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1.
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
2.
Properties of large conductance Ca2+-activated K+ channels were studied in the soma of motoneurones visually identified in thin slices of neonatal rat spinal cord. The channels
had a conductance of 82 ± 5 pS in external Ringer solution (5.6 mm K+
o
//155 mm K+
i
) and 231 ± 4 pS in external high-K
o
solution (155 mm K+
o
//155 mm K+
i
). The channels were activated by depolarization and by an increase in internal Ca2+ concentration. Potentials of half-maximum channel activation (E50) were −13, −34, −64 and −85 mV in the presence of 10−6, 10−5, 10−4 and 10−3
m internal Ca2+, respectively. Using an internal solution containing 10−4
m Ca2+, averaged KCa currents showed fast activation within 2–3 msec after a voltage step to +50 mV. Averaged KCa currents did not inactivate during 400 msec voltage pulses. External TEA reduced the apparent single-channel amplitude with
a 50% blocking concentration (IC50) of 0.17 ± 0.02 mm. KCa channels were completely suppressed by externally applied 100 mm charybdotoxin. It is concluded that KCa channels activated by Ca2+ entry during the action potential play an important role in the excitability of motoneurones.
Received: 7 November 1996/Revised: 29 October 1997 相似文献
3.
An amiloride-sensitive, Ca2+-activated nonselective cation (NSC) channel in the apical membrane of fetal rat alveolar epithelium plays an important role
in stimulation of Na+ transport by a beta adrenergic agonist (beta agonist). We studied whether Ca2+ has an essential role in the stimulation of the NSC channel by beta agonists. In cell-attached patches formed on the epithelium,
terbutaline, a beta agonist, increased the open probability (P
o
) of the NSC channel to 0.62 ± 0.07 from 0.03 ± 0.01 (mean ±se; n= 8) 30 min after application of terbutaline in a solution containing 1 mm Ca2+. The P
o
of the terbutaline-stimulated NSC channel was diminished in the absence of extracellular Ca2+ to 0.26 ± 0.05 (n= 8). The cytosolic Ca2+ concentration ([Ca2+]
c
) in the presence and absence of extracellular Ca2+ was, respectively, 100 ± 6 and 20 ± 2 nm (n= 7) 30 min after application of terbutaline. The cytosolic Cl− concentration ([Cl−]
c
) in the presence and absence of extracellular Ca2+ was, respectively, 20 ± 1 and 40 ± 2 mm (n= 7) 30 min after application of terbutaline. The diminution of [Ca2+]
c
from 100 to 20 nm itself had no significant effects on the P
o
if the [Cl−]
c
was reduced to 20 mm; the P
o
was 0.58 ± 0.10 at 100 nm [Ca2+]
c
and 0.55 ± 0.09 at 20 nm [Ca2+]
c
(n= 8) with 20 mm [Cl−]
c
in inside-out patches. On the other hand, the P
o
(0.28 ± 0.10) at 20 nm [Ca2+]
c
with 40 mm [Cl−]
c
was significantly lower than that (0.58 ± 0.10; P < 0.01; n= 8) at 100 nm [Ca2+]
c
with 20 mm [Cl−]
c
, suggesting that reduction of [Cl−]
c
is an important factor stimulating the NSC channel. These observations indicate that the extracellular Ca2+ plays an important role in the stimulatory action of beta agonist on the NSC channel via reduction of [Cl−]
c
.
Received: 11 August 2000/Revised: 4 December 2000 相似文献
4.
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
5.
