共查询到20条相似文献,搜索用时 15 毫秒
1.
Henning K. Andersen Valerie Urbach Emmy Van Kerkhove Ena Prosser Brian J. Harvey 《Pflügers Archiv : European journal of physiology》1995,431(1):52-65
With the single-channel patch-clamp technique we have identified Ca2+-sensitive, high-conductance (maxi) K+ channels in the basolateral membrane (BLM) of exocrine gland cells in frog skin. Under resting conditions, maxi K+ channels were normally quiescent, but they were activated by muscarinic agonists or by high serosal K+. In excised inside-out patches and with symmetrical 140 mmol/l K+, single-channel conductance was 200 pS and the channel exhibited a high selectivity for K+ over Na+. Depolarization of the BLM increased maxi K+ channel activity. Increasing cytosolic free Ca2+ (by addition of 100 nmol/l thapsigargin to the bathing solution of cell-attached patches also increased channel activity, whereas thapsigargin had no effect when added to excised inside-out patches. An increase in cytosolic free Ca2+ directly activated channel activity in a voltage-dependent manner. Maxi K+ channel activity was sensitive to changes in intracellular pH, with maximal activity at pH 7.4 and decreasing activities following acidification and alkalinization. Maxi K+ channel outward current was reversibly blocked by micromolar concentrations of Ba2+ from the cytosolic and extracellular site, and was irreversibly blocked by micromolar concentrations of charybdotoxin and kaliotoxin from the extracellular site in outside-out patches. 相似文献
2.
Ove Christensen Thomas Zeuthen 《Pflügers Archiv : European journal of physiology》1987,408(3):249-259
We have studied a Ca2+-activated K+ channel in the ventricular membrane of the epithelium of choroid plexus by means of the patch-clamp technique, using excised inside-out patches. The channel was highly K+ selective. It had a conductance of 200 pS with 112 mM KCl on both sides of the membrane. The probability for the channel being open increased with intracellular Ca2+, pH and with membrane potential. The channel shows two gating modes. The primary gating mode has open and closed times which depend strongly on membrane potential, intracellular Ca2+ and pH. It accounts for the variation of the channel open probability. Lowering intracellular pH from 7.4 to 6.4 reduced the channel open probability mainly by increasing the channel closed time. It appears, that H+ can compete with Ca2+ in binding to the same site, thereby preventing channel opening. A second gating mode consisted of short-lived closures, or flickers. The open and closed time for this process were largely independent of membrane potential, intracellular Ca2+ and pH. The channel density was 0.4 m–2 corresponding to a K+-permeability of 2.2 10–5 cm s–1 if the channels were fully open. In cell-attached patches we measured the open probability of the channel in the intact cell membrane. The channel is almost totally closed under normal cellular conditions. This type of channel is therefore not the membrane component that forms the electrodiffusive pathway for K+-ions. 相似文献
3.
Experiments were designed to characterize the cellular mechanisms of action of endothelium-derived vasodilator substances in the rabbit femoral artery. Acetylcholine (ACh, 10(-8)-10(-5) M) induced a concentration-dependent relaxation of isolated endothelium-intact arterial rings precontracted with norepinephrine (NE, 10(-6) M). The ACh-induced response was abolished by the removal of endothelium. NG-nitro-L-arginine (L-NAME, 10(-4) M), an inhibitor of NO synthase, partially inhibited ACh-induced endothelium-dependent relaxation, whereas indomethacin (10(-5) M) showed no effect on ACh-induced relaxation. 25 mM KCl partially inhibited ACh-induced relaxation by shifting the concentration-response curve and abolished the response when combined with L-NAME and NE. In the presence of L-NAME, ACh-induced relaxation was unaffected by glibenclamide (10(-5) M) but significantly reduced by apamin (10(-6) M), and almost completely blocked by tetraethylammonium (TEA, 10(-3) M), iberiotoxin (10(-7) M) and 4-aminopyridine (4-AP, 5 x 10(-3) M). The cytochrome P450 inhibitors, 7-ethoxyresorufin (7-ER, 10(-5) M) and miconazole (10(-5) M) also significantly inhibited ACh-induced relaxation. Ouabain (10(-6) M), an inhibitor of Na+, K(+)-ATPase, or K(+)-free solution, also significantly inhibited ACh-induced relaxation. ACh-induced relaxation was not significantly inhibited by 18-alpha-glycyrrhetinic acid (18 alpha-GA, 10(-4) M). These results of this study indicate that ACh-induced endothelium-dependent relaxation of the rabbit femoral artery occurs via a mechanism that involves activation of Na+, K(+)-ATPase and/or activation of both the voltage-gated K+ channel (Kv) and the large-conductance, Ca(2+)-activated K+ channel (BKCa). The results further suggest that EDHF released by ACh may be a cytochrome P450 product. 相似文献
4.
