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1.
Francisco Barros Carlos Villalobos Javier García-Sancho Donato del Camino Pilar de la Peña 《Pflügers Archiv : European journal of physiology》1994,426(3-4):221-230
Exposure of GH3 rat anterior pituitary cells to cholera toxin for 2–4 h significantly increased the thyrotropin-releasing-hormone(TRH)-induced inhibition of the inwardly rectifying K+ current studied in patchperforated voltage-clamped cells. On the other hand, the current reduction became almost totally irreversible after washout of the neuropeptide. Comparison of the effects elicited by the toxin with those of 8-(4-chlorophenylthio)-cAMP or forskolin plus isobutylmethylxanthine indicated that, although the irreversibility may be due, at least in part, to elevations of cAMP levels, the enhancement of the TRH-induced inhibition of the current is not mediated by the cyclic nucleotide. Only reductions on the inwardly rectifying K+ current, but not those elicited by TRH on voltage-dependent Ca2+ currents, were increased by the treatment with cholera toxin. In current-clamped cells showing similar rates of firing, the second phase of enhanced action-potential frequency induced by TRH was also significantly potentiated by cholera toxin. Measurements of [Ca2+]i oscillations associated with electrical activity, using video imaging with fura-2-loaded cells, demonstrated that cholera toxin treatment causes a clear reduction of spontaneous [Ca2+]i oscillations. However, this did not prevent the stimulatory effect of TRH on oscillations due to the action potentials. In cholera-toxin-treated cells, the steady-state, voltage dependence of inactivation of the inward rectifier was shifted by nearly 20 mV to more negative values. These data suggest that the inwardly rectifying K+ current plays an important role in maintenance of the resting K+ conductance in GH3 cells. Furthermore, the TRH-induced reductions on this current may be an important factor contributing to the increased cell excitability promoted by the neuropeptide. 相似文献
2.
F. Barros Donato del Camino Luis A. Pardo Teresa Palomero Teresa Giráldez Pilar de la Peña D. del Camino 《Pflügers Archiv : European journal of physiology》1997,435(1):119-129
Reduction of an inwardly rectifying K+ current by thyrotropin-releasing hormone (TRH) and caffeine has been considered to be an important determinant of electrical
activity increases in GH3 rat anterior pituitary cells. However, the existence of an inwardly rectifying K+ current component was recently regarded as a misidentification of an M-like outward current, proposed to be the TRH target
in pituitary cells, including GH3 cells. In this report, an inwardly rectifying component of K+ current is indeed demonstrated in perforated-patch voltage-clamped GH3 cells. The degree of rectification varied from cell to cell, but both TRH and caffeine specifically blocked a fraction of
current with strong rectification in the hyperpolarizing direction. Use of ramp pulses to continuously modify the membrane
potential demonstrated a prominent blockade even in cells with no current reduction at voltages at which M-currents are active.
Depolarization steps to positive voltages at the maximum of the inward current induced a caffeine-sensitive instantaneous
outward current followed by a single exponential decay. The magnitude of this current was modified in a biphasic way according
to the duration of the previous hyperpolarization step. The kinetic characteristics of the current are compatible with the
possibility that removal from inactivation of a fast-inactivating delayed rectifier causes the hyperpolarization-induced current.
Furthermore, the inwardly rectifying current was blocked by astemizole, a potent and selective inhibitor of human ether-á-go-go -related gene (HERG) K+ channels. Along with other pharmacological and kinetic evidence, this indicates that the secretagogue-regulated current is
probably mediated by a HERG-like K+ channel. Addition of astemizole to current-clamped cells induced clear increases in the frequency of action potential production.
Thus, an inwardly-rectifying K+ current and not an M-like outward current seems to be involved in TRH and caffeine modulation of electrical activity in GH3 cells.
Received: 15 May 1997 / Received after revision and accepted: 24 July 1997 相似文献
3.
