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1.
Inward-rectifying potassium channels in plant cells provide important mechanisms for low-affinity K+ uptake and membrane potential control in specific cell types, including guard cells, pulvinus cells, aleurone cells and root
hair cells. K+ channel blockers are potent tools for studying the physiological functions and structural properties of K+ channels. In the present study the structural and biophysical mechanisms of Cs+ and TEA+ block of a cloned Arabidopsis inward-rectifying K+ channel (KAT1) were analyzed. Effects of the channel blockers Cs+ and TEA+ were characterized both extracellularly and intracellularly. Both external Cs+ and TEA+ block KAT1 currents. A mutant of KAT1 (``m2KAT1'; H267T, E269V) was produced by site-directed mutagenesis of two amino acid
residues in the C-terminal portion of the putative pore (P) domain. This mutant channel was blocked less by external Cs+ and TEA+ than the wild-type K+ channel. Internal TEA+ and Cs+ did not significantly block either m2KAT1 or KAT1 channels. Other properties, such as cation selectivity, voltage-dependence
and proton activation did not show large changes between m2KAT1 and KAT1, demonstrating the specificity of the introduced
mutations. These data suggest that the amino acid positions mutated in the inward-rectifying K+ channel, KAT1, are accessible to external blockers and may be located on the external side of the membrane, as has been suggested
for outward-rectifying K+ channels.
Received: 31 July 1995/Revised: 5 January 1996 相似文献
2.
3.
O. Ortiz-Carranza M.E. Miller N.C. Adragna P.K. Lauf 《The Journal of membrane biology》1997,156(3):287-295
We examined the effects of pH, internal ionized Ca (Ca2+
i
), cellular ATP, external divalent cations and quinine on Cl-independent ouabain-resistant K+ efflux in volume-clamped sheep red blood cells (SRBCs) of normal high (HK) and low (LK) intracellular K+ phenotypes. In LK SRBCs the K+ efflux was higher at pH 9.0 (350%) than at pHs 7.4 and 6.5, and was inhibited by external divalent cations, quinine, and
cellular ATP depletion. The above findings suggest that the increased K+ efflux at alkaline pH is due to the opening of ion channels or specific transporters in the cell membrane. In addition, K+ efflux was activated (100%) when Ca2+
i
was increased (+A23187, +Ca2+
o
) into the μm range. However, in comparison to human red blood cells, the Ca2+
i
-induced increase in K+ efflux in LK SRBCs was fourfold smaller and insensitive to quinine and charybdotoxin. The Na+ efflux was also higher at pH 9.0 than at pH 7.4, and activated (about 40%) by increasing Ca2+
i
. In contrast, in HK SRBCs the K+ efflux at pH 9.0 was neither inhibited by quinine nor activated by Ca2+
i
. These studies suggest the presence in LK SRBCs, of at least two pathways for Cl−-independent K+ and Na+ transport, of which one is unmasked by alkalinization, and the other by a rise in Ca2+
i
.
Received: 23 May 1996/Revised: 6 December 1996 相似文献
4.
The depolarization-activated, high-conductance ``maxi' cation channel in the plasma membrane of rye (Secale cereale L.) roots is permeable to a wide variety of monovalent and divalent cations. The permeation of K+, Na+, Ca2+ and Ba2+ through the pore could be simulated using a model composed of three energy barriers and two ion binding sites (a 3B2S model),
which assumed single-file permeation and the possibility of double cation occupancy. The model had an asymmetrical free energy
profile. Differences in permeation between cations were attributed primarily to differences in their free energy profiles
in the regions of the pore adjacent to the extracellular solution. In particular, the height of the central free energy peak
differed between cations, and cations differed in their affinities for ion binding sites. Significant ion repulsion occurred
within the pore, and the mouths of the pore had considerable surface charge. The model adequately described the diverse current
vs. voltage (I/V) relationships obtained over a wide variety of experimental conditions. It described the phenomena of non-Michaelian unitary
conductance vs. activity relationships for K+, Na+ and Ca2+, differences in selectivity sequences obtained from measurements of conductance and permeability ratios, changes in relative
cation permeabilities with solution composition, and the complex effects of Ba2+ and Ca2+ on K+ currents through the channel. The model enabled the prediction of unitary currents and ion fluxes through the maxi cation
channel under physiological conditions. It could be used, in combination with data on the kinetics of the channel, as input
to electrocoupling models allowing the relationships between membrane voltage, Ca2+ influx and Ca2+ signaling to be studied theoretically.