Willy Van Driessche Mrs. J. De Beir-Simaels 《Pflügers Archiv : European journal of physiology》1987,410(3):243-249
Previously (Van Driessche et al. 1987) we showed that small inward (mucosa towards serosa) oriented shortcircuit currents (I
sc) were recorded through the toad urinary bladder when the mucosal side was exposed to Ca2+ free solutions containing K+, Na+ (+amiloride), Cs+ or Rb+ as main cation. This current component is inhibitable by micromolar concentrations of mucosal La3+ and divalent cations (Ca2+, Cd2+) and is considerably elevated by oxytocin (0.1 U/ml). The present study demonstrates that the addition of 50 nmol/l Ag+ to the mucosal medium during oxytocin treatment caused an additional large increase of the La3+-sensitiveI
sc component. The power density spectrum of the fluctuation in current contained a Lorentzian component which was enhanced by oxytocin treatment. The Lorentzian component disappeared as a consequence of the administration of mucosal Ag+. In experiments with Ca2+, Ba2+ or Mg2+ as principal mucosal cation, the La3+-sensitiveI
sc component was negligible under control conditions and during oxytocin treatment. Mucosal Ag+ (40 nmol/l) elicited a large inward oriented current which was blockable by the calcium channel blockers, La3+ and Cd2+. Also the organic calcium entry blockers, nicardipine and verapamil (10 mol/l) depressed the inward current considerably. Noise analysis of the currents carried by divalent cations showed a La3+-sensitive noise component. Oxytocin-Ag+ activated currents could not be recorded in the absence of the divalent cations or small inorganic cations, e.g. with solutions which contained N-methyld-glucamine (NMDG) as main mucosal cation. 相似文献
5.
Impalement studies in isolated perfused cortical collecting ducts (CCD) of rats have shown that the basolateral membrane possesses a K+ conductive pathway. In the present study this pathway was investigated at the single-channel level using the patch-clamp technique. Patch-clamp recordings were obtained from enzymatically isolated CCD segments and freshly isolated CCD cells with the conventional cell-free, cell-attached and the cell-attached nystatin method. Two K+ channels were found which were highly active on the cell with a conductance of 67±5 pS (n=18) and 148±4 pS (n=21) with 145 mmol/l K+ in the pipette. In excised patches the first channel had a conductance of 28±2 pS (n=15), whereas the second one had a conductance of 85±1 pS (n=53) at 0 mV clamp voltage with 145 mmol/l K+ on one side and 3.6 mmol/l K+ on the other side of the membrane. So far it has not been possible to characterize the smaller channel further. Excised, and with symmetrical K+ concentrations of 145 mmol/l, the intermediate channel had a linear conductance of 198±19 pS (n=5). After excision in the inside-out configuration the open probability (P
o) of this channel was low (0.18±0.05, n=13) whereas in the outside-out configuration this channel had a threefold higher P
o (0.57±0.04, n=12). Several inhibitors were tested in excised membranes. Ba2+ (1 mmol/l), tetraethylammonium (TEA+, 10 mmol/l) and verapamil (0.1 mmol/l) all blocked this channel reversibly. Furthermore P
o was reversibly reduced by 10 nmol/l charybdotoxin (outside-out). This K+ channel of the basolateral membrane was regulated by cellular pH. P
o was reduced to 26±3% at pH 6.5 (n=6) and increased to 216±18% at pH 8.5 (n=7) compared to pH 7.4. Half-maximal inhibition was reached at pH 7.0. The channel had its highest P
o at a Ca2+ activity of less than 10–8 mol/l (n=13). Increasing the Ca2+ activity to 1 mmol/l on the cytosolic side of the membrane resulted in a reduction of P
o to 13±3% (n=11). Half-maximal inhibition was reached at a Ca2+ activity of 10–5 mol/l. The high activity of both K+ channels of the basolateral membrane on the cell indicates that they may serve for K+ recirculation across the basolateral membrane. 相似文献
6.