Francisco Barros Donato del Camino Luis A. Pardo Pilar de la Peña 《Pflügers Archiv : European journal of physiology》1996,431(3):443-451
Treatment of rat anterior pituitary GH3 cells with caffeine causes a reversible enhancement of electrical activity superimposed over a depolarization of the plasma membrane potential. Similar results are obtained with theophylline, but not with isobutylmethylxanthine or forskolin. The effects of caffeine are not related to Ca2+ liberation from intracellular stores since they are not affected by incubation of the cells with ryanodine or thapsigargin. Furthermore, caffeine-induced hyperpolarization of the membrane is not detectable even in cells in which Ca2+ liberation from inositol 1,4,5-trisphosphate-sensitive compartments produces a prominent transient hyperpolarization in response to thyrotropin-releasing hormone. Reductions of Ca2+-dependent K+ currents caused by partial block of L-type Ca2+ channels by caffeine are not sufficient to explain the effects of the xanthine, since the results obtained with caffeine are not mimicked by direct blockade of Ca2+ channels with nisoldipine. GH3 cell inwardly rectifying K+ currents are inhibited by caffeine. Studies on the voltage dependence of the caffeine-induced effects indicate a close correlation between alterations of electrical parameters and reported values of steady-state voltage dependence of inactivation of these currents. We conclude that, as previously shown for thyrotropin-releasing hormone, modulation of inwardly rectifying K+ currents plays a major role determining the firing rate of GH3 cells and its enhancement by caffeine. 相似文献
4.
P. Komwatana A. D. Conigrave L. Delbridge J. A. Young D. I. Cook 《Pflügers Archiv : European journal of physiology》1994,426(3-4):320-327
We have used whole-cell patch-clamp techniques to study the conductances in the plasma membranes of human parathyroid cells. With a KCl-rich pipette solution containing Ca2+ buffered to a concentration of 0.1 mol/l, the zero current potential was –71.1±0.5 mV (n=19) and the whole-cell current/ voltage (I/V) relation had an inwardly rectifying and an outwardly rectifying component. The inwardly rectifying current activated instantaneously on hyperpolarization of the plasma membrane to potentials more negative than –80 mV, and a semi-logarithmic plot of the reversal potential of the inward current (estimated by extrapolation from the range in which it was linear) as a function of extracellular K+ concentration ([K+]o) revealed a linear relation with a slope of 64 mV per decade change in [K+]o, which is not significantly different from the Nernstian slope, demonstrating that the current was carried by K+ ions. The conductance exhibited a square root dependence on [K+]o as has been observed for inward rectifiers in other tissues. The current was blocked by the presence of Ba2+ (1 mmol/l) or Cs+ (1.5 mmol/l) in the bath. The outwardly rectifying current was activated by depolarization of the membrane potential to potentials more positive than –20 mV. It was inhibited by replacement of pipette K+ with Cs+, indicating that it also was a K+ current: it was partially (42%) blocked when tetraethylammonium (TEA+, 10 mmol/l) was added to the bath. The outwardly rectifying, but not the inwardly rectifying K+ current, was regulated by intracellular free Ca2+ concentration ([Ca2+]i) such that increasing [Ca2+]i above 10 nmol/l inhibited the outwardly rectifying current, the half-maximum effect being seen at 1 mol/l. Since it is known that increases in [Ca2+]o produce increases in [Ca2+]i, and that they depolarize parathyroid cells by reducing the membrane K+ conductance, we suggest that it is the reduction of the outwardly rectifying K+ conductance by increases in [Ca2+]i which is responsible for the reduction in K+ conductance seen when [Ca2+]o is increased. 相似文献
5.
Akiko Itoh Shunji Ueda Yasunobu Okada 《Pflügers Archiv : European journal of physiology》1994,428(5-6):468-475
K+ conductance species in a human intestinal epithelial cell line (Intestine 407) were studied in connection with their sensitivities to an intestinal secretagogue, histamine, using the tight-seal whole-cell patch-clamp technique. Applications of positive command pulses rapidly induced outward K+ currents. The conductance became progressively larger with increasing command voltages, exhibiting an outwardly rectifying current voltage relation. Inward K+ currents were also rapidly activated upon applications of hyperpolarizing pulses at potentials negative to the equilibrium potential of K+ (E K), and the conductance inwardly rectified. Application of a Ca2+ ionophore, ionomycin, brought about activation of additional K+ currents. An inhibitor of protein kinase C, polymyxin B, did not affect the ionomycin-induced response. Histamine (10–200 μM) also activated a similar K+ current which was abolished by cytosolic Ca2+ chelation. Under conditions where Ca2+ mobilization was minimized, histamine was found to significantly augment inwardly rectifying K+, but suppress outwardly rectifying K+, currents. Polymyxin B blocked these effects of histamine. An activator of protein kinase C, 1-oleoyl-2-acetylglycerol, mimicked the histamine effects. It is concluded that the intestinal epithelial cell has three distinct types of K+ conductance and that histamine modulates not only Ca2+-activated K+ conductance via Ca2+ mobilization, but also inward- and outward-rectifier K+ conductances via activation of protein kinase C. 相似文献
6.