Received: 29 April 1998/Revised: 20 November 1998 相似文献
5.
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
6.
Calcium channels in the plasma membrane of root cells fulfill both nutritional and signaling roles. The permeability of these
channels to different cations determines the magnitude of their cation conductances, their effects on cell membrane potential
and their contribution to cation toxicities. The selectivity of the rca channel, a Ca2+-permeable channel from the plasma membrane of wheat (Triticum aestivum L.) roots, was studied following its incorporation into planar lipid bilayers. The permeation of K+, Na+, Ca2+ and Mg2+ through the pore of the rca channel was modeled. It was assumed that cations permeated in single file through a pore with three energy barriers and two
ion-binding sites. Differences in permeation between divalent and monovalent cations were attributed largely to the affinity
of the ion binding sites. The model suggested that significant negative surface charge was present in the vestibules to the
pore and that the pore could accommodate two cations simultaneously, which repelled each other strongly. The pore structure
of the rca channel appeared to differ from that of L-type calcium channels from animal cell membranes since its ion binding sites had
a lower affinity for divalent cations. The model adequately accounted for the diverse permeation phenomena observed for the
rca channel. It described the apparent submillimolar K
m
for the relationship between unitary conductance and Ca2+ activity, the differences in selectivity sequences obtained from measurements of conductance and permeability ratios, the
changes in relative cation permeabilities with solution ionic composition, and the complex effects of Ca2+ on K+ and Na+ currents through the channel. Having established the adequacy of the model, it was used to predict the unitary currents that
would be observed under the ionic conditions employed in patch-clamp experiments and to demonstrate the high selectivity of
the rca channel for Ca2+ influx under physiological conditions.
Received: 23 August 1999/Revised: 12 November 1999 相似文献
7.
P.R. Andjus M.R. Djurišić Z. Žujović N. Begović R. Srejić D. Vučelić 《The Journal of membrane biology》1999,167(3):267-274
The NMR (nuclear magnetic resonance) method of Conlon and Outhred (1972) was used to measure diffusional water permeability
of the nodal cells of the green alga Chara gymnophylla. Two local minima at 15 and 30°C of diffusional water permeability (P
d
) were observed delimiting a region of low activation energy (E
a
around 20 kJ/mol) indicative of an optimal temperature region for membrane transport processes. Above and below this region
water transport was of a different type with high E
a
(about 70 kJ/mol). The triphasic temperature dependence of the water transport suggested a channel-mediated transport at
15–30°C and lipid matrix-mediated transport beyond this region. The K+ channel inhibitor, tetraethylammonium as well as the Cl− channel inhibitor, ethacrynic acid, diminished P
d
in the intermediate temperature region by 54 and 40%, respectively. The sulfhydryl agent p-(chloromercuri-benzensulfonate)
the water transport inhibitor in erythrocytes also known to affect K+ transport in Chara, only increased P
d
below 15°C. In high external potassium (`K-state') water transport minima were pronounced. The role of K+ channels as sensors of the optimal temperature limits was further emphasized by showing a similar triphasic temperature dependence
of the conductance of a single K+ channel also known to cotransport water, which originated from cytoplasmic droplets (putatively tonoplast) of C. gymnophylla. The minimum of K+ single channel conductance at around 15°C, unlike the one at 30°C, was sensitive to changes of growth temperature underlining
membrane lipid involvement. The additional role of intracellular (membrane?) water in the generation of discontinuities in
the above thermal functions was suggested by an Arrhenius plot of the cellular water relaxation rate which showed breaks at
13 and 29°C.
