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1.
The voltage-gated potassium channel, Kv1.3, which is highly expressed in a number of immune cells, contains concensus sites
for phosphorylation by protein kinase C (PKC). In lymphocytes, this channel is involved in proliferation—through effects on
membrane potential, Ca2+ signalling, and interleukin-2 secretion—and in cytotoxic killing and volume regulation. Because PKC activation (as well as
increased intracellular Ca2+) is required for T-cell proliferation, we have studied the regulation of Kv1.3 current by PKC in normal (nontransformed) human T lymphocytes. Adding intracellular ATP to support phosphorylation, shifted the voltage dependence of activation by
+8 mV and inactivation by +17 mV, resulting in a 230% increase in the window current. Inhibiting ATP production and action
with ``death brew' (2-deoxyglucose, adenylylimidodiphosphate, carbonyl cyanide-m-chlorophenyl hydrazone) reduced the K+ conductance (G
K
) by 41 ± 2%. PKC activation by 4β-phorbol 12,13-dibutyrate, increased G
K
by 69 ± 6%, and caused a positive shift in activation (+9 mV) and inactivation (+9 mV), which resulted in a 270% increase
in window current. Conversely, several PKC inhibitors reduced the current. Diffusion into the cell of inhibitory pseudosubstrate
or substrate peptides reduced G
K
by 43 ± 5% and 38 ± 8%, respectively. The specific PKC inhibitor, calphostin C, potently inhibited Kv1.3 current in a dose-
and light-dependent manner (IC50∼ 250 nm). We conclude that phosphorylation by PKC upregulates Kv1.3 channel activity in human lymphocytes and, as a result of shifts
in voltage dependence, this enhancement is especially prevalent at physiologically relevant membrane potentials. This increased
Kv1.3 current may help maintain a negative membrane potential and a high driving force for Ca2+ entry in the presence of activating stimuli.
Received: 12 July 1996/Revised: 21 October 1996 相似文献
2.
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
3.
J.I. Kourie 《The Journal of membrane biology》1999,172(1):37-45
The lipid bilayer technique is used to examine the biophysical properties of anion and cation channels frequently formed
by platypus (Ornithorhynchus anatinus) venom (OaV). The OaV-formed anion channel in 250/50 mm KCl cis/trans has a maximum conductance of 857 ± 23 pS (n= 5) in 250/50 mm KCl cis/trans. The current-voltage relationship of this channel shows strong inward rectification. The channel activity undergoes time-dependent
inactivation that can be removed by depolarizing voltage steps more positive than the reversal potential for chloride, E
Cl
, (+40 mV).
The reversal potential of the OaV-formed slow current activity in 250/50 mm KCl cis/trans is close to the potassium equilibrium potential (E
K
) of −40 mV. The conductance values for the slow channel are 22.5 ± 2.6 pS and 41.38 ± 4.2 pS in 250/50 and 750/50 mm
cis/trans, respectively. The gating kinetics of the slow ion channels are voltage-dependent. The channel open probability (P
o
) is between 0.1 and 0.8 at potentials between 0 and +140 mV. The channel frequency (F
o
) increases with depolarizing voltages between 0 and +140 mV, whereas mean open time (T
o
) and mean closed time (T
c
) decrease. Ion substitution experiments of the cis solution show that the channel has conductance values of 21.47 ± 2.3 and 0.53 ± 0.1 pS in 250 mm KCl and choline Cl, respectively. The amplitude of the single channel current is dependent on [K+]
cis
and the current reversal potential (E
rev
) responds to increases in [K+]
cis
by shifting to more negative voltages. The increase in current amplitude as a function of increasing [K+]
cis
can be best described by a third order polynomial fit. At +140 mV, the values of the maximal single channel conductance (γ
max
) and the concentration for half maximal γ (K
s
) are 38.6 pS and 380 mm and decline to 15.76 pS and 250 mm at 0 mV, respectively. The ion selectivity of the channel to K+, Na+, Cs+ and choline+ was determined in ion substitution experiments. The permeability values for P
K+
:P
Na+
:P
Cs+
:P
choline+
were 1:1:0.63:0.089, respectively. On the other hand, the activity of the slow channel was eliminated (Fig. 7B). The slow channel was reversibly inhibited by [TEA+]
trans
and the half-maximal inhibitory concentration (K
i
) was ∼48 mm.