C. K. Bauer I. Davison I. Kubasov J. R. Schwarz W. T. Mason 《Pflügers Archiv : European journal of physiology》1994,428(1):17-25
In rat anterior pituitary tumour cells (GH3/B6) thyrotropin-releasing hormone (TRH) elicits a biphasic response. First, a release of intracellularly stored Ca2+ induces a hyperpolarization of the cell. Second, a depolarization thought to be induced by a reduction of the inward-rectifying K+ current (KIR) causes an increase in action potential frequency and a plateau-like increase in [Ca2+]i. It has been proposed that the two phases are induced by the actions of inositol 1,4,5-trisphosphate (InsP
3) and protein kinase C (PKC), respectively, but we demonstrate here that PKC is not responsible for the second phase increase in [Ca2+]i and suggest that the pathways diverge at the level of receptor and G protein coupling. Both phases of the TRH response were insensitive to pertussis toxin, but cholera toxin (CTX) selectively affected the second phase. After CTX pretreatment cells had a high spontaneous spiking frequency and smaller KIR amplitude. In these cells TRH failed to increase the action potential frequency after the first phase hyperpolarization, elicited only a transient peak increase in [Ca2+]i with no plateau phase and could only slightly reduce KIR. These effects of CTX are not mediated by its ability to increase cAMP via activation of GS, as increased cAMP levels neither inhibit KIR nor prevent its reduction by TRH. In addition, inhibition of protein kinase A activation did not block the second phase increase in [Ca2+]i induced by TRH, suggesting that the CTX-sensitive G protein mediating the second phase of the TRH response is not GS.On leave from Uchtomsky-Institute, St. Petersburg University, Russia 相似文献
7.
Di-8-ANEPPS (4-{2-[6-(dibutylamino)-2-naphthalenyl]-ethenyl}-1-(3-sulfopropyl)pyridinium inner salt) has been used as a fast-response
voltage-sensitive styrylpyridinium probe. However, little is known regarding the mechanism of di-8-ANEPPS actions on ion currents.
In this study, the effects of this dye on ion currents were investigated in pituitary GH3 cells. In whole-cell configuration, di-8-ANEPPS (10 μM) reversibly increased the amplitude of Ca2+-activated K+ current. In inside-out configuration, di-8-ANEPPS (10 μM) applied to the intracellular surface of the membrane caused no
change in single-channel conductance; however, it did enhance the activity of large-conductance Ca2+-activated K+ (BKCa) channels with an EC50 value of 7.5 μM. This compound caused a left shift in the activation curve of BKCa channels with no change in the gating charge of these channels. A decrease in mean closed time of the channels was seen in
the presence of this dye. In the cell-attached mode, di-8-ANEPPS applied on the extracellular side of the membrane also activated
BKCa channels. However, neither voltage-gated K+ nor ether-à-go-go-related gene (erg)-mediated K+ currents in GH3 cells were affected by di-8-APPNES. Under current-clamp configuration, di-8-ANEPPS (10 μM) decreased the firing of action
potentials in GH3 cells. In pancreatic βTC-6 cells, di-8-APPNES (10 μM) also increased BKCa-channel activity. Taken together, this study suggests that during the exposure to di-8-ANEPPS, the stimulatory effects on
BKCa channels could be one of potential mechanisms through which it may affect cell excitability. 相似文献
8.