W. Berger R. Grygorcyk W. Schwarz 《Pflügers Archiv : European journal of physiology》1984,402(1):18-23
Attempts have been made to apply the patch-clamp technique to enzymatically dispersed smooth muscle cells of frog and toad stomach. The rate of successful gigaseal formation has been extremely low, but better results can be obtained when patches are taken from membrane evaginations which develop on single cells after mechanical agitation and incubation in Ca2+-containing solutions at 25° C. Also ball-shaped single cells formed by the confluence of membrane evaginations were found to be equally useful for patch-clamp studies. Giga-seal formation was obtained in more than 80% of all attempts. Electron micrographs indicate that the myofilaments in membrane evaginations an in ball-shaped cells are separated from the cell membrane. Channel activity in membrane patches of such myoballs or evaginations is similar to the channel activity as found in intact cells. Two types of K+ channels (100 and 200 pS) have been observed that can be blocked by tetraethylammonium. Channels with the conductance of 200 pS are activated by intracellular Ca2+. The formation of evaginations has also been observed in other cells and may help to apply the patch-clamp technique to cells contaminated with surface coats. 相似文献
7.
Ingrid De Wolf Willy Van Driessche 《Pflügers Archiv : European journal of physiology》1988,413(2):111-117
The voltage-dependence of the inhibitory effect of mucosal Cs+ on the inward K+ current through the apical membrane of frog skin (Rana temporaria) was studied by recording transepithelial current-voltage relations. Experiments were performed with skins exposed to NaCl and KCl Ringer solutions on the serosal and mucosal side respectively (contron skins), as well as with tissues incubated with K2SO4 Ringer solutions on both sides (depolarized skins). Studies of the dose-depedence of the Cs+ block showed that under both experimental conditions the apparent affinity of Cs+ increased as the transepithelial potential was clamped at higher mucosal positive voltages. Under control conditions, the concentration of Cs+ required to block 50% of the K+ current (KCs) recorded while the transepithelial voltage was clamped at zero mV was 16 mmol/1. KCs decreased exponentially with muscosal positive voltages. The dependence of KCs on the membrane potential was analyzed with Eyring rate theory in which Cs+ was assumed to block the K+ transport by binding to a site within the channel. The analysis showed that this site is located at a relative electrical distance =0.32 of the voltage drop across the apical membrane, measured from the cytosolic side. The Hill coefficient obtained from this analysis wasn=3.1. Experiments with K+-depolarized tissues showed that only inward K+ currents recorded with positive transepithelial voltages were depressed by external Cs+. Also under these conditions KCs showed an exponential dependence on the transepithelial potential. The analysis of these data with the rate theory revealed =0.09 andn=1.7. The difference in found in control and depolarized tissues can be explained by the influence of the basolateral membrane resistance on theI–V relations. 相似文献
8.
Impaired absorption of sodium (Na+) and water is a major factor in the pathogenesis of diarrhoea in ulcerative colitis (UC). Electrogenic Na+ absorption, present mainly in human distal colon and rectum, is defective in UC, but the molecular basis for this is unclear. The effect of UC on the expression of apical Na+ channels (ENaC) and basolateral Na+, K+-ATPase, the critical determinants of electrogenic Na+ transport, was therefore investigated in this study. Sigmoid colonic and/or proximal rectal mucosal biopsies were obtained from patients with mild to moderate UC, and patients with functional abdominal pain (controls). ENaC subunit expression was studied by immunohistochemistry, western blot analysis, and in situ hybridization, and Na+, K+-ATPase isoform expression was studied by immunohistochemistry, western blotting, and northern blot analysis. UC was associated with substantial decreases in the expression of the ENaC beta- and gamma-subunit proteins and mRNAs, whereas the decrease in ENaC alpha-subunit protein detected by immunolocalization was less marked. The levels of expression of Na+, K+-ATPase alpha1- and beta1-isoform proteins were also lower in UC patients than in controls, although there were no differences in Na+, K+-ATPase alpha1- and beta1-isoform mRNA levels between the two groups. Taken together, these results show that UC results mainly in decreased expression of the apical ENaC beta- and gamma-subunits, as well as the basolateral Na+, K+-ATPase alpha1- and beta1-isoforms. In conclusion, these changes provide a basis for the low/negligible levels of electrogenic Na+ absorption seen in the distal colon and rectum of UC patients, which contribute to the pathogenesis of diarrhoea in this disease. 相似文献
9.