Received: 26 April 1999/Revised: 19 July 1999 相似文献
4.
We have obtained and modeled the electrical characteristics of the plasma membrane of Chara internodal cells: intact, without turgor and perfused with and without ATP. The cells were voltage and space-clamped to obtain
the I/V (current-voltage) and G/V (conductance-voltage) profiles of the cell membrane.
The intact cells yielded similar I/V characteristics with resting p.d.s of −221 ± 12 mV (cytoplasmic clamp, 5 cells) and −217 ± 12 mV (vacuolar clamp, 5 cells).
The cut unperfused cells were depolarized at −169 ± 12 mV (7 cells) compared to the vacuole-clamped intact cells. The cells
perfused with ATP fell into three groups: hyperpolarized group with resting p.d. −175 ± 12 mV (4 cells) and I/V profile similar to the intact and cut unperfused cells; depolarized group with resting p.d. of −107 ± 12 mV (6 cells) and
I/V profiles close to linear; and excited cells with profiles showing a negative conductance region and resting p.d. at −59 ±
12 mV (5 cells). The cells perfused with medium containing no ATP showed upwardly concave I/V characteristics and resting p.d. at −81 ± 12 mV (6 cells).
The I/V curves were modeled employing the ``Two-state' model for the H+ pump (Hansen et al., 1981). The inward and outward rectifiers were fitted to exponential functions and combined with a linear
background current. The excitation state in perfused cells was modeled by including an inward current, i
excit, with p.d.-dependence described by a combination of hyperbolic tangent functions. An inward current, i
no-ATP, with a smaller amplitude, but very similar p.d.-dependence was also included in the simulation of the I/V curves from cells without ATP. This approach avoided I/V curve subtraction.
The modeling of the total I/V and G/V characteristics provided more information about the parameters of the ``Two-state' pump model, as well as more quantitative
understanding of the interaction of the major transport systems in the plasmalemma in generation of the resting potential
under a range of circumstances. ATP had little effect on nonpump currents except the excitation current; depolarization profoundly
affected the pump characteristics.
Received: 23 January/Revised: 10 October 1995 相似文献
5.
An ion channel activated by hyperpolarization was identified in excised patches of bovine pigmented ciliary epithelial cells
using the single channel patch clamp technique. In symmetrical NaGluconate, the channel had a maximum conductance of 285 pS.
The channel was characterized by frequent flickery transitions between the fully open and closed levels. The channel did not
discriminate very clearly between anions and cations; when the cytoplasmic face of excised patches was bathed in a dilute
NaCl solution, the channel had a chloride-to-sodium permeability ratio (P
Cl/P
Na) of 1.3. However, the channel showed a small anion selectivity (P
Cl/P
Na= 3.7) when bathed in a concentrated NaCl solution. Gd3+ blocked the channel reversibly. Channel kinetics were characterised by slow (≈ min) voltage-dependent activation and inactivation
rate constants. The channel was most active in the range −60 to −140 mV and showed a peak at −120 mV. A similar time- and
voltage-dependent activation was also observed in cell-attached recordings. In conclusion, hyperpolarization of pigmented
ciliary epithelial cell membrane patches activated a large conductance, nonselective ion channel. This combination of nonselectivity
and hyperpolarizing activation is consistent with the involvement of this channel in ion loading from the blood into pigmented
ciliary epithelial cells—the first phase in the secretion of aqueous humor.
Received: 30 June 1995/Revised: 16 November 1995 相似文献
6.
