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1.
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   

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.
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   

4.
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   

5.
In frog red blood cells, K-Cl cotransport (i.e., the difference between ouabain-resistant K fluxes in Cl and NO3) has been shown to mediate a large fraction of the total K+ transport. In the present study, Cl-dependent and Cl-independent K+ fluxes via frog erythrocyte membranes were investigated as a function of external and internal K+ ([K+] e and [K+] i ) concentration. The dependence of ouabain-resistant Cl-dependent K+ (86Rb) influx on [K+] e over the range 0–20 mm fitted the Michaelis-Menten equation, with an apparent affinity (K m ) of 8.2 ± 1.3 mm and maximal velocity (V max ) of 10.4 ± 1.6 mmol/l cells/hr under isotonic conditions. Hypotonic stimulation of the Cl-dependent K+ influx increased both K m (12.8 ± 1.7 mm, P < 0.05) and V max (20.2 ± 2.9 mmol/l/hr, P < 0.001). Raising [K+] e above 20 mm in isotonic media significantly reduced the Cl-dependent K+ influx due to a reciprocal decrease of the external Na+ ([Na+] e ) concentration below 50 mm. Replacing [Na+] e by NMDG+ markedly decreased V max (3.2 ± 0.7 mmol/l/hr, P < 0.001) and increased K m (15.7 ± 2.1 mm, P < 0.03) of Cl-dependent K+ influx. Moreover, NMDG+ Cl substitution for NaCl in isotonic and hypotonic media containing 10 mm RbCl significantly reduced both Rb+ uptake and K+ loss from red cells. Cell swelling did not affect the Na+-dependent changes in Rb+ uptake and K+ loss. In a nominally K+(Rb+)-free medium, net K+ loss was reduced after lowering [Na+] e below 50 mm. These results indicate that over 50 mm [Na+] e is required for complete activation of the K-Cl cotransporter. In nystatin-pretreated cells with various intracellular K+, Cl-dependent K+ loss in K+-free media was a linear function of [K+] i , with a rate constant of 0.11 ± 0.01 and 0.18 ± 0.008 hr−1 (P < 0.001) in isotonic and hypotonic media, respectively. Thus K-Cl cotransport in frog erythrocytes exhibits a strong asymmetry with respect to transported K+ ions. The residual, ouabain-resistant K+ fluxes in NO3 were only 5–10% of the total and were well fitted to linear regressions. The rate constants for the residual influxes were not different from those for K+ effluxes in isotonic (∼0.014 hr−1) and hypotonic (∼0.022 hr−1) media, but cell swelling resulted in a significant increase in the rate constants. Received: 19 November 1998/Revised: 23 August 1999  相似文献   

6.
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   

7.
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   

8.
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   

9.
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   

10.
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.
One of the main effects of abscisic acid (ABA) is to induce net loss of potassium salts from guard cells enabling the stomata to close. K+ is released from the vacuole into the cytosol and then to the extracellular space. The effects of increasing cytosolic K+ on the voltage- and time-dependence of the outwardly rectifying K+-current (I K,out) in guard cell protoplasts (GCP) was examined in the whole-cell configuration of the patch-clamp technique. The same quantitative analysis was performed in the presence of ABA at different internal K+ concentrations ([K+] i ). Varying [K+] i in the patch pipette from 100 to 270 mm increased the magnitude of I K,out in a nonlinear manner and caused a negative shift in the midpoint (V 0.5) of its steady-state activation curve. External addition of ABA (10–20 μm) also increased the magnitude of I K,out at all [K+] i , but caused a shift in V 0.5 of the steady-state activation curve only in those GCP loaded with 150 mm internal K+ or less. Indeed, V 0.5 did not shift upon addition of ABA when the [K+] i was above 150 mm and up to 270 mm, i.e., the shift in V 0.5 caused by ABA depended on the [K+] i . Both increase in [K+] i and external addition of ABA, decreased (by ≈ 20%) the activation time constant (τ n ) of I K,out. The small decrease in τ n , in both cases, was found to be independent of the membrane voltage. The results indicate that ABA mimics the effect of increasing cytoplasmic K+, and suggest that ABA may increase I K,out and alter V 0.5 of its steady-state activation curve via an enhancement in cytosolic K+. This report describes for the first time the effects of [K+] i on the voltage- and time-dependence of I K,out in guard cells. It also provides an explanation for the quantitative (total membrane current) and qualitative (current kinetics) differences found between intact guard cells and their protoplasts. Received: 1 December 1995/Revised: 8 May 1996  相似文献   