Received: 12 August 1998/Revised: 13 November 1998 相似文献
8.
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
9.
10.
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
11.
The permeation properties of KAT1, an inward rectifying potassium channel from plant cells, were investigated with different
ions in the external medium. With either K+, NH+
4 or methylammonium (MA) in the external solution, the channel, expressed in Xenopus oocytes, appeared permeable to K+ and, to a lesser extent, to NH+
4 but not to the slightly bigger, methylated analogue of NH+
4, MA. Substituting NH+
4 for K+ shifted the voltage dependency of channel activation further negative and hastened activation kinetics. This suggests that
channel operation depends on the transported substrate. In mixed solution (50 mm K+, 50 mm MA) MA inhibited K+ current in a voltage-independent manner. The maximum block did not exceed 50% of the K+ current. In contrast, when NH+
4 was the permeant ion (50 mm NH+
4, 50 mm MA) MA caused a voltage-dependent, slowly developing open channel block, achieving complete inhibition at very negative voltages.
The latter block could be partially overcome by the addition of K+ in the external solution. The data support a model in which ions, after entering the channel pore, compete with different
affinities for binding sites on their permeation pathway.
Received: 6 October 1997/Revised: 28 January 1998 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.
J.R. Clay 《The Journal of membrane biology》1996,153(3):195-201
An increase in extracellular potassium ion concentration, K
o
, significantly slows the potassium channel deactivation rate in squid giant axons, as previously shown. Surprisingly, the
effect does not occur in all preparations which, coupled with the voltage independence of this result in preparations in which
it does occur, suggests that it is mediated at a site outside of the electric field of the channel, and that this site is
accessible to potassium ions in some preparations, but not in others. In other words, the effect does not appear to be related
to occupancy of the channel by potassium ions. This conclusion is supported by a four-barrier, three-binding site model of
single file diffusion through the channel in which one site, at most, is unoccupied by a potassium ion (single-vacancy model).
The model is consistent with current-voltage relations with various levels of K
o
, and, by definition, with multiple occupancy by K+. The model predicts that occupancy of any given site is essentially independent of K
o
(or K
i
). The effects of extracellular Rb+ and Cs+ on gating are strongly voltage dependent, and they were observed in all preparations investigated. Consequently, the mechanism
underlying these results would appear to be different from that which underlies the effect of K+ on gating. In particular, the effect of Rb+ on gating is reduced by strong hyperpolarization, which in the context of the occupancy hypothesis, is consistent with the
voltage dependence of the current-voltage relation in the presence of Rb+. The primary, novel, finding in this study is that the effects of Cs+ are counterintuitive in this regard. Specifically, the slowing of channel deactivation rate by Cs+ is also reduced by hyperpolarization, similar to the Rb+ results, whereas blockade is enhanced, which is seemingly inconsistent with the concept that occupancy of the channel by
Cs+ underlies the effect of this ion on gating. This result is further elucidated by barrier modeling of the current-voltage
relation in the presence of Cs+.
Received: 19 December 1995/Revised: 10 June 1996 相似文献
14.
The current through TOK1 (YKC1), the outward-rectifying K+ channel in Saccharomyces cerevisiae, was amplified by expressing TOK1 from a plasmid driven by a strong constitutive promoter. TOK1 so hyper-expressed could overcome the K+ auxotrophy of a mutant missing the two K+ transporters, TRK1 and TRK2. This trk1Δtrk2Δ double mutant hyperexpressing the TOK1 transgene had a higher internal K+ content than one expressing the empty plasmid. We examined protoplasts of these TOK1-hyperexpressing cells under a patch clamp. Besides the expected K+ outward current activating at membrane potential (V
m
) above the K+ equilibrium potential (E
K+
), a small inward current was consistently observed when the V
m
was slightly below E
K+
. The inward and the outward currents are similar in their activation rates, deactivation rates, ion specificities and Ba2+ inhibition, indicating that they flow through the same channel. Thus, the yeast outwardly rectifying K+ channel can take up K+ into yeast cells, at least under certain conditions.
