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1.
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   

2.
K+ channels, membrane voltage, and intracellular free Ca2+ are involved in regulating proliferation in a human melanoma cell line (SK MEL 28). Using patch-clamp techniques, we found an inwardly rectifying K+ channel and a calcium-activated K+ channel. The inwardly rectifying K+ channel was calcium independent, insensitive to charybdotoxin, and carried the major part of the whole-cell current. The K+ channel blockers quinidine, tetraethylammonium chloride and Ba2+ and elevated extracellular K+ caused a dose-dependent membrane depolarization. This depolarization was correlated to an inhibition of cell proliferation. Charybdotoxin affected neither membrane voltage nor proliferation. Basic fibroblast growth factor and fetal calf serum induced a transient peak in intracellular Ca2+ followed by a long-lasting Ca2+ influx. Depolarization by voltage clamp decreased and hyperpolarization increased intracellular Ca2+, illustrating a transmembrane flux of Ca2+ following its electrochemical gradient. We conclude that K+ channel blockers inhibit cell-cycle progression by membrane depolarization. This in turn reduces the driving force for the influx of Ca2+, a messenger in the mitogenic signal cascade of human melanoma cells. Received: 9 May 1995/Revised: 30 January 1996  相似文献   

3.
Previous reports have indicated that Plasmodium falciparum-infected red cells (pRBC) have an increased Ca2+ permeability. The magnitude of the increase is greater than that normally required to activate the Ca2+-dependent K+ channel (K Ca channel) of the red cell membrane. However, there is evidence that this channel remains inactive in pRBC. To clarify this discrepancy, we have reassessed both the functional status of the K Ca channel and the Ca2+ permeability properties of pRBC. For pRBC suspended in media containing Ca2+, K Ca channel activation was elicited by treatment with the Ca2+ ionophore A23187. In the absence of ionophore the channel remained inactive. In contrast to previous claims, the unidirectional influx of Ca2+ into pRBC in which the Ca2+ pump was inhibited by vanadate was found to be within the normal range (30–55 μmol (1013 cells · hr)−1), provided the cells were suspended in glucose-containing media. However, for pRBC in glucose-free media the Ca2+ influx increased to over 1 mmol (1013 cells · hr)−1, almost an order of magnitude higher than that seen in uninfected erythrocytes under equivalent conditions. The pathway responsible for the enhanced influx of Ca2+ into glucose-deprived pRBC was expressed at approximately 30 hr post-invasion, and was inhibited by Ni2+. Possible roles for this pathway in pRBC are considered. Received: 12 May 1999/Revised: 8 July 1999  相似文献   

4.
The calcium indicator fura-2 was used to study the effect of hypotonic solutions on the intracellular calcium concentration, [Ca2+] i , in a human osteoblast-like cell line. Decreasing the tonicity of the extracellular solution to 50% leads to an increase in [Ca2+] i from ∼150 nm up to 1.3 μm. This increase in [Ca2+] i was mainly due to an influx of extracellular Ca2+ since removing of extracellular Ca2+ reduced this increase to ∼250 nm. After cell swelling most of the cells were able to regulate their volume to the initial level within 800 sec. The whole-cell recording mode of the patch-clamp technique was also used to study the effect of an increase in [Ca2+] i on membrane currents in these cells. An increase in [Ca2+] i revealed two types of Ca2+-activated K+ channels, K(Ca) channels. Current through both channel types could not be observed below voltage of +80 mV with [Ca2+] i buffered to 100 nm or less. With patch-electrodes filled with solutions buffering [Ca2+] i to 10 μm both channels types could be readily observed. The activation of the first type was apparently voltage-independent since current could be observed over the entire voltage range used from −160 to +100 mV. In addition, the current was also blocked by charybdotoxin (CTX). The second type of K(Ca) channels in these cells could be activated with depolarizations more positive than −40 mV from a holding potential of −80 mV. This type was blocked by CTX and paxilline. Adding paxilline to the extracellular solution inhibited regulatory volume decrease (RVD), but could not abolish RVD. We conclude that two K(Ca) channel types exist in human osteoblasts, an intermediate conductance K(Ca) channel and a MaxiK-like K(Ca) channel. MaxiK channels might get activated either directly or by an increase in [Ca2+] i elicited through hypotonic solutions. In combination with the volume-regulated Cl conductance in the same cells this K+ channel seems to play a vital role in volume regulation in human osteoblasts. Received: 8 February 2000/Revised: 13 July 2000  相似文献   