Mario Díaz Francisco V. Sepúlveda 《Pflügers Archiv : European journal of physiology》1995,430(2):168-180
The whole-cell configuration of the patch-clamp technique was used to examine K+ currents in HeLa cells. Under quasi-physiological ionic gradients, using an intracellular solution containing 10–7 mol/l free Ca2+, mainly outward currents were observed. Large inwardly rectifying currents were elicited in symmetrical 145 mmol/l KCl. Replacement of all extracellular K+ by isomolar Na+, greatly decreased inward currents and shifted the reversal potential as expected for K+ selectivity. The inwardly rectifying K+ currents exhibited little or no apparent voltage dependence within the range of from –120 mV to 120 mV. A square-root relationship between chord conductance and [K+]0 at negative potentials could be established. The inwardly rectifying nature of the currents was unaltered after removal of intracellular Mg2+ and chelation with ATP and ethylenediaminetetraacetic acid (EDTA). Permeability ratios for other monovalent cations relative to K+ were: K+ (1.0)>Rb+ (0.86)>Cs+ (0.12)>Li+ (0.08)>Na+ (0.03). Slope conductance ratios measured at –100 mV were: Rb+ (1.66)>K+ (1.0)>Na+ (0.09)>Li+ (0.08)>Cs+ (0.06). K+ conductance was highly sensitive to intracellular free Ca2+ concentration. The relationship between conductance at 0 mV and Ca2+ concentration was well described by a Hill expression with a dissociation constant, K
D, of 70 nmol/l and a Hill coefficient, n, of 1.81. Extracellular Ba2+ blocked the currents in a concentration- and voltage-dependent manner. The dependence of the K
D for the blockade was analysed using a Woodhull-type treatment, locating the ion interaction site at 19 % of the distance across the electrical field of the membrane and a K
D (0 mV) of 7 mmol/l. Tetraethylammonium and 4-aminopyridine were without effect whilst quinine and quinidine blocked the currents with concentrations for half-maximum effects equal to 7 mol/l and 3.5 mol/l, respectively. The unfractionated venom of the scorpion Leiurus quinquestriatus (LQV) blocked the K+ currents of HeLa cells. The toxins apamin and scyllatoxin had no detectable effect whilst charybdotoxin, a component of LQV, blocked in a voltage-dependent manner with half-maximal concentrations of 40 nmol/l at –120 mV and 189 nmol/l at 60 mV; blockade by charybdotoxin accounts for the effect of LQV. Application of ionomycin (5–10 mol/l), histamine (1 mmol/l) or bradykinin (1–10 mol/l) to cells dialysed with low-buffered intracellular solutions induced K+ currents showing inward rectification and a lack of voltage dependence. 相似文献
9.
Stephen M. Sims Melanie E. M. Kelly S. Jeffrey Dixon 《Pflügers Archiv : European journal of physiology》1991,419(3-4):358-370
Membrane electrical properties of freshly isolated rat osteoclasts were studied using patch-clamp recording methods. Characterization of the passive membrane properties indicated that the osteoclast cell membrane behaved as an isopotential surface. The specific membrane capacitance was 1.2±0.3 F/cm2 (mean ±SD), with no difference between cells plated on glass and those adhering to a permeable collagen substrate. The current/voltage (I/V) relationship of all cells showed inward rectification and I/V curves shifted 51 mV positive per tenfold increase of [K+]out, indicating an inwardly rectifying K+ conductance. The voltage dependence of the K+ chord conductance (g
K) also shifted positive along the voltage axis, and the maximum conductance increased, with elevation of [K+]out. g
K for cells bathed in 4.7 mM [K+]out increased e-fold per 12mV hyperpolarization, and half-maximal activation was at –89 mV. Approximately 18% (50 pS/pF) of the maximum g
K was active at –70 mV. Inward single-channel currents were recorded in cell-attached patches at hyperpolarizing potentials. With symmetrical K+, channel conductance was 25±3 pS and reversal was close to the K+ equilibrium potential, consistent with this K+ channel underlying the whole-cell K+ currents. With both conventional whole-cell and perforated-patch recording, no voltage-activated Ca2+ current was detected. In approximately 30% of osteoclasts studied, an outwardly rectifying current was observed, which was reversibly blocked by 4,4-diisothiocyanostilbene-2,2-disulphonic acid (DIDS) and 4-acetamido-4-isothiocyanostilbene2,2-disulphonic acid (SITS). This DIDS- and SITS-sensitive current reversed direction at the chloride equilibrium potential. We conclude that an inwardly rectifying K+ current is present in all rat osteoclasts and that some osteoclasts also exhibit an outwardly rectifying Cl– current. Both these membrane conductances may play an important physiological role by dissipating the potential that arises from the electrogenic transport of H+ across the ruffled membrane of the osteoclast. 相似文献
10.