Received: 1 October 1998/Revised: 9 December 1998 相似文献
15.
V.M. Baizabal-Aguirre S. Clemens N. Uozumi J.I. Schroeder 《The Journal of membrane biology》1999,167(2):119-125
The Arabidopsis thaliana cDNA, KAT1 encodes a hyperpolarization-activated K+ (K+
in
) channel. In the present study, we identify and characterize dominant negative point mutations that suppress K+
in
channel function. Effects of two mutations located in the H5 region of KAT1, at positions 256 (T256R) and 262 (G262K), were
studied. The co-expression of either T256R or G262K mutants with KAT1 produced an inhibition of K+ currents upon membrane hyperpolarization. The magnitude of this inhibition was dependent upon the molar ratio of cRNA for
wild-type to mutant channel subunits injected. Inhibition of KAT1 currents by the co-expression of T256R or G262K did not
greatly affect the ion selectivity of residual currents for Rb+, Na+, Li+, or Cs+. When T256R or G262K were co-expressed with a different K+ channel, AKT2, an inhibition of the channel currents was also observed. Voltage-dependent Cs+ block experiments with co-expressed wild type, KAT1 and AKT2, channels further indicated that KAT1 and AKT2 formed heteromultimers.
These data show that AKT2 and KAT1 are able to co-assemble and suggest that suppression of channel function can be pursued
in vivo by the expression of the dominant negative K
+
in
channel mutants described here.
Received: 2 July 1998/Revised: 23 October 1998 相似文献
16.
L-lactate transport mechanism across rat jejunal enterocyte was investigated using isolated membrane vesicles. In basolateral
membrane vesicles l-lactate uptake is stimulated by an inwardly directed H+ gradient; the effect of the pH difference is drastically reduced by FCCP, pCMBS and phloretin, while furosemide is ineffective.
The pH gradient effect is strongly temperature dependent. The initial rate of the proton gradient-induced lactate uptake is
saturable with respect to external lactate with a K
m
of 39.2 ± 4.8 mm and a J
max of 8.9 ± 0.7 nmoles mg protein−1 sec−1. A very small conductive pathway for l-lactate is present in basolateral membranes. In brush border membrane vesicles both Na+ and H+ gradients exert a small stimulatory effect on lactate uptake. We conclude that rat jejunal basolateral membrane contains
a H+-lactate cotransporter, whereas in the apical membrane both H+-lactate and Na+-lactate cotransporters are present, even if they exhibit a low transport rate.
Received: 22 October 1996/Revised: 11 March 1997 相似文献
17.
T.-H. Hwang D.-J. Suh H.-R. Bae S.-H. Lee J.-S. Jung 《The Journal of membrane biology》1996,154(3):251-257
To study K+ channels in the basolateral membrane of chloride-secreting epithelia, rat tracheal epithelial monolayers were cultured on
permeable filters and mounted into an Ussing chamber system. The mucosal membrane was permeabilized with nystatin (180 μg/ml)
in the symmetrical high K+ (145 mm) Ringer solution. During measurement of the macroscopic K+ conductance properties of the basolateral membrane under a transepithelial voltage clamp, we detected at least two types
of K+ currents: one is an inwardly rectifying K+ current and the other is a slowly activating outwardly rectifying K+ current. The inwardly rectifying K+ current is inhibited by Ba2+. The slowly activating K+ current was potentiated by cAMP and inhibited by clofilium, phorbol 12-myristae 13-acetate (PMA) and lowering temperature.
This is consistent with the biophysical characteristics of I
SK channel. RT-PCR analysis revealed the presence of I
SK cDNA in the rat trachea epithelia. Although 0.1 mm Ba2+ only had minimal affect on short-circuit current (I
sc) induced by cAMP in intact epithelia, 0.1 mm clofilium strongly inhibited it. These results indicate that I
SK might be important for maintaining cAMP-induced chloride secretion in the rat trachea epithelia.