5.
Human aortic endothelial cells (HAEC) respond to flow with Ca2+ entry, activation of a nonselective cation channel, activation of a chloride channel, and activation of a calcium-activated potassium channel. Conversely, human capillary endothelial cells were unaffected by similar flow rates. In HAEC the flow induced cytosolic free calcium increase ([Ca2+] i ) and the ionic currents associated with it were sustained for up to 15 min after perfusion was stopped. In the absence of extracellular Ca2+, fluid flow was unable to evoke the [Ca2+] i increase or the increase in membrane currents but the response could be restored by addition of extracellular Ca2+. Surprisingly, the flow response was inhibited in 50% of the cells by inhibitors of nitric oxide production. The results suggest that the sustained flow response in HAEC may be partially mediated by nitric oxide production and release. Received: 29 January 1999/Revised: 2 June 1999  相似文献   

6.
Patients with cerebral tumors often present with elevated levels of acetylcholine (ACh) in their cerebrospinal fluid. This motivated us to investigate physiological effects of ACh on cultured human astrocytoma cells (U373) using a combination of videomicroscopy, calcium microspectrofluorimetry and perforated patch-clamp recording. Astrocytoma cells exhibited the typical morphological changes associated with cell migration; polarized cells displayed prominent lamellipodia and associated membrane ruffling at the anterior of the cell, and a long tail region that periodically contracted into the cell body as the cell moved forward. Bath application of the ACh receptor agonist, muscarine, reversibly inhibited cell migration. In conjunction with this inhibition, ACh induced a dose-dependent, biphasic increase in resting intracellular free calcium concentration ([Ca2+] i ) associated with periodic Ca2+ oscillations during prolonged ACh applications. The early transient rise in [Ca2+] i was abolished by ionomycin and thapsigargin but was insensitive to caffeine and ryanodine while the plateau phase was strictly dependent on external calcium. The Ca2+ response to ACh was mimicked by muscarine and abolished by the muscarinic antagonists, atropine or 4-DAMP, but not by pirenzepine. Using perforated patch-clamp recordings combined with fluorescent imaging, we demonstrated that ACh-induced [Ca2+] i oscillations triggered membrane voltage oscillations that were due to the activation of voltage-dependent, Ca2+-sensitive K+ currents. These K+ currents were blocked by intracellular injection of EGTA, or by extracellular application of TEA, quinine, or charybdotoxin, but not by apamin. These studies suggest that activation of muscarinic receptors on glioma cells induce the release of Ca2+ from intracellular stores which in turn activate Ca2+-dependent (BK-type) K+ channels. Furthermore, this effect was associated with inhibition of cell migration, suggesting an interaction of this pathway with glioma cell migration. Received: 17 December/Revised: 17 March 2000  相似文献   

7.
Large Conductance Ca2+-Activated K+ Channels in Human Meningioma Cells   总被引:2,自引:0,他引:2  
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   

8.
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   

9.
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   

10.
A new type of nonselective cation channel was identified and characterized in pheochromocytoma (PC12) cells using inside-out and cell-attached patch-clamp recordings. The channel shows a large unitary conductance (274 pS in symmetric 145 mm K+) and selectivity for Na+≈ K+ > Li+, and is practically impermeable to Cl. The channel activity-voltage relationship is bell-shaped, showing maximal activation at ≈−10 mV. The overall activity of this channel is unmodified by [Na+] ic , or [Ca++] ic . However, increases in [Ca++] ic lead to a decrease in the unitary current amplitude. In addition, overall activity is mildly increased when suction is applied to the back of the patch pipette. Together, these characteristics distinguish the present channel from all other large conductance nonselective cation channels reported so far in a variety of preparations. The frequency of appearance of this channel type is similar in undifferentiated and NGF-treated PC12 cells (≈8–27% of patches). The combination of large conductance, permeability to Na+, and existence of conducting states at negative potentials, may provide a significant pathway for inward current and depolarization in PC12 cells. Received: 14 February 1997/Revised: 28 July 1997  相似文献   