Voltage-clamp experiments were performed on single bovine adrenal fasciculata cells in short-term primary culture using either
standard (broken membrane) or perforated whole-cell patch clamp recording. The membrane current measured with the perforated
method was dominated by a very stable transient outward current. By contrast, the transient outward current recorded using
the standard method was unstable. The reversal potential of the transient outward current varied linearly with the logarithm
of [K+]
e
with a slope of 47 mV per decade. The onset of activation was sigmoidal and was fitted with a power function where n= 4. Time constants ranged from 1 to 4 msec with a maximum at −25 mV. The steady-state activation curve spanned the voltage
range −50 to +80 mV without reaching a clear maximum. During a pulse, the current decayed in a biexponential manner. Time
constants τ1 and τ2 were voltage-dependent and ranged from 50 to 200 msec respectively for a voltage step at +50 mV. The steady-state inactivation
was dependent on the conditioning pulse duration. Using short conditioning pulses (1.2 sec), the curve which spanned the voltage
range −40 to −20 mV, was 15 mV more positive than that obtained with longer conditioning pulses (60 sec). Time constants of
this ``very slow inactivation' process (τvs) determined for voltage steps at −60 and −50 mV were 15 and 10 sec respectively. A ``facilitation process' of the peak current
was observed when the duration or the amplitude of conditioning pulses were increased in the voltage range −100 to −50 mV.
Recovery from inactivation followed a biexponential time course which seemed a mixture of both inactivation processes. In
some experimental conditions, isolated cells were able to produce overshooting action potentials. These results are discussed
in relation with the membrane electrogenesis of this cell type.
Received: 14 November 1994/Revised: 24 October 1995 相似文献
7.
E. Modesto P. D. Lampe M. C. Ribeiro D. C. Spray A. C. Campos de Carvalho 《The Journal of membrane biology》1996,154(3):239-249
Membrane fractions highly enriched in chicken lens MIP (MIP28) were found to form ion channels when incorporated into planar
lipid bilayers. The channels displayed prominent unitary conductances of about 60 and 290 pS in symmetric 150 mm KCl solution and were slightly anion selective. For both depolarizing and hyperpolarizing voltages, voltage sensitivity of
the MIP28-induced conductance could be fit by a Boltzmann relation, symmetric around zero mV, with V
0
= 18.5 mV, n= 4.5 and g
min/g
max= 0.17. Channel properties were not appreciably altered by pH in the range of 5.8 to 7, although channel incorporation was
observed to occur more frequently at lower pH values. Calcium, at millimolar concentrations, decreased the channel mean open
time. Partial proteolysis of MIP28 to yield MIP21 did not appreciably affect single-channel conductance or voltage sensitivity
of the reconstituted channels. MIP28 was not phosphorylated by cAMP dependent protein kinase (PKA). Although unitary conductance
and selectivity of the chicken MIP channel are similar to those reported for the bovine MIP (MIP26), the voltage sensitivity
of MIP28 was higher than that of the bovine homologue, and voltage sensitivity of MIP28 was not modulated by treatments previously
shown to affect MIP26 voltage gating (partial proteolysis and protein phosphorylation by PKA: (Ehring et al., 1990). The existence
of such strikingly different functional properties in highly homologous channel isoforms may provide a useful system for exploration
of the structure-function relations of MIP channels.
Received: 27 March 1996/Revised: 5 August 1996 相似文献
8.
The anion conductance of the plasma membrane of Coffea arabica protoplasts was isolated and characterized using the whole-cell patch clamp technique. Voltage pulse protocols revealed two
components: a voltage-gated conductance (G
s
) and a voltage-independent one (G
l
). G
s
is activated upon depolarization (e-fold activation every +36 mV) with time constants of 1 sec and 5 sec at all potentials.
G
l
and G
s
also differ by their kinetic and biophysical properties. In bi-ionic conditions the current associated with G
s
shows strong outward rectification and its permeability sequence is F− > NO3
− > Cl−. In the same conditions the current associated with G
l
does not rectify and its permeability sequence is F−≫ NO3
−= Cl−. Furthermore, at potentials over +50 mV G
s
, but not G
l
, increases with a time constant of several minutes. Finally the gating of G
s
is affected by stretch of the membrane, which leads to an increased activation and a reduced voltage sensitivity. Anion conductances
similar to the ones described here have been found in many plant preparations but G
l
-type components have been generally interpreted as the background activation of the slow voltage-gated channels (corresponding
to G
s
). We show that in coffee protoplasts G
l
and G
s
are kinetically and biophysically distinct, suggesting that they correspond to two different molecular entities.