Received: 1 March 1996/Revised: 5 August 1996 相似文献
18.
A large conductance, Ca2+-activated K+ channel of the BK type was examined in cultured pituitary melanotrophs obtained from adult male rats. In cell-attached recordings
the slope conductance for the BK channel was ≈190 pS and the probability (P
o
) of finding the channel in the open state at the resting membrane potential was low (<<0.1). Channels in inside-out patches
and in symmetrical 150 mm K+ had a conductance of ≈260 pS. The lower conductance in the cell-attached recordings is provisionally attributed to an intracellular
K+ concentration of ≈113 mm. The permeability sequence, relative to K+, was K+ > Rb+ (0.87) > NH+
4 (0.17) > Cs+≥ Na+ (≤0.02). The slope conductance for Rb+ was much less than for K+. Neither Na+ nor Cs+ carried measurable currents and 150 mm internal Cs+ caused a flickery block of the channel. Internal tetraethylammonium ions (TEA+) produced a fast block for which the dissociation constant at 0 mV (K
D
(0 mV)) was 50 mm. The K
D
(0 mV) for external TEA+ was much lower, 0.25 mm, and the blocking reaction was slower as evidenced by flickery open channel currents. With both internal and external TEA+ the blocking reaction was bimolecular and weakly voltage dependent. External charybdotoxin (40 nm) caused a large and reversible decrease of P
o
. The P
o
was increased by depolarization and/or by increasing the concentration of internal Ca2+. In 0.1 μm Ca2+ the half-maximal P
o
occurred at ≈100 mV; increasing Ca2+ to 1 μm shifted the voltage for the half-maximal P
o
to −75 mV. The Ca2+ dependence of the gating was approximated by a fourth power relationship suggesting the presence of four Ca2+ binding sites on the BK channel.
Received: 23 October/Revised: 15 December 1995 相似文献
19.
T.E. DeCoursey S.Y. Kim M.R. Silver F.N. Quandt 《The Journal of membrane biology》1996,152(2):141-157
Ion channel expression was studied in THP-1 human monocytic leukemia cells induced to differentiate into macrophage-like cells
by exposure to the phorbol ester, phorbol 12-myristate 13-acetate (PMA). Inactivating delayed rectifier K+ currents, I
DR, present in almost all undifferentiated THP-1 monocytes, were absent from PMA-differentiated macrophages. Two K+ channels were observed in THP-1 cells only after differentiation into macrophages, an inwardly rectifying K+ channel (I
IR) and a Ca2+-activated maxi-K channel (I
BK). I
IR was a classical inward rectifier, conducting large inward currents negative to E
K and very small outward currents. I
IR was blocked in a voltage-dependent manner by Cs+, Na+, and Ba2+, block increasing with hyperpolarization. Block by Na+ and Ba2+ was time-dependent, whereas Cs+ block was too fast to resolve. Rb+ was sparingly permeant. In cell-attached patches with high [K+] in the pipette, the single I
IR channel conductance was ∼30 pS and no outward current could be detected. I
BK channels were observed in cell-attached or inside-out patches and in whole-cell configuration. In cell-attached patches the
conductance was ∼200–250 pS and at potentials positive to ∼100 mV a negative slope conductance of the unitary current was
observed, suggesting block by intracellular Na+. I
BK was activated at large positive potentials in cell-attached patches; in inside-out patches the voltage-activation relationship
was shifted to more negative potentials by increased [Ca2+]. Macroscopic I
BK was blocked by external TEA+ with half block at 0.35 mm. THP-1 cells were found to contain mRNA for Kv1.3 and IRK1. Levels of mRNA coding for these K+ channels were studied by competitive PCR (polymerase chain reaction), and were found to change upon differentiation in the
same direction as did channel expression: IRK1 mRNA increased at least 5-fold, and Kv1.3 mRNA decreased on average 7-fold.
Possible functional correlates of the changes in ion channel expression during differentiation of THP-1 cells are discussed.
Received: 19 September 1995/Revised: 14 March 1996 相似文献
20.
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