Daniel G. Lang Aileen K. Ritchie 《Pflügers Archiv : European journal of physiology》1987,410(6):614-622
Single Ca2+-activated K+ channels were studied in membrane patches from the GH3 anterior pituitary cell line. In excised inside-out patches exposed to symmetrical 150 mM KCl, two channel types with conductances in the ranges of 250–300 pS and 9–14 pS were routinely observed. The activity of the large conductance channel is enhanced by internal Ca2+ and by depolarization of the patch membrane. This channel contributes to the repolarization of Ca2+ action potentials but has a Ca2+ sensitivity at –50 mV that is too low for it to contribute to the resting membrane conductance. The small conductance channel is activated by much lower concentrations of Ca2+ at –50 mV, ad its open probability is not strongly voltage sensitive. In cell-attached patches from voltage-clamped cells, the small conductance channels were found to be active during slowly decaying Ca2+-activated K+ tails currents and during Ca2+-activated K+ currents stimulated by thyrotropin-releasing hormone induced elevations of cytosolic calcium. In cell-attached patches on unclamped cells, the small conductance channels were also active at negative membrane potentials when the frequency of spontaneously firing action potentials was high or during the slow afterhyperpolarization following single spontaneous action potentials of slightly prolonged duration. The small conductance channel may thus contribute to the regulation of membrane excitability. 相似文献
11.
Calcitonin-gene-related peptide activates the muscarinic-gated K+ current in atrial cells 总被引:3,自引:0,他引:3
Donghee Kim 《Pflügers Archiv : European journal of physiology》1991,418(4):338-345
A high density of nerve fibers containing calcitonin-gene-related peptide (CGRP) is present in the atria. Recently CGRP was reported to open ATP-sensitive K channels in arterial smooth muscle cells. This study examines whether CGRP activates a similar K+ channel in cardiac cells. In voltage-clamped whole cells loaded with GTP and ATP, CGRP reversibly evoked an inwardly rectifying K+ current. To identify the K+ channel that gives rise to this current, three types of K+ channel (resting, ATP-sensitive and acetylcholine-activated) were examined. CGRP failed to activate or inhibit the ATP-sensitive or the resting K+ channel. However, CGRP (0.1–1 M) caused activation of single channels with kinetics similar to that of the muscarinic K+ channel (35–40 pS conductance and approx. 1 ms mean open time in symmetrical 140 mM K+). In excised, inside-out (CGRP in pipette) or in outside-out (GTP in pipette) patches, the K+ current was activated by perfusion with GTP or CGRP, respectively, suggesting that CGRP activated the muscarinic K+ channel via GTP-binding protein. Treatment with pertussis toxin inhibited the activation of the K+ channel, suggesting that CGRP receptor may be coupled to a Gi or a Go type of GTP-binding protein. Together with previous findings, these results suggest that CGRP modulates several types of ion channels to produce its cellular effects.This work was supported in part by HL40586 and American Heart Association grant-in-aid. 相似文献
12.
Rodney L. Parsons Laura A. Merriam 《Pflügers Archiv : European journal of physiology》1993,422(4):410-412
Galanin- and bethanechol-activated K+ currents have been studied in mudpuppy atrial myocytes. The galanin and bethanechol K+ currents were time-dependent and inwardly rectifying. In GTPS, the galanin and bethanechol currents were reduced progressively as G-protein gated K+ channels became activated. GDPS inhibited agonist-induced outward currents. We conclude that galanin and bethanechol activate the same or a very similar inwardly rectifying K+ conductance and that activation of a G protein is required. 相似文献
13.