Junichi Taniguchi Ken -Ichi Furukawa Munekazu Shigekawa 《Pflügers Archiv : European journal of physiology》1993,423(3-4):167-172
By using a patch clamp technique, we examined the effect of cyclic guanosine monophosphate (cGMP)-dependent protein kinase (G kinase) on Ca2+-activated maxi K+ channels in canine coronary artery smooth muscle cells. Maxi K+ channels (274±4 pS in symmetrical 140 mM KCl at 24–26°C) were activated by cytoplasmic Ca2+ and were completely blocked by 100 nM charybdotoxin (CTX). G kinase (300 U/ml) added to the cytoplasmic face of the membrane patch shifted the voltage dependence of these channels by about 25 mV in the negative direction in the presence of 1 M Ca2+, 50 M cGMP and 1 mM magnesium adenosine triphosphate. At –50 mV and 1 M Ca2+, G kinase treatment increased the mean number of open channels 4.5-fold compared with the control. -Human atrial natriuretic peptide (ANP, 100 nM) reduced the isometric tension of coronary arterial rings elicited by 14 or 24 mM KCl, but failed to relax the artery contracted by 34 mM KCl. Addition of 100 nM CTX augmented tension development elicited by 24 mM KCl and totally prevented ANP from relaxing the arterial rings. These results indicate that G kinase-dependent protein phosphorylation activates maxi K+ channels in canine coronary smooth muscle, and further suggest that the G kinase-induced activation of maxi K+ channels may cause hyperpolarization and relaxation of coronary artery. 相似文献
10.
Stimulation-induced transport of K+ in the submandibular salivary gland of cats and dogs anesthetized with pentobarbital was studied with an extracellular K+-specific microelectrode. Electrical stimulation of the para-sympathetic chorda-lingual nerve caused a rapid transient increase in extracellular K+ concentration from 2.2 to 18.7 meq/liter in the cat and from 2.3 to 15.2 meq/liter in the dog. Eventually the K+ concentration fell below the prestimulatory level, indicating uptake of K+ by the gland cells. In case of prolonged stimulation (2-10 min), the uptake began during stimulation. However, a further reduction in extracellular K+ concentration occurred upon cessation of stimulation, a result that demonstrated that the cells did not fully recover their K+ ,content during stimulation. The latency of the release of K+, defined as the time from the beginning of stimulation to the point at which, the K+-specific microelectrode signal had increased by 2 mV, was 0.6 s in the cat and 0.8 s in the dog. Because these are overestimates of the "true" latencies, we conclude that the K+ release begins simultaneously with the hyperpolarization of the acinar cell membrane. 相似文献
11.
12.
Ambriz-Rivas M Islas LD Gomez-Lagunas F 《Pflügers Archiv : European journal of physiology》2005,450(4):255-261
K+ depletion exerts dramatically variable effects on different potassium channels. Here we report that Shab channels are rather stable in the absence of either internal or external K+ alone; however, its stability is greater with K+ outside the cell. In contrast, with 0 K+ (non-added) solutions on both sides of the membrane, the conductance (GK) is rapidly and irreversibly lost. GK is lost with the channels closed and regardless of the composition of the 0 K+ solutions. In comparison, it is known that the Shaker B GK collapses only if the channels are gated in 0 K+, Na+-containing solutions. In order to compare the behavior of Shab to that of Shaker, we show that after extensively gating the channels in 0 K+ N-methyl-D-glucamine solutions, most Shaker channels remain stable, and in a conformation where GK collapses as soon as there is Na+ in the solutions. Regarding ion conduction, in contrast to Kv2.1 and Shaker A463C that have a sizable GNa in 0 K+, Shab, which shares a 463-cysteine and an identical signature sequence with these channels, does not appreciably conduct Na+, although it presents a significant Cs+ conductance. The observations suggest that there are at least two sites where K+ binds and thus maintains Shab GK stable, one internal and the other(s) most likely located outside the selectivity filter. 相似文献
13.