The Ca2+-activated maxi K+ channel was found in the apical membrane of everted rabbit connecting tubule (CNT) with a patch-clamp technique. The mean
number of open channels (NP
o
) was markedly increased from 0.007 ± 0.004 to 0.189 ± 0.039 (n= 7) by stretching the patch membrane in a cell-attached configuration. This activation was suggested to be coupled with the
stretch-activation of Ca2+-permeable cation channels, because the maxi K+ channel was not stretch-activated in both the cell-attached configuration using Ca2+-free pipette and in the inside-out one in the presence of 10 mm EGTA in the cytoplasmic side. The maxi K+ channel was completely blocked by extracellular 1 μm charybdotoxin (CTX), but was not by cytoplasmic 33 μm arachidonic acid (AA). On the other hand, the low-conductance K+ channel, which was also found in the same membrane, was completely inhibited by 11 μm AA, but not by 1 μm CTX. The apical K+ conductance in the CNT was estimated by the deflection of transepithelial voltage (ΔV
t
) when luminal K+ concentration was increased from 5 to 15 mEq. When the tubule was perfused with hydraulic pressure of 0.5 KPa, the ΔV
t
was only −0.7 ± 0.4 mV. However, an increase in luminal fluid flow by increasing perfusion pressure to 1.5 KPa markedly enhanced
ΔV
t
to −9.4 ± 0.9 mV. Luminal application of 1 μm CTX reduced the ΔV
t
to −1.3 ± 0.6 mV significantly in 6 tubules, whereas no significant change of ΔV
t
was recorded by applying 33 μm AA into the lumen of 5 tubules (ΔV
t
=−7.2 ± 0.5 mV in control vs.ΔV
t
=−6.7 ± 0.6 mV in AA). These results suggest that the Ca2+-activated maxi K+ channel is responsible for flow-dependent K+ secretion by coupling with the stretch-activated Ca2+-permeable cation channel in the rabbit CNT.
Received: 21 August 1997/Revised: 20 March 1998 相似文献
6.
The effect of extracellular cation concentration and membrane voltage on the current carried by outward-rectifying K+ channels was examined in stomatal guard cells of Vicia faba L. Intact guard cells were impaled with double-barrelled microelectrodes and the K+ current was monitored under voltage clamp in 0.1–30 mm K+ and in equivalent concentrations of Rb+, Cs+ and Na+. From a conditioning voltage of −200 mV, clamp steps to voltages between −150 and +50 mV in 0.1 mm K+ activated current through outward-rectifying K+ channels (I
K,
out) at the plasma membrane in a voltage-dependent fashion. Increasing [K+]
o
shifted the voltage-sensitivity of I
K,
out in parallel with the equilibrium potential for K+ across the membrane. A similar effect of [K+]
o
was evident in the kinetics of I
K,
out activation and deactivation, as well as the steady-state conductance- (g
K
−) voltage relations. Linear conductances, determined as a function of the conditioning voltage from instantaneous I-V curves, yielded voltages for half-maximal conductance near −130 mV in 0.1 mm K+, −80 mV in 1.0 mm K+, and −20 mV in 10 mm K+. Similar data were obtained with Rb+ and Cs+, but not with Na+, consistent with the relative efficacy of cation binding under equilibrium conditions (K+≥ Rb+ > Cs+ > > Na+). Changing Ca2+ or Mg2+ concentrations outside between 0.1 and 10 mm was without effect on the voltage-dependence of g
K
or on I
K,
out activation kinetics, although 10 mm [Ca2+]
o
accelerated current deactivation at voltages negative of −75 mV. At any one voltage, increasing [K+]
o
suppressed g
K
completely, an action that showed significant cooperativity with a Hill coefficient of 2. The apparent affinity for K+ was sensitive to voltage, varying from 0.5 to 20 mm with clamp voltages near −100 to 0 mV, respectively. These, and additional data indicate that extracellular K+ acts as a ligand and alters the voltage-dependence of I
K,
out gating; the results implicate K+-binding sites accessible from the external surface of the membrane, deep within the electrical field, but distinct from the
channel pore; and they are consistent with a serial 4-state reaction-kinetic model for channel gating in which binding of
two K+ ions outside affects the distribution between closed states of the channel.
Received: 27 November 1996/Revised: 4 March 1997 相似文献
7.
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
8.