Received: 25 November 1996/Revised: 9 April 1997 相似文献
9.
These experiments were conducted to determine the membrane K+ currents and channels in human urinary bladder (HTB-9) carcinoma cells in vitro. K+ currents and channel activity were assessed by the whole-cell voltage clamp and by either inside-out or outside-out patch
clamp recordings. Cell depolarization resulted in activation of a Ca2+-dependent outward K+ current, 0.57 ± 0.13 nS/pF at −70 mV holding potential and 3.10 ± 0.15 nS/pF at 30 mV holding potential. Corresponding patch
clamp measurements demonstrated a Ca2+-activated, voltage-dependent K+ channel (KCa) of 214 ± 3.0 pS. Scorpion venom peptides, charybdotoxin (ChTx) and iberiotoxin (IbTx), inhibited both the activated current
and the KCa activity. In addition, on-cell patch recordings demonstrated an inwardly rectifying K+ channel, 21 ± 1 pS at positive transmembrane potential (V
m
) and 145 ± 13 pS at negative V
m
. Glibenclamide (50 μm), Ba2+ (1 mm) and quinine (100 μm) each inhibited the corresponding nonactivated, basal whole-cell current. Moreover, glibenclamide inhibited K+ channels in inside/out patches in a dose-dependent manner, and the IC50= 46 μm. The identity of this K+ channel with an ATP-sensitive K+ channel (KATP) was confirmed by its inhibition with ATP (2 mm) and by its activation with diazoxide (100 μm). We conclude that plasma membranes of HTB-9 cells contain the KCa and a lower conductance K+ channel with properties consistent with a sulfonylurea receptor-linked KATP.
Received: 12 June 1997/Revised: 21 October 1997 相似文献
10.
D.P. Lotshaw 《The Journal of membrane biology》1997,156(3):261-277
Nystatin perforated-patch clamp and single-channel recording methods were used to characterize macroscopic and single-channel
K+ currents and the effects of angiotensin II (AngII) in cultured rat adrenal glomerulosa cells. Two basic patterns of macroscopic
current-voltage relationships were observed: type 1 exhibited a rapidly activating, noninactivating, voltage-dependent outward
current and type 2 exhibited an inactivating voltage-dependent outward current attributed to charybdotoxin sensitive Ca++-dependent K+ channels. Most cells exhibited the type 1 pattern and experiments focused on this cell type. Cell-attached and inside-out
patches were dominated by a single K+ channel class which exhibited an outward conductance of 12 pS (20 mm K+ pipette in cell-attached and inside-out configurations, 145 mm K+
in), a mean open time of 2 msec, and a weakly voltage-dependent low open probability that increased with depolarization. Channel
open probability was reversibly inhibited by bath stimulation with AngII. At the macroscopic level, type 1 cell macroscopic
K+ currents appeared comprised of two components: a weakly voltage-dependent current controlling the resting membrane potential
(−85 mV) which appeared mediated by the 12 pS K+ channel and a rapidly activating, noninactivating voltage-dependent current activated above −50 mV. The presence of the second
voltage-dependent K+ channel class was suggested by the effects of AngII, the blocking effects of quinidine and Cs+, and the properties of the weakly voltage-dependent K+ channel described. The K+ selectivity of the macroscopic current was demonstrated by the dependence of current reversal potentials on the K+ equilibrium potential and by the effects of K+ channel blockers, Cs+ and quinidine. AngII (10 pm to 1 nm) reversibly inhibited macroscopic K+ currents and this effect was blocked by the AT1 receptor antagonist losartin.
Received: 6 August 1996/Revised: 15 November 1996 相似文献
11.