Microelectrode study of voltage-dependent Ba2+ and Cs+ block of apical K+ channels in the skin of Rana temporaria 总被引:1,自引:0,他引:1
Willy Van Driessche Ingrid De Wolf 《Pflügers Archiv : European journal of physiology》1991,418(4):400-407
The blockage of the apical K+ channels in frog species Rana temporaria by Ba2+ and Cs+ is strongly voltage-dependent. The interaction of both blockers with the K+ channels was studied by recording relations between the K+ currents (I
K) and the transepithelial and intracellular potential. Mucosal Ba2+ and Cs+ depress I
K, hyperpolarize the cell and induce pronounced nonlinearities in the current/voltage (I/V) relations. The nonlinearities are caused by the voltage-dependent interaction of Ba2+ and Cs+ with the binding site. Consequently, the apical membrane resistance not only depends on the blocker concentration but also on the apical membrane potential. Also the fractional resistance, fR
a, and the voltage divider ratio, fV
a, will change with blocker concentration and voltage. Owing to this non-ohmic behaviour, measurements of fV
a in the presence of Ba2+ deviate markedly from the expected fR
a values. The inhibitory effect of Ba2+ and Cs+ was analysed at different transepithelial and apical membrane voltages. The relation between the Michaelis-Menten constants and the voltage could be fitted with equations based on Eyring rate theory with the assumption of a single binding site. With this model we calculated the relative electrical position of the binding site for the blocker (), referred to the extracellular side of the channel. We obtained for Ba2+, =0.34±0.05 and for Cs+, =0.81±0.01. Comparison of the results from apical and transepithelial I/V relations demonstrates that the analysis of the transepithelial data provides overestimated values of the Hill coefficient and results in an underestimation of . 相似文献
14.
Using the patch-clamp technique, Na+ selective channels were observed in the luminal membrane of rabbit straight proximal tubule segments. In the cell-attached configuration (NaCl-Ringers in pipette and bath) influx of Na+ ions from the pipette into the cell through fluctuating channels was observed was observed. The current-voltage curve of these Na+ channels yielded a zero-current potential of 84.3±30.9 mV (n=10), reflecting the electrochemical driving force for Na+ influx under resting conditions. The single channel conductance was 12.0±2.1 pS (n=13). In inside-out oriented cell-excised patches the single channel conductance was not significantly different with NaCl-Ringers on both sides. At clamp potentials ranging from +50 mV to –50 mV the single channel current was ohmic and channel kinetics were independent of the voltage. With KCl-Ringers on the bath side (corresponding to cell interior), the zero current potential was 62±19 mV (n=4), indicating a high selectivity of Na+ over K+ ions. Addition of 10–5 mol/l amiloride to the bathing solution decreased the mean channel open time slightly. This effect was more pronounced with 10–4 mol/l amiloride, whereas the single channel conductance was unaffected by the diuretic. 10–3 mol/l amiloride caused a complete block of the channel. It is concluded that amiloride sensitive Na+ channels, with similar properties to those observed in tight epithelia, contribute to Na+ reabsorbtion in the straight portion of proximal tubules. 相似文献
15.