12.
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   

13.
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   

14.
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   

15.
The rabbit Na+/glucose cotransporter (SGLT1) exhibits a presteady-state current after step changes in membrane voltage in the absence of sugar. These currents reflect voltage-dependent processes involved in cotransport, and provide insight on the partial reactions of the transport cycle. SGLT1 presteady-state currents were studied as a function of external Na+, membrane voltage V m , phlorizin and temperature. Step changes in membrane voltage—from the holding V h to test values, elicited transient currents that rose rapidly to a peak (at 3–4 msec), before decaying to the steady state, with time constants τ≈4–20 msec, and were blocked by phlorizin (K i ≈30 μm). The total charge Q was equal for the application of the voltage pulse and the subsequent removal, and was a function of V m . The Q-V curves obeyed the Boltzmann relation: the maximal charge Q max was 4–120 nC; V 0.5, the voltage for 50% Q max was −5 to +30 mV; and z, the apparent valence of the moveable charge, was 1. Q max and z were independent of V h (between 0 and −100 mV) and temperature (20–30°C), while increasing temperature shifted V 0.5 towards more negative values. Decreasing [Na+] o decreased Q max, and shifted V 0.5 to more negative voltages 9by −100 mV per 10-fold decrease in [Na+] o ). The time constant τ was voltage dependent: the τ-V relations were bell-shaped, with maximal τmax 8–20 msec. Decreasing [Na+] o decreased τmax, and shifted the τ-V curves towards more negative voltages. Increasing temperature also shifted the τ-V curves, but did not affect τmax. The maximum temperature coefficient Q 10 for τ was 3–4, and corresponds to an activation energy of 25 kcal/mole. Simulations of a 6-state ordered kinetic model for rabbit Na+/glucose cotransport indicate that charge-movements are due to Na+-binding/dissociation and a conformational change of the empty transporter. The model predicts that (i) transient currents rise to a peak before decay to steady-state; (ii) the τ-V relations are bell-shaped, and shift towards more negative voltages as [Na+] o is reduced; (iii) τmax is decreased with decreasing [Na+] o ; and (iv) the Q-V relations are shifted towards negative voltages as [Na+] o is reduced. In general, the kinetic properties of the presteady-state currents are qualitatively predicted by the model. Received: 12 August 1996/Revised: 30 September 1996  相似文献   

16.
Transport Pathways for Therapeutic Concentrations of Lithium in Rat Liver   总被引:1,自引:0,他引:1  
Although both amiloride- and phloretin-sensitive Na+/Li+ exchange activities have been reported in mammalian red blood cells, it is still unclear whether or not the two are mediated by the same pathway. Also, little is known about the relative contribution of these transport mechanisms to the entry of therapeutic concentrations of Li+ (0.2–2 mm) into cells other than erythrocytes. Here, we describe characteristics of these transport systems in rat isolated hepatocytes in suspension. Uptake of Li+ by hepatocytes, preloaded with Na+ and incubated in the presence of ouabain and bumetanide, comprised three components. (a) An amiloride-sensitive component, with apparent K m 1.2 mm Li+, V max 40 μmol · (kg dry wt · min)−1, showed increased activity at low intracellular pH. The relationship of this component to the concentration of intracellular H+ was curvilinear suggesting a modifier role of [H+] i . This system persisted in Na+-depleted cells, although with apparent K m 3.8 mm. (b) A phloretin-sensitive component, with K m 1.2 mm, V max 21 μmol · (kg · min)−1, was unaffected by pH but was inactive in Na+-depleted cells. Phloretin inhibited Li+ uptake and Na+ efflux in parallel. (c) A residual uptake increased linearly with the external Li+ concentration and represented an increasing proportion of the total uptake. The results strongly suggest that the amiloride-sensitive and the phloretin-sensitive Li+ uptake in rat liver are mediated by two separate pathways which can be distinguished by their sensitivity to inhibitors and intracellular [H+]. Received: 8 April 1999/Revised: 19 July 1999  相似文献   

17.
Hyperthermia induces transient changes in [Na+] i and [K+] i in mammalian cells. Since Cl flux is coupled with Na+ and K+ in several processes, including cell volume control, we have measured the effects of heat on [Cl] i using the chloride indicator, MQAE, with flow cytometry. The mean basal level of [Cl] i in Chinese hamster ovary cells was 12 mm. Cells heated at 42.0° or 45.0°C for 30 min had about a 2.5-fold increase in [Cl] i above unheated control values when measured immediately after heating. There was about a 3-fold decrease in [Na+] i under the same conditions, as measured by Sodium Green. The magnitude of the increase in [Cl] i depended upon time and temperature. The [Cl] i recovered in a time-dependent fashion to control values by 30 min after heating. When cells were heated at 45.0°C for 30 min in the presence of 1.5 mm furosemide, the heat-induced [Cl] i increase was completely blocked. Since furosemide inhibits the Na+/K+/2Cl cotransporter, Cl channels, and even ClHCO3 exchange, these ion transporters may be involved in the heat-induced increase in [Cl] i . Received: 15 June 1995/Revised: 9 April 1996  相似文献   

18.
In cystic fibrosis airway epithelia, mutation of the CFTR protein causes a reduced response of Cl secretion to secretagogues acting via cAMP. Using a Ca2+ imaging system, the hypothesis that CFTR activation may permit ATP release and regulate [Ca2+] i via a receptor-mediated mechanism, is tested in this study. Application of external nucleotides produced a significant increase in [Ca2+] i in normal (16HBE14o cell line and primary lung culture) and in cystic fibrosis (CFTE29o cell line) human airway epithelia. The potency order of nucleotides on [Ca2+] i variation was UTP ≫ ATP > UDP > ADP > AMP > adenosine in both cell types. The nucleotide [Ca2+] i response could be mimicked by activation of CFTR with forskolin (20 μm) in a temperature-dependent manner. In 16HBE14o cells, the forskolin-induced [Ca2+] i response increased with increasing temperature. In CFTE29o cells, forskolin had no effect on [Ca2+] i at body temperature-forskolin-induced [Ca2+] i response in CF cells could only be observed at low experimental temperature (14°C) or when cells were cultured at 26°C instead of 37°C. Pretreatment with CFTR channel blockers glibenclamide (100 μm) and DPC (100 μm), with hexokinase (0.5 U/mg), and with the purinoceptor antagonist suramin (100 μm), inhibited the forskolin [Ca2+] i response. Together, these results demonstrate that once activated, CFTR regulates [Ca2+] i by mediating nucleotide release and activating cell surface purinoceptors in normal and CF human airway epithelia. Received: 3 April 2000/Revised: 30 June 2000  相似文献   

19.
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   

20.
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   

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