Carlos Villalobos Javier García-Sancho 《Pflügers Archiv : European journal of physiology》1995,430(6):923-935
Treatment of GH3 cells with either hypothalamic peptide thyrotropin-releasing hormone (TRH), the endomembrane Ca2+-ATPase inhibitor thapsigargin or the Ca2+ ionophore ionomycin mobilized, with different kinetics, essentially all of the Ca2+ pool from the intracellular Ca2+ stores. Any of the above-described treatments induced a sustained increase in intracellular Ca2+ concentration ([Ca2+]i), which was dependent on extracellular Ca2+ and was prevented by Ni2+ but not by dihydropyridines (DHPs), suggesting that it was due to capacitative Ca2+ entry via activation of a plasma membrane pathway which opened upon the emptying of the intracellular Ca2+ stores. The increase of the plasma membrane permeability to Ca2+ correlated negatively with the filling degree of the intracellular Ca2+ stores and was reversed by refilling of the stores. The mechanism of capacitative Ca2+ entry into GH3 cells differed from similar mechanisms described in several types of blood cells in that the pathway was poorly permeable to Mn2+ and not sensitive to cytochrome P450 inhibitors. In GH3 cells, TRH induced a transient [Ca2+]i increase due to Ca2+ release from the stores (phase 1) followed by a sustained [Ca2+]i increase due to Ca2+ entry (phase 2). At the single-cell level, phase 2 was composed of a DHP-insensitive sustained [Ca2+]i increase, due to activation of capacitative Ca2+ entry, superimposed upon which DHP-sensitive [Ca2+]i oscillations took place. The two components of the TRH-induced Ca2+ entry differed also in that [Ca2+]i oscillations remained for several minutes after TRH removal, whereas the sustained [Ca2+]i increase dropped quickly to prestimulatory levels, following the same time course as the refilling of the stores. The drop was prevented when the refilling was inhibited by thapsigargin. It is concluded that, even though the mechanisms of capacitative Ca2+ entry may show differences from cell to cell, it is also present and may contribute to the regulation of physiological functions in excitable cells such as GH3. There, capacitative Ca2+ entry cooperates with voltage-gated Ca2+ channels to generate the [Ca2+]i increase seen during phase 2 of TRH action. This contribution of capacitative Ca2+ entry may be relevant to the enhancement of prolactin secretion induced by TRH. 相似文献
14.
Antonio R. Artalejo Antonio G. García Erwin Neher 《Pflügers Archiv : European journal of physiology》1993,423(1-2):97-103
Simultaneous whole-cell patch-clamp and fura-2 fluorescence [Ca2+]i measurements were used to characterize Ca2+-activated K+ currents in cultured bovine chromaffin cells. Extracellular application of histamine (10 M) induced a rise of [Ca2+]i concomitantly with an outward current at holding potentials positive to –80 mV. The activation of the current reflected an increase in conductance, which did not depend on membrane potential in the range –80 mV to –40 mV. Increasing the extracellular K+ concentration to 20 mM at the holding potential of –78 mV was associated with inwardly directed currents during the [Ca2+]i elevations induced either by histamine (10 M) or short voltage-clamp depolarizations. The current reversal potential was close to the K+ equilibrium potential, being a function of external K+ concentration. Current fluctuation analysis suggested a unit conductance of 3–5 pS for the channel that underlies this K+ current. The current could be blocked by apamin (1 M). Whole-cell current-clamp recordings snowed that histamine (10 M) application caused a transient hyperpolarization, which evolved in parallel with the [Ca2+]i changes. It is proposed that a small-conductance Ca2+-activated K+ channel is present in the membrane of bovine chromaffin cells and may be involved in regulating catecholamine secretion by the adrenal glands of various species. 相似文献
15.