Ca2+-and voltage activated K+ channel in apical cell membrane of gallbladder epithelium fromTriturus
Y. Maruyama H. Matsunaga T. Hoshi 《Pflügers Archiv : European journal of physiology》1986,406(6):563-567
The presence of Ca2+- and voltage-activated K+ channels was directly demonstrated in the apical cell membrane of gallbladder epithelium by patch-clamp single-channel current recording. In K+-depolarized epithelial cells, negative pipette potentials induced outward current steps when the patch-pipette was filled with Na+-rich solution and these current steps were not affected by the presence or absence of Cl–. When K+-rich solution was in the pipette and K+-depolarized cells were examined, the current-voltage relations were linear with a single-channel conductance of 140 pS and polarity was reversed at 0 mV. In excised inside-out membrane patches, raising the free Ca2+ concentration of the medium facing the inner side of the membrane from 10–7 to 10–6 M evoked a marked increase in open state probability of the channels without affecting the elementary current steps. This suggests that intracellular Ca2+ as a second messenger plays a crucial role in the regulatory mechanism of the membrane potential by modulating the high-conductance apical K+ channels. 相似文献
16.
S. Yoshida S. Plant A. I. McNiven C. R. House 《Pflügers Archiv : European journal of physiology》1990,415(4):516-518
The properties of the Ca2+-activated K+ channel in unfertilized hamster oocytes were investigated at the single-channel level using inside-out excised membrane patches. The results indicate a new type of Ca2+-activated K+ channel which has the following characteristics: (1) single-channel conductance of 40–85 pS for outward currents in symmetrical K+ (150 mM) solutions, (2) inward currents of smaller conductance (10–50 pS) than outward currents, i.e. the channel is outwardly rectified in symmetrical K+ solutions, (3) channel activity dependent on the internal concentration of free Ca+ and the membrane potential, (4) modification of the channel activity by internal adenosine 5 diphosphate (0.1 mM) producing a high open probability regardless of membrane potential. 相似文献
17.
Fatima Atia Wolfgang Zeiske W. Van Driessche 《Pflügers Archiv : European journal of physiology》1999,438(3):344-353
Distal kidney cells (A6) from Xenopus laevis were cultured to confluency on porous supports. Tissues were mounted in Ussing-type chambers to measure short-circuit current
(I
sc), transepithelial conductance and capacitance, and to analyse the fluctuation in I
sc. In the absence of apical NaCl, but with normal basolateral NaCl Ringer’s solution, extracellular addition of ATP, oxytocin,
a membrane-permeant cAMP derivative, and forskolin produced a transient increase of the electrical parameters. Noise analysis
revealed a spontaneous Lorentzian component. All responses depend strictly on the presence of basolateral Cl– and are caused by the activation of an apical (CFTR type) Cl– permeability. Repetitive treatment with ATP (or oxytocin) resulted in refractoriness. ATP and oxytocin acted antagonistically,
whereas cAMP and ATP had additive effects. Incubation with the vesicular Ca2+ pump inhibitor thapsigargin or application of the Ca2+ channel blocker nifedipine elicited finite but variable Cl– channel activity. After treatment with nifedipine or thapsigargin, the response to oxytocin was severely impaired. We speculate
that not only cAMP but also cell Ca2+ plays a crucial role in the activation of CFTR in A6. Ca2+ may be multifunctional but the rise in capacitance (apical area) observed with all stimulants strongly suggests its involvement
in, and contribution to, exocytosis in the process of the CFTR-mediated transcellular Cl– movements.
Received: 30 November 1998 / Received after revision: 23 February 1999 / Accepted: 12 March 1999 相似文献
18.
In patch-clamp records of K+
ATP channels in an insulin-secreting cell line (RINm5F) inhibition evoked by exposing the internal surface of the membrane to ATP is followed not just by the recovery of K+
ATP channel activity when the ATP is removed but by a marked activation of K+
ATP channels. This phenomenon is not a direct consequence of channel closure as inhibition induced by quinidine and quinine is followed upon the removal of the drug only by the recovery of K+
ATP channel activity and not by post-inhibitory activation. If ATP is applied to the exposed internal surface of a membrane patch when all of its K+
ATP channel have run down subsequent removal of the ATP causes their activation. The magnitude and duration of the reactivation of K+
ATP channels is shown to depend upon both the concentration of ATP and the length of time for which the membrane is exposed to ATP. We therefore have a paradoxical situation in that K+ channels which are inhibited by intracellular ATP require intracellular ATP to retain the ability to open. 相似文献
19.