Melanoma cells are transformed melanocytes of neural crest origin. K+ channel blockers have been reported to inhibit melanoma cell proliferation. We used whole-cell recording to characterize
ion channels in four different human melanoma cell lines (C8161, C832C, C8146, and SK28). Protocols were used to identify
voltage-gated (KV), Ca2+-activated (KCa), and inwardly rectifying (KIR) K+ channels; swelling-sensitive Cl− channels (Clswell); voltage-gated Ca2+ channels (CaV) and Ca2+ channels activated by depletion of intracellular Ca2+ stores (CRAC); and voltage-gated Na+ channels (NaV). The presence of Ca2+ channels activated by intracellular store depletion was further tested using thapsigargin to elicit a rise in [Ca2+]
i
. The expression of K+ channels varied widely between different cell lines and was also influenced by culture conditions. KIR channels were found in all cell lines, but with varying abundance. Whole-cell conductance levels for KIR differed between C8161 (100 pS/pF) and SK28 (360 pS/pF). KCa channels in C8161 cells were blocked by 10 nm apamin, but were unaffected by charybdotoxin (CTX). KCa channels in C8146 and SK28 cells were sensitive to CTX (K
d
= 4 nm), but were unaffected by apamin. KV channels, found only in C8146 cells, activated at ∼−20 mV and showed use dependence. All melanoma lines tested expressed
CRAC channels and a novel Clswell channel. Clswell current developed at 30 pS/sec when the cells were bathed in 80% Ringer solution, and was strongly outwardly rectifying (4:1
in symmetrical Cl−). We conclude that different melanoma cell lines express a diversity of ion channel types.
Received: 2 April 1996/Revised: 22 August 1996 相似文献
9.
Smooth muscle cells isolated from the secondary and tertiary branches of the rabbit mesenteric artery contain large Ca2+-dependent channels. In excised patches with symmetrical (140 mm) K+ solutions, these channels had an average slope conductance of 235 ± 3 pS, and reversed in direction at −6.1 ± 0.4 mV. The
channel showed K+ selectivity and its open probability (P
o
) was voltage-dependent. Iberiotoxin (50 nm) reversibly decreased P
o
, whereas tetraethylammonium (TEA, at 1 mm) reduced the unitary current amplitude. Apamin (200 nm) had no effect. The channel displayed sublevels around 1/3 and 1/2 of the mainstate level. The effect of [Ca2+] on P
o
was studied and data fitted to Boltzmann relationships. In 0.1, 0.3, 1.0 and 10 μm Ca2+, V
1/2 was 77.1 ± 5.3 (n= 18), 71.2 ± 4.8 (n= 16), 47.3 ± 10.1 (n= 11) and −14.9 ± 10.1 mV (n= 6), respectively. Values of k obtained in 1 and 10 μm [Ca2+] were significantly larger than that observed in 0.1 μm [Ca2+]. With 30 μm NS 1619 (a BKCa channel activator), V
1/2 values were shifted by 39 mV to the left (hyperpolarizing direction) and k values were not affected. TEA applied intracellularly, reduced the unitary current amplitude with a K
d
of 59 mm. In summary, BKCa channels show a particularly weak sensitivity to intracellular TEA and they also display large variation in V
1/2 and k. These findings suggest the possibility that different types (isoforms) of BKCa channels may exist in this vascular tissue.
Received: 22 December 1997/Revised: 27 March 1998 相似文献
10.
The current-voltage (I/V) profiles of Ventricaria (formerly Valonia) membranes were measured at a range of external potassium concentrations, [K+]
o
, from 0.1 to 100 mm. The conductance-voltage (G/V) characteristics were computed to facilitate better resolution of the profile change with time after exposure to different
[K+]
o
. The resistance-voltage (R/V) characteristics were computed to attempt resolution of plasmalemma and tonoplast. Four basic electrophysiological stages
emerged: (1) Uniform low resistance between −60 and +60 mV after the cell impalement. (2) High resistance between +50 and
+150 for [K+]
o
from 0.1 to 1.0 mm and hypotonic media. (3) High resistance between −150 and −20 mV for [K+]
o
of 10 mm (close to natural seawater) and hypertonic media. (4) High resistance between −150 and +170 mV at [K+]
o
of 100 mm.
