Ca2+ caused cell shrinkage by diminishing the activity of the amiloride-sensitive Na+ channel and the bumetanide-sensitive Na+/K+/2Cl− cotransporter, and that removal of extracellular Ca2+ abolished the activity of the quinine-sensitive K+ channel. We conclude that the cell shrinkage induced by removal of extracellular Ca2+ results from diverse effects on the cotransporter and Na+ and K+ channels.
Received: 2 September 1998/Revised: 30 November 1998 相似文献
19.
J.I. Kourie 《The Journal of membrane biology》1999,167(1):73-83
The understanding of the role of cytoplasmic pH in modulating sarcoplasmic reticulum (SR) ion channels involved in Ca2+ regulation is important for the understanding of the function of normal and adversely affected muscles. The dependency of
the SR small chloride (SCl) channel from rabbit skeletal muscle on cytoplasmic pH (pH
cis
) and luminal pH (pH
trans
) was investigated using the lipid bilayer-vesicle fusion technique. Low pH
cis
6.75–4.28 modifies the operational mode of this multiconductance channel (conductance levels between 5 and 75 pS). At pH
cis
7.26–7.37 the channel mode is dominated by the conductance and kinetics of the main conductance state (65–75 pS) whereas
at low pH
cis
6.75–4.28 the channel mode is dominated by the conductance and kinetics of subconductance states (5–40 pS). Similarly, low
pH
trans
4.07, but not pH
trans
6.28, modified the activity of SCl channels. The effects of low pH
cis
are pronounced at 10−3 and 10−4
m [Ca2+]
cis
but are not apparent at 10−5
m [Ca2+]
cis
, where the subconductances of the channel are already prominent. Low pH
cis
-induced mode shift in the SCl channel activity is due to modification of the channel proteins that cause the uncoupling of
the subconductance states. The results in this study suggest that low pH
cis
can modify the functional properties of the skeletal SR ion channels and hence contribute, at least partly, to the malfunction
in the contraction-relaxation mechanism in skeletal muscle under low cytoplasmic pH levels.
Received: 20 May 1998/Revised: 24 September 1998 相似文献
20.
Nonselective Cation and BK Channels in Apical Membrane of Outer Sulcus Epithelial Cells 总被引:5,自引:0,他引:5
The outer sulcus epithelium was recently shown to absorb cations from the lumen of the gerbil cochlea. Patch clamp recordings
of excised apical membrane were made to investigate ion channels that participate in this reabsorptive flux. Three types of
channel were observed: (i) a nonselective cation (NSC) channel, (ii) a BK (large conductance, maxi K or K
Ca
) channel and (iii) a small K+ channel which could not be fully characterized. The NSC channel found in excised insideout patch recordings displayed a linear
current-voltage (I-V) relationship (27 pS) and was equally conductive for Na+ and K+, but not permeable to Cl− or N-methyl-d-glucamine. Channel activity required the presence of Ca2+ at the cytosolic face, but was detected at Ca2+ concentrations as low as 10−7
m (open probability (P
o
) = 0.11 ± 0.03, n= 8). Gadolinium decreased P
o
of the NSC channel from both the external and cytosolic side (IC50∼ 0.6 μm). NSC currents were decreased by amiloride (10 μm− 1 mm) and flufenamic acid (0.1 mm). The BK channel was also frequently (38%) observed in excised patches. In symmetrical 150 mm KCl conditions, the I-V relationship was linear with a conductance of 268 pS. The Goldman-Hodgkin-Katz equation for current carried solely by K+ could be fitted to the I-V relationship in asymmetrical K+ and Na+ solutions. The channel was impermeable to Cl− and N-methyl-d-glucamine. P
o
of the BK channel increased with depolarization of the membrane potential and with increasing cytosolic Ca2+. TEA (20 mm), charybdotoxin (100 nm) and Ba2+ (1 mm) but not amiloride (1 mm) reduced P
o
from the extracellular side. In contrast, external flufenamic acid (100 μm) increased P
o
and this effect was inhibited by charybdotoxin (100 nm). Flufenamic acid inhibited the inward short-circuit current measured by the vibrating probe and caused a transient outward
current. We conclude that the NSC channel is Ca2+ activated, voltage-insensitive and involved in both constitutive K+ and Na+ reabsorption from endolymph while the BK channel might participate in the K+ pathway under stimulated conditions that produce an elevated intracellular Ca2+ or depolarized membrane potential.
Received: 14 October 1999/Revised: 10 December 1999 相似文献