Single-channel properties of a delayed rectifier voltage-gated K+ channel (I-type) were investigated in peripheral myelinated axons from Xenopus laevis. Channels activated between −60 and −40 mV with a potential of half-maximal activation, E50, at −47.5 mV. Averaged single-channel currents activated with a time delay at all membrane potentials tested. Time to half-maximal
activation decreased from 80 to 1.6 msec between −60 and +40 mV. The channel inactivated monoexponentially with a time constant
of 10.9 sec at −40 mV. The time constant of deactivation was 126 msec at −80 mV and 16.9 msec at −110 mV. In symmetrical 105
mm K+, the single-channel conductance (γ) was 22 and 13 pS at negative and positive membrane potentials, respectively, at 13–15°C.
In Na+-rich solution with 2.5 mm extracellular K+γ was 7 pS and the reversal potential was negative to −80 mV, indicating a high selectivity for K+ over Na+. γ depended on extracellular K+ concentration (K
D
= 19.6 mm) and temperature (Q
10= 1.45). External tetraethylammonium (TEA) reduced the apparent single-channel current amplitude at all potentials tested
with a half-maximal inhibiting concentration (IC50) of 0.6 mm. Open probability of the channel, but not single-channel current amplitude was decreased by extracellular dendrotoxin (DTX,
IC50= 6.8 nm) and mast cell degranulating peptide (MCDP, IC50= 41.9 nm). In Ringer solution the membrane potential of macroscopic I-channel patches was about −65 mV and depolarized under TEA and
DTX. It is concluded that besides their activation during action potentials, I-channels may also stabilize the resting membrane
potential.
Received: 2 June 1995/Revised: 13 October 1995 相似文献
12.
M. Iino S. Ciani K. Tsuzuki S. Ozawa Y. Kidokoro 《The Journal of membrane biology》1997,155(2):143-156
Ion permeation properties of the mouse e2/ζ1 NMDA receptor channel expressed in Xenopus oocytes were studied using the outside-out patch-clamp technique. In symmetrical Na+ solutions, the single-channel I-V relations were almost linear at low electrolyte concentrations, but rectified inwardly for Na+ concentrations above 50 mm. In symmetrical Na+ solutions, the ``zero-current conductance' increased with Na+ concentration and saturated according to a hyperbolic curve, the half-maximal saturating activity, K
M
(Na), being 14.2 mm and the maximal conductance, G
max(Na), 53.9 pS. When Ca2+ was present with Na+ in the external solution, the single-channel current was lower than in pure Na+, although the reversal potential indicated a higher permeability for Ca2+ than for Na+. Using ion activities, PCa/PNa was found to be about 17. The I-V data were fitted with a model based on the Eyring's rate theory, assuming a one-ion pore with three energy barriers and two
sites. The K
M
(Ca) and G
max (Ca) were 76.5 μm and 21.2 pS, respectively. According to the estimated rate constants, K
M
for Ca2+ is mainly determined by the binding strength of a site located 80% away from the channel opening at the external membrane-solution
interface, a position similar to that postulated previously for the Mg2+ blocking site.
Received: 3 May 1996/Revised: 25 September 1996 相似文献
13.
Protection from Cell Death by mcl-1 Is Mediated by Membrane Hyperpolarization Induced By K+ Channel Activation 总被引:3,自引:0,他引:3
Mcl-1, a member of the Bcl-2 family, has been identified as an inhibitor of apoptosis induced by anticancer agents and radiation in myeloblastic leukemia
cells. The molecular mechanism underlying this phenomenon, however, is not yet understood. In the present study, we report
that hyperpolarization of the membrane potential is required for prevention of mcl-1 mediated cell death in murine myeloblastic FDC-P1 cells. In cells transfected with mcl-1, the membrane potential, measured by the whole-cell patch clamp, was hyperpolarized more than −30 mV compared with control
cells. The membrane potential was repolarized by increased extracellular K+ concentration (56 mV per 10-fold change in K+ concentration). Using the cell-attached patch-clamp technique, K+ channel activity was 1.7 times higher in mcl-1 transfected cells (NP
o
= 22.7 ± 3.3%) than control cells (NP
o
= 13.2 ± 1.9%). Viabilities of control and mcl-1 transfected cells after treatment with the cytotoxin etoposide (20 μg/ml), were 37.9 ± 3.9% and 78.2 ± 2.0%, respectively.