The changes between these states were slow, requiring minutes to hours and sometimes exhibiting spontaneous oscillations of
the membrane p.d. (potential difference). Our analysis of the I/V data supports a previous hypothesis, that Ventricaria tonoplast is the more resistive membrane containing a pump, which transports K+ into the vacuole to regulate turgor. We associate state (1) with the plasmalemma conductance being dominant and the K+ pump at the tonoplast short-circuited probably by a K+ channel, state (2) with the K+ pump ``off' or short-circuited at p.d.s more negative than +50 mV, state (3) with the K+ pump ``on,' and state (4) with the pump dominant, but affected by high K+. A model for the Ventricaria membrane system is proposed.
Received: 5 November 1998/Revised: 11 May 1999 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.
T. Miyashita H. Tatsumi H. Furuta N. Mori M. Sokabe 《The Journal of membrane biology》2001,182(2):113-122
We identified a Ca2+-sensitive cation channel in acutely dissociated epithelial cells from the endolymphatic sac (ES) of guinea pigs using the
patch-clamp technique. Single-channel recordings showed that the cation channel had a conductance of 24.0 ± 1.3 pS (n= 8) in our standard solution. The relative ionic permeability of the channel was in the order K+= Na+ > Ca2+≫ Cl−. This channel was weakly voltage-dependent but was strongly activated by Ca2+ on the cytosolic side at a concentration of around 1 mm in inside-out excised patches. With cell-attached patches, however, the channel was activated by much lower Ca2+ concentrations. Treatment of the cells, under cell-attached configuration, with ionomycin (10 μm), carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 20 μm), or ATP (1 mm), which increased intracellular Ca2+ concentration ([Ca2+]i), activated the channel at an estimated [Ca2+]i from 0.6 μm to 10 μm. It is suggested that some activators of the channel were deteriorated or washed out during the formation of excised patches.
Based on this Ca2+ sensitivity, we speculated that the channel contributes to the regulation of ionic balance and volume of the ES by absorbing
Na+ under certain pathological conditions that will increase [Ca2+]i. This is the first report of single-channel recordings in endolymphatic sac epithelial cells.
Received: 24 October 2000/Revised: 10 April 2001 相似文献
14.
It has previously been shown that osmotic cell shrinkage activates a nonselective cation (NSC) channel in M-1 mouse cortical
collecting duct cells [54] and in a variety of other cell types [20]. In the present study we further characterized the shrinkage-activated
NSC channel in M-1 cells and its mechanism of activation using whole-cell current recordings. Osmotic cell shrinkage induced
by addition of 100 mm sucrose to the bath solution caused a 20-fold increase in whole-cell inward currents from −10.8 ± 1.5 pA to −211 ± 10.2 pA
(n= 103). A similar response was observed when cell shrinkage was elicited using a hypo-osmotic pipette solution. This indicates
that cell shrinkage and not extracellular osmolarity per se is the signal for current activation. Cation substitution experiments revealed that the activated channels discriminate poorly
between monovalent cations with a selectivity sequence NH4 (1.2) ≥ Na+ (1) ≈ K+ (0.9) ≈ Li+ (0.9). In contrast there was no measurable permeability for Ca2+ or Ba2+ and the cation-to-anion permeability ratio was about 14. The DPC-derivatives flufenamic acid, 4-methyl-DPC and DCDPC were
the most effective blockers followed by LOE 908, while amiloride and bumetanide were ineffective. The putative channel activator
maitotoxin had no effect. Current activation was dependent upon the presence of intracellular ATP and Mg2+ and was inhibited by staurosporine (1 μm) and calphostin C (1 μm). Moreover, cytochalasin D (10 μm) and taxol (2 μm) reduced the current response to cell shrinkage. These findings suggest that the activation mechanism of the shrinkage-activated
NSC channel involves protein kinase mediated phosphorylation steps and cytoskeletal elements.
Received: 3 May 2000/Revised: 6 July 2000 相似文献
15.
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
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.