Carlos Villalobos Javier García-Sancho 《Pflügers Archiv : European journal of physiology》1996,431(3):371-378
Caffeine, a well known facilitator of Ca2+-induced Ca2+ release, induced oscillations of cytosolic free Ca2+ ([Ca2+]i) in GH3 pituitary cells. These oscillations were dependent on the presence of extracellular Ca2+ and blocked by dihydropyridines, suggesting that they are due to Ca2+ entry through L-type Ca2+ channels, rather than to Ca2+ release from the intracellular Ca2+ stores. Emptying the stores by treatment with ionomycin or thapsigargin did not prevent the caffeine-induced [Ca2+]i oscillations. Treatment with caffeine occluded phase 2 ([Ca2+]i oscillations) of the action of thyrotropin-releasing hormone (TRH) without modifying phase 1 (Ca2+ release from the intracellular stores). Caffeine also inhibited the [Ca2+]i increase induced by depolarization with high-K+ solutions (56% at 20 mM), suggesting direct inhibition of the Ca2+ entry through voltage-gated Ca2+ channels. We propose that the [Ca2+]i increase induced by caffeine in GH3 cells takes place by a mechanism similar to that of TRH, i.e. membrane depolarization that increases the firing frequency of action potentials. The increase of the electrical activity overcomes the direct inhibitory effect on voltage-gated Ca2+ channels with the result of increased Ca2+ entry and a rise in [Ca2+]i. Consideration of this action cautions interpretation of previous experiments in which caffeine was assumed to increase [Ca2+]i only by facilitating the release of Ca2+ from intracellular Ca2+ stores. 相似文献
16.
ATP-sensitive K+ channels regulated by intracellular Ca2+ and phosphorylation in normal (T84) and cystic fibrosis (CFPAC-1) epithelial cells 总被引:1,自引:0,他引:1
Brigitte Roch Isabelle Baró Anne -Sophie Hongre Denis Escande 《Pflügers Archiv : European journal of physiology》1995,429(3):355-363
The elementary K+ conductance activated by the cAMP or the Ca2+ second messenger pathways was investigated in the model salt-secreting epithelium, the human T84 cell line. Under Cl–-free conditions, an inwardly rectifying whole-cell K+ current was evoked by either forskolin 10 (mol/l) or acetylcholine 1 (mol/l) and blocked by extracellular charybdotoxin 10 (nmol/l). In the cell-attached mode, both secretory agonists induced the opening of a channel showing inward rectification with a unitary chord conductance of 36.8±2.5 pS (n=26) for inward currents. In inside-out patches, a 35-pS inwardly rectifying K+ channel that corresponded to the channel recorded in the cell-attached configuration was recorded in the presence of 0.3 mol/l free Ca2+ at the inner side of the membrane. This channel was blocked by Ba2+ (5 mol/l) and by charybdotoxin (50 nmol/l). Its open probability was enhanced by intracellular Ca2+ with and EC50 of 0.25 mol/l and strongly reduced by intracellular MgATP with an IC50 of 600 mol/l. In the continuous presence of ATP, the channel activity was consistently increased by 125 kU/l catalytic subunit of cAMP-dependent protein kinase. In the cystic fibrosis pancreatic duct cell line CFPAC-1, a K+ channel was also recorded, with similar characterictics and regulation as the 35-pS channel in T84 cells. We conclude that an ATP-sensitive K+ channel regulated by intracellular Ca2+ and phosphorylation supports the main K+ current activated by secretory agonists in normal cystic fibrosis cell lines. This conductance possibly represents the major pathway for K+ recycling at the basolateral membrane during transepithelial fluid secretion. 相似文献
17.
C. Alzheimer G. ten Bruggencate 《Pflügers Archiv : European journal of physiology》1991,419(3-4):288-295
The properties of the current underlying the membrane hyperpolarization evoked by adenosine (50–100 m) were investigated in hippocampal CA3 neurons in vitro using current-clamp and single-electrode voltage-clamp techniques. In voltage-clamp measurements, the adenosine-induced current (I
Ado) was outward at rest and reversed at membrane potentials close to the equilibrium potential of K+ (E
K), indicating that I
Ado was carried by K+ ions. Determination of I
Ado at several membrane potentials revealed a nonlinear current/voltage (I/V) relationship of the current displaying inward rectification in the hyperpolarizing direction. Similarly, adenosine increased the membrane slope conductance only at membrane potentials negative to rest, whereas the slope of the neuronal I/V curve remained unchanged when determined at potentials positive to rest. Since the electrophysiological properties of I
Ado were very similar to those described for K+ conductances activated by other neuroactive substances like serotonin, opioid peptides and -aminobutyric acid B receptor (GABAB) agonists, we conclude that I
Ado belongs to a family of ligand-operated, inwardly rectifying K+ currents which apparently share a common mechanism to reduce postsynaptic excitability. As an additional feature, the postsynaptic adenosine response was reduced by bath application of Co2+ or Ni2+. The adenosine-induced membrane hyperpolarization was not affected by low-Ca2+ or low-Mg2+ solutions, nor by buffering of intra-cellular Ca2+, but a gradual decline of I
Ado was observed following superfusion with Co2+ or Ni2+. In contrast, Mn2+ caused only a weak attenuation of the adenosine response. 相似文献
18.