11.
2-Methoxyestradiol, an endogenous metabolite of 17β-estradiol, is known to have antitumor and antiangiogenic actions. The effects of 2-methoxyestradiol on ionic currents were investigated in an endothelial cell line (HUV-EC-C) originally derived from human umbilical vein. In the whole-cell patch-clamp configuration, 2-methoxyestradiol (0.3–30 μm) reversibly suppressed the amplitude of K+ outward currents. The IC 50 value of the 2-methoxyestradiol-induced decrease in outward current was 3 μm. Evans blue (30 μm) or niflumic acid (30 μm), but not diazoxide (30 μm), reversed the 2-methoxyestradiol-induced decrease in outward current. In the inside-out configuration, application of 2-methoxyestradiol (3 μm) to the bath did not modify the single-channel conductance of large-conductance Ca2+-activated K+ (BKCa) channels; however, it did suppress the channel activity. 2-Methoxyestradiol (3 μm) produced a shift in the activation curve of BKCa channels to more positive potentials. Kinetic studies showed that the 2-methoxyestradiol-induced inhibition of BKCa channels is primarily mediated by a decrease in the number of long-lived openings. 2-Methoxyestradiol-induced inhibition of the channel activity was potentiated by membrane stretch. In contrast, neither 17β-estradiol (10 μm) nor estriol (10 μm) affected BKCa channel activity, whereas 2-hydroxyestradiol (10 μm) slightly suppressed it. Under current-clamp condition, 2-methoxyestradiol (10 μm) caused membrane depolarization and Evans blue (30 μm) reversed 2-methoxyestradiol-induced depolarization. The present study provides evidence that 2-methoxyestradiol can suppress the activity of BKCa channels in endothelial cells. These effects of 2-methoxyestradiol on ionic currents may contribute to its effects on functional activity of endothelial cells. Received: 27 November 2000/Revised: 13 April 2001  相似文献   

12.
The pharmacological profile of a voltage-independent Ca2+-activated potassium channel of intermediate conductance (IK(Ca2+)) present in bovine aortic endothelial cells (BAEC) was investigated in a series of inside-out and outside-out patch-clamp experiments. Channel inhibition was observed in response to external application of ChTX with a half inhibition concentration of 3.3 ± 0.3 nm (n= 4). This channel was insensitive to IbTX, but channel block was detected following external application of MgTX and StK leading to the rank order toxin potency ChTX > StK > MgTX >>IbTX. A reduction of the channel unitary current amplitude was also measured in the presence of external TEA, with half reduction occurring at 23 ± 3 mm TEA (n= 3). The effect of TEA was voltage insensitive, an indication that TEA may bind to a site located on external side of the pore region of this channel. Similarly, the addition of d-TC to the external medium caused a reduction of the channel unitary current amplitude with half reduction at 4.4 ± 0.3 mm (n= 4). In contrast, application of d-TC to the bathing medium in inside-out experiments led to the appearance of long silent periods, typical of a slow blocking process. Finally, the IK(Ca2+) in BAEC was found to be inhibited by NS1619, an activator of the Ca2+-activated potassium channel of large conductance (Maxi K(Ca2+)), with a half inhibition value of 11 ± 0.8 μm (n= 4). These results provide evidence for a pharmacological profile distinct from that reported for the Maxi K(Ca2+) channel, with some features attributed to the voltage-gated KV1.2 potassium channel. Received: 6 November 1997/Revised: 19 February 1998  相似文献   

13.
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   

14.
Human capillary endothelial cells (HCEC) in normal media contain noninactivating outwardly rectifying chloride currents, TEA-sensitive delayed rectifier K+ currents and an inward rectifier K+ current. Two additional ionic currents are induced in HCEC when the media are allowed to become conditioned: A Ca2+-activated K+ current (BKCA) that is sensitive to iberiotoxin is induced in 23.5% of the cells, a transient 4-AP-sensitive K+ current (A current) is induced in 24.7% of the cells, and in 22.3% of the cells both the transient and BKCA currents are coinduced. The EC50 for Ca2+ activation of the BKCA current in HCEC from conditioned media is 213 nM. RNA message for BKCA (hSlo clone) is undetecable after PCR amplification in control cells but is seen in those from conditioned cells. The induction of BKCA current is not blocked by conditioning with inhibitors of nitric oxide synthase, cyclo-oxgenase or lypo-oxygenase pathways. Apparently the characteristics of human endothelial cells are highly malleable and can be easily modified by their local environment. Received: 21 May 1998/Revised: 23 September 1998  相似文献   