We studied the characteristics of the basal and antidiuretic hormone (arginine vasotocin, AVT)-activated whole cell currents
of an aldosterone-treated distal nephron cell line (A6) at two different cytosolic Ca2+ concentrations ([Ca2+]
c
, 2 and 30 nm). A6 cells were cultured on a permeable support filter for 10 ∼ 14 days in media with supplemental aldosterone (1 μm). At 30 nm [Ca2+]
c
, basal conductances mainly consisted of Cl− conductances, which were sensitive to 5-nitro-2-(3-phenylpropylamino)-benzoate. Reduction of [Ca2+]
c
to 2 nm abolished the basal Cl− conductance. AVT evoked Cl− conductances at 2 as well as 30 nm [Ca2+]
c
. In addition to Cl− conductances, AVT induced benzamil-insensitive nonselective cation (NSC) conductances. This action on NSC conductances was
observed at 30 nm [Ca2+]
c
but not at 2 nm [Ca2+]
c
. Thus, cytosolic Ca2+ regulates NSC and Cl− conductances in a distal nephron cell line (A6) in response to AVT. Keeping [Ca2+]
c
at an adequate level seems likely to be an important requirement for AVT regulation of ion conductances in aldosterone-treated
A6 cells.
Received: 6 May 1996/Revised: 28 June 1996 相似文献
18.
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
19.
The reactive disulfide 4,4′-dithiodipyridine (4,4′DTDP) was added to single cardiac ryanodine receptors (RyRs) in lipid bilayers.
The activity of native RyRs, with cytoplasmic (cis) [Ca2+] of 10−7
m (in the absence of Mg2+ and ATP), increased within ∼1 min of addition of 1 mm 4,4′-DTDP, and then irreversibly ceased 5 to 6 min after the addition. Channels, inhibited by either 1 mm
cis Mg2+ (10−7
m
cis Ca2+) or by 10 mm
cis Mg2+ (10−3
m
cis Ca2+), or activated by 4 mm ATP (10−7
m
cis Ca2+), also responded to 1 mm
cis 4,4′-DTDP with activation and then loss of activity. P
o
and mean open time (T
o
) of the maximally activated channels were lower in the presence of Mg2+ than in its absence, and the number of openings within the long time constant components of the open time distribution was
reduced. In contrast to the reduced activation by 1 mm 4,4′-DTDP in channels inhibited by Mg2+, and the previously reported enhanced activation by 4,4′-DTDP in channels activated by Ca2+ or caffeine (Eager et al., 1997), the activation produced by 1 mm
cis 4,4′-DTDP was the same in the presence and absence of ATP. These results suggest that there is a physical interaction between
the ATP binding domain of the cardiac RyR and the SH groups whose oxidation leads to channel activation.
Received: 8 September 1997/Revised: 20 January 1998 相似文献
20.
We have investigated the interaction of two peptides (ShB — net charge +3 and ShB:E12KD13K — net charge +7) derived from the NH2-terminal domain of the Shaker K+ channel with purified, ryanodine-modified, cardiac Ca2+-release channels (RyR). Both peptides produced well resolved blocking events from the cytosolic face of the channel. At a
holding potential of +60 mV the relationship between the probability of block and peptide concentration was described by a
single-site binding scheme with 50% saturation occurring at 5.92 ± 1.06 μm for ShB and 0.59 ± 0.14 nm for ShB:E12KD13K. The association rates of both peptides varied with concentration (4.0 ± 0.4 sec−1μm
−1 for ShB and 2000 ± 200 sec−1μm
−1 for ShB:E12KD13K); dissociation rates were independent of concentration. The interaction of both peptides was influenced by applied
potential with the bulk of the voltage-dependence residing in Koff. The effectiveness of the inactivation peptides as blockers of RyR is enhanced by an increase in net positive charge. As
is the case with inactivation and block of K+ channels, this is mediated by a large increase in Kon. These observations are consistent with the proposal that the conduction pathway of RyR contains negatively charged sites
which will contribute to the ion handling properties of this channel.
Received: 15 December 1997/Revised: 13 March 1998 相似文献