Suppression of K+ channel activity by 4-aminopyridine (4-AP) before etoposide treatment significantly reduced the viability of mcl-1 transfected cells to 49.0 ± 4.6%. These results indicate that as part of the prevention of cell death, mcl-1 causes a hyperpolarization of membrane potential through activation of K+ channel activity.
Received: 30 March 1999/Revised: 20 July 1999 相似文献
14.
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
15.
A large-conductance (or maxi-) chloride channel was identified in bovine pigmented ciliary epithelial (PCE) cells using inside-out
excised patch clamp recording. The channel had a mean conductance of 293 pS when excised patches were bathed in symmetrical
130 mm NaCl although the conductance decreased to 209 pS when the solution bathing the cytoplasmic face of the patch contained only
33 mm NaCl. The channel was highly selective for chloride, with a P
Cl/P
Na= 24. A flickery, reversible block was produced by the diuretic stilbene 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic
acid (SITS), while 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) produced a permanent block. The channel was rarely
active in cell-attached patches and usually required several minutes of polarization before activity could be detected in
excised patches, a process known as metagenesis. Once activated, the channel was voltage-dependent and was mainly open within
the voltage range −30 to +30 mV closing when the membrane was polarized to larger values. GTPγS (100 μm) activated the channel with a latency of 170 sec when applied to the cytoplasmic face of patches. This activation was not
reversible upon return to control solution within the duration of the experiment. We assess the available evidence and suggest
a role for this channel in volume regulation.
Received: 24 June 1996/Revised: 18 February 1997 相似文献
16.
A detailed temperature dependence study of a well-defined plant ion channel, the Ca2+-activated K+ channel of Chara corallina, was performed over the temperature range of their habitats, 5–36°C, at 1°C resolution. The temperature dependence of the
channel unitary conductance at 50 mV shows discontinuities at 15 and 30°C. These temperatures limit the range within which
ion diffusion is characterized by the lowest activation energy (E
a
= 8.0 ± 1.6 kJ/mol) as compared to the regions below 15°C and above 30°C. Upon reversing membrane voltage polarity from 50
to −50 mV the pattern of temperature dependence switched from discontinuous to linear with E
a
= 13.6 ± 0.5 kJ/mol. The temperature dependence of the effective number of open channels at 50 mV showed a decrease with increasing
temperature, with a local minimum at 28°C. The mean open time exhibited a similar behavior. Changing the sign of membrane
potential from 50 to −50 mV abolished the minima in both temperature dependencies. These data are discussed in the light of
higher order phase transitions of the Characean membrane lipids and corresponding change in the lipid-protein interaction,
and their modulation by transmembrane voltage.
Received: 14 June 2000/Revised: 20 September 2000 相似文献
17.
P.M. Dunn 《The Journal of membrane biology》1998,165(2):133-143
The actions of clotrimazole and cetiedil, two drugs known to inhibit the Gardos channel, have been studied on single intermediate
conductance calcium-activated potassium (IKCa) channels in inside out patches from human red blood cells, and compared with those of TEA and Ba2+ applied to the cytoplasmic face of the membrane. TEA produced a fast block which was observed as a reduction in the amplitude
of the single channel current. This effect was weakly voltage dependent with the fraction of the membrane potential sensed
by TEA at its binding site (δ) of 0.18 and a K
d
at 0 mV of 20.5 mm. Ba2+ was a very potent blocker of the channel, breaking the single channel activity up into bursts, interspersed with silent periods
lasting several seconds. The effect of Ba2+ was very voltage sensitive, δ= 0.44, and a K
d
at 0 mV of 0.15 μm. Clotrimazole applied to the inner face of the membrane at a concentration ≤1 μm produced a slow block resulting in bursts of channel activity separated by quiescent periods lasting many seconds. The effect
of clotrimazole was mimicked by a quaternary derivative UCL 1559, in keeping with an action at the cytoplasmic face of the
channel. A high concentration of cetiedil (100 μm) produced only a weak block of the channel. The kinetics of this action were very slow, with burst and inter-burst intervals
lasting several minutes. While inhibition of the Gardos channel by cetiedil is unlikely to involve an intracellular site of
action, if clotrimazole is able to penetrate the membrane, part of its effect may result from binding to an intracellular
site on the channel.