15.
In our previous studies on sheep parotid secretory cells, we showed that the K+ current evoked by acetylcholine (ACh) was not carried by the high-conductance voltage- and Ca2+-activated K+ (BK) channel which is so conspicuous in unstimulated cells, notwithstanding that the BK channel is activated by ACh. Since several studies from other laboratories had suggested that the BK channel did carry the ACh-evoked K+ current in the secretory cells of the mouse mandibular gland, and that the current could be blocked with tetraethylammonium (TEA), a known blocker of BK channels, we decided to investigate the ACh-evoked K+ current in mouse cells more closely. We studied whether the ACh-evoked K+ current in the mouse is inhibited by TEA and quinine. Using the whole-cell patch-clamp technique and microspectrofluorimetric measurement of intracellular Ca2+, we found that TEA and quinine do inhibit the ACh-evoked K+ current but that the effect is due to inhibition of the increase in intracellular Ca2+ evoked by ACh, not to blockade of a K+ conductance. Furthermore, we found that the K+ conductance activated when ionomycin is used to increase intracellular free Ca2+ was inhibited only by quinine and not by TEA. We conclude that the ACh-evoked K+ current in mouse mandibular cells does not have the blocker sensitivity pattern that would be expected if it were being carried by the high-conductance, voltage- and Ca2+-activated K+ (BK) channel. The properties of this current are, however, consistent with those of a 40 pS K+ channel that we have reported to be activated by ACh in these cells [16]. Received: 9 January 1996/Revised: 17 April 1996  相似文献   

16.
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   

17.
Effects of the extracellular Ca2+ concentration ([Ca2+] o ) on whole cell membrane currents were examined in mouse osteoclastic cells generated from bone marrow/stromal cell coculture. The major resting conductance in the presence of 1 mm Ca2+ was mediated by a Ba2+-sensitive, inwardly rectifying K+ (IRK) current. A rise in [Ca2+] o (5–40 mm) inhibited the IRK current and activated an 4,4′-diisothiocyano-2,2′-stilbenedisulfonate (DIDS)-sensitive, outwardly rectifying Cl (ORCl) current. The activation of the ORCl current developed slowly and needed higher [Ca2+] o than that required to inhibit the IRK current. The inhibition of the IRK current consisted of two components, initial and subsequent late phases. The initial inhibition was not affected by intracellular application of guanosine 5′-O-(3-thiotriphosphate) (GTPγS) or guanosine 5′-O-(2-thiodiphosphate) (GDPβS). The late inhibition, however, was enhanced by GTPγS and attenuated by GDPβS, suggesting that GTP-binding proteins mediate this inhibition. The activation of the ORCl current was suppressed by pretreatment with pertussis toxin, but not potentiated by GTPγS. An increase in intracellular Ca2+ level neither reduced the IRK current nor activated the ORCl current. Staurosporine, an inhibitor for protein kinase C, did not modulate the [Ca2+] o -induced changes in the IRK and ORCl conductances. These results suggest that high [Ca2+] o had a dual action on the membrane conductance of osteoclasts, an inhibition of an IRK conductance and an activation of an ORCl conductance. The two conductances modulated by [Ca2+] o may be involved in different phases of bone resorption because they differed in Ca2+ sensitivity, temporal patterns of changes and regulatory mechanisms. Received: 28 May 1996/Revised: 28 January 1997  相似文献   

18.
The K+ channel KCNQ1 (KVLQT1) is a voltage-gated K+ channel, coexpressed with regulatory subunits such as KCNE1 (IsK, mink) or KCNE3, depending on the tissue examined. Here, we investigate regulation and properties of human and rat KCNQ1 and the impact of regulators such as KCNE1 and KCNE3. Because the cystic fibrosis transmembrane conductance regulator (CFTR) has also been suggested to regulate KCNQ1 channels we studied the effects of CFTR on KCNQ1 in Xenopus oocytes. Expression of both human and rat KCNQ1 induced time dependent K+ currents that were sensitive to Ba2+ and 293B. Coexpression with KCNE1 delayed voltage activation, while coexpression with KCNE3 accelerated current activation. KCNQ1 currents were activated by an increase in intracellular cAMP, independent of coexpression with KCNE1 or KCNE3. cAMP dependent activation was abolished in N-terminal truncated hKCNQ1 but was still detectable after deletion of a single PKA phosphorylation motif. In the presence but not in the absence of KCNE1 or KCNE3, K+ currents were activated by the Ca2+ ionophore ionomycin. Coexpression of CFTR with either human or rat KCNQ1 had no impact on regulation of KCNQ1 K+ currents by cAMP but slightly shifted the concentration response curve for 293B. Thus, KCNQ1 expressed in Xenopus oocytes is regulated by cAMP and Ca2+ but is not affected by CFTR. Received: 13 December 2000/Revised: 30 March 2001  相似文献   

19.
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   

20.
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   

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