Drosophila mutants provide an ideal model to study channel-type specificity of ion channel regulation in situ. In this study, the effects of divalent cations on voltage-gated K+ currents were investigated in acutely dissociated central neurons of Drosophila third instar larvae using the whole-cell patch-clamp recording. Our data showed that micromolar Cd2+ enhanced the peak inactivating current (IA) without affecting the delayed component (IK). The same results were obtained in Ca2+-free external solution, and from slo1 mutation, which eliminates transient Ca2+-activated K+ current. Micromolar Cd2+and Zn2+, and millimolar Ca2+and Mg2+ all shifted the steady-state inactivation curve of IA without affecting the voltage-dependence of IA activation, whereas millimolar Cd2+markedly affected both the activation and steady-state inactivation curves for IA. Divalent cations affected IA with different potency; the sequence was: Zn2+ > Cd2+ > Ca2+ > Mg2+. The modulation of IA by Cd2+ was partially inhibited in ShM, a null Shaker (one of IA-encoding genes) mutation. Taken together, the channel-type specificity, the asymmetric effects on IA activation and inactivation kinetics, and the diverse potency of divalent cations all strongly support the idea that physiological divalent cations modulate A-type K+ channels through specific binding to extracellular sites of the channels. 相似文献
19.
B. Belles J. Hescheler G. Trube 《Pflügers Archiv : European journal of physiology》1987,409(6):582-588
Single isolated myocytes were obtained from the ventricles of adult guinea pig hearts. The whole-cell recording configuration of the patch-clamp technique was used to measure membrane currents. A decrease (run-down) of the Ca2+ inward current and an increase of a time-independent K+ outward current were observed during long lasting (1–3 h) recordings. The time at which the outward current developed depended on the intracellular ATP concentration in the pipette, suggesting that this current is identical to the ATP-dependent K+ current described by Noma and Shibasaki (1985). However, the maximum outward current reached in the experiments was independent of the ATP concentration indicating a limited diffusion of ATP in the cell interior. In single-channel experiments on isolated patches of cell membrane and in whole-cell recordings the ATP-dependent K+ current could be blocked by the hypoglycaemic sulphonylurea tolbutamide. The IC50 of 0.38 mM was about 50 times higher than that reported for pancreatic -cells (Trube et al. 1986). The Ca2+ inward current and the inwardly retifying K+ current were not affected by tolbutamide (3 mM). 相似文献
20.
Daniel G. Lang Aileen K. Ritchie 《Pflügers Archiv : European journal of physiology》1990,416(6):704-709
Single Ca2+-activated K+ channels were studied in membrane patches from the GH3 anterior pituitary cell line. We have previously demonstrated the coexistence of large-conductance and small-conductance (280 pS and 11 pS in symmetrical 150 mM K+, respectively) Ca2+-activated K+ channels in this cell line (Lang and Ritchie 1987). Here we report the existence of a third type of Ca2+-activated K+ channel that has a conductance of about 35 pS under similar conditions. In excised inside-out patches, this channel can be activated by elevations of the internal free Ca2+ concentration, and the open probability increases as the membrane potential is made more positive. In excised patches, the sensitivity of this 35-pS channel to internal Ca2+ is low; at positive membrane potentials, this channel requires Ca2+ concentrations greater than 10 M for activation. However, 35-pS channels have a much higher sensitivity to Ca2+ in the first minute after excision (activated by 1 M Ca2+ at –50 mV). Therefore, it is possible that the Ca2+ sensitivity of this channel is stabilized by intracellular factors. In cell-attached patches, this intermediate conductance channel can be activated (at negative membrane potentials) by thyrotropin-releasing hormone-induced elevations of the intracellular Ca2+ concentration and by Ca2+ influx during action potentials. The intermediate conductance channel is inhibited by high concentrations of external tetraethylammonium ions (K
d=17 mM) and is relatively resistant to inhibition by apamin. 相似文献