Received; 18 February 1998/Received: 5 June 1998 相似文献
18.
The THP-1 human monocytic leukemia cell line is a useful model of macrophage differentiation. Patch clamp methods were used
to identify five types of ion channels in undifferentiated THP-1 monocytes. (i) Delayed rectifier K+ current, I
DR, was activated by depolarization to potentials positive to −50 mV, inactivated with a time constant of several hundred msec,
and recovered from inactivation with a time constant ∼21 sec. I
DR was inhibited by 4-aminopyridine (4-AP), tetraethylammonium (TEA+), and potently by charybdotoxin (ChTX). (ii) Ca-activated K+ current (I
SK) dominated whole-cell currents in cells studied with 3–10 μm [Ca2+]
i
. I
SK was at most weakly voltage-dependent, with reduced conductance at large positive potentials, and was inhibited by ChTX and
weakly by TEA+, Cs+, and Ba2+, but not 4-AP or apamin. Block by Cs+ and Ba2+ was enhanced by hyperpolarization. (iii) Nonselective cation current, I
cat, appeared at voltages above +20 mV. Little time-dependence was observed, and a panel of channel blockers was without effect.
(iv) Chloride current, I
Cl, was present early in experiments, but disappeared with time. (v) Voltage-activated H+ selective current is described in detail in a companion paper (DeCoursey & Cherny, 1996. J. Membrane Biol.
152:2). The ion channels in THP-1 cells are compared with channels described in other macrophage-related cells. Profound changes
in ion channel expression that occur during differentiation of THP-1 cells are described in a companion paper (DeCoursey et
al., 1996. J. Membrane Biol.
152:2).
Received: 19 September 1995/Revised: 14 March 1996 相似文献
19.
Muscarinic receptor-linked G protein, G
i
, can directely activate the specific K+ channel (I
K(ACh)) in the atrium and in pacemaker tissues in the heart. Coupling of G
i
to the K+ channel in the ventricle has not been well defined. G protein regulation of K+ channels in isolated human ventricular myocytes was examined using the patch-clamp technique. Bath application of 1 μm acetylcholine (ACh) reversibly shortened the action potential duration to 74.4 ± 12.1% of control (at 90% repolarization,
mean ±sd, n= 8) and increased the whole-cell membrane current conductance without prior β-adrenergic stimulation in human ventricular
myocytes. The ACh effect was reversed by atropine (1 μm). In excised inside-out patch configurations, application of GTPγS (100 μm) to the bath solution (internal surface) caused activation of I
K(ACh) and/or the background inwardly-rectifying K+ channel (I
K1) in ventricular cell membranes. I
K(ACh) exhibited rapid gating behavior with a slope conductance of 44 ± 2 pS (n= 25) and a mean open lifetime of 1.8 ± 0.3 msec (n= 21). Single channel activity of GTPγS-activated I
K1 demonstrated long-lasting bursts with a slope conductance of 30 ± 2 pS (n= 16) and a mean open lifetime of 36.4 ± 4.1 msec (n= 12). Unlike I
K(ACh), G protein-activated I
K1 did not require GTP to maintain channel activity, suggesting that these two channels may be controlled by G proteins with
different underlying mechanisms. The concentration of GTP at half-maximal channel activation was 0.22 μm in I
K(ACh) and 1.2 μm in I
K1. Myocytes pretreated with pertussis toxin (PTX) prevented GTP from activating these channels, indicating that muscarinic
receptor-linked PTX-sensitive G protein, G
i
, is essential for activation of both channels. G protein-activated channel characteristics from patients with terminal heart
failure did not differ from those without heart failure or guinea pig. These results suggest that ACh can shorten the action
potential by activating I
K(ACh) and I
K1 via muscarinic receptor-linked G
i
proteins in human ventricular myocytes.
Received: 23 September 1996/Revised: 18 December 1996 相似文献
20.
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