Toru Ishikawa Masataka Murakami Yoshiteru Seo 《Pflügers Archiv : European journal of physiology》1994,428(5-6):516-525
The involvement of large-conductance, voltage- and Ca2+-activated K+ channels (maxi-K+ channels) in basolateral Ca2+-dependent K+-efflux pathways and fluid secretion by the rat submandibular gland was investigated. Basolateral K+ efflux was monitored by measuring the change in K+ concentration in the perfusate collected from the vein of the isolated, perfused rat submandibular gland every 30 s. Under conditions in which the Na+/K+-ATPase and Na+-K+-2Cl– cotransporter were inhibited by ouabain (1 mmol/l) and bumeta-nide (50 mol/l) respectively, continuous stimulation with acetylcholine (ACh) (1 mol/l) caused a transient large net K+ efflux, followed by a smaller K+ efflux, which gradually returned to the basal level within 10 min. These two components of the K+ efflux appear to be dependent on an increase in cytosolic Ca2+ concentration. The initial transient K+ efflux was not affected by charybdotoxin (100 nmol/l) or tetraethylammonium (TEA) (5 mmol/l) but the smaller second component was strongly and reversibly inhibited by charybdotoxin (100 nmol/l) and TEA (0.1 and 5 mmol/l). The initial K+ efflux transient induced by ACh was inhibited by quinine (0.1–3 mmol/l), quinidine (1–3 mmol/l) and Ba2+ (5 mmol/l), but not by verapamil (0.1 mmol/l), lidocaine (1 mmol/l), 4-aminopyridine (1 mmol/l) or apamin (1 mol/l). Ca2+-dependent transient large K+ effluxes induced by substance P (0.01 mol/l) and A23187 (3 mol/l) were not inhibited by TEA (5 mmol/l or 10 mmol/l). A23187 (3 mol/l) evoked a biphasic fluid-secretory response, which was not inhibited by TEA (5 mmol/l). Patch-clamp studies confirmed that the whole-cell outward K+ current attributable to maxi-K+ channels obtained from rat submandibular endpiece cells was strongly inhibited by the addition of TEA (1–10 mmol/l) to the bath. It is concluded that maxi-K+ channels are not responsible for the major part of the Ca2+-dependent basolateral K+ efflux and fluid secretion by the rat submandibular gland. 相似文献
20.
The basolateral membrane of rabbit straight proximal tubules, which were cannulated and perfused on one side, was investigated with the patch clamp technique. Properties of inward and outward directed single K+ channel currents were studied in cell-attached and insideout oriented cell-excised membrane patches. In cell-attached patches with NaCl Ringer solution both in pipette and bath, outward K+ currents could be detected after depolarization of the membrane patch by about 20–30 mV. The current-voltage (i/V) relationship could be fitted by the Goldman-Hodgkin-Katz (GHK) current equation, with the assumption that these channels were mainly permeable for K+ ions. A permeability coefficientP
K of (0.17±0.04) · 10–12 cm3/s was obtained, the single channel slope conductance at infinite positive potentialg(V
) was 50±12 pS and the single channel conductance at the membrane resting potentialg(V
bl) was 12±3 pS (n=4). In cell-excised patches, with NaCl in the pipette and KCl in the bath, the data could also be fitted to the GHK equation and yieldedP
K = (0.1 ±0.01) ·10–12 cm3/s,g(V
) = 40 ± 4 pS andg(V
bl) = 7 ± 1 pS (n=8). In cell-attached patches with KCl in the pipette and NaCl in the bath, inward K+ channels occurred at clamp potentials 60 mV, whereas outward K+ channel current was detected at more positive voltages. The current-voltage curves showed slight inward rectification. The single channel conductance, obtained from the linear part of the i/V curve by linear regression, was 46±3 pS and the reversal potential was 59±6 mV (n=9). In cell-excised patches with KCl in the pipette and NaCl in the bath, inward directed K+ channel currents could again be described by the GHK equation. The single channel parameters were similar to those recorded for outward K+ currents (see above). In inside-out oriented cell-excised patches with NaCl in the pipette and KCl in the bath, reducing bath (i.e. cytosolic) Ca2+ concentration from 10–6 mol/l to less than 10–9 mol/l did not affect the open state probability of single channel currents. These results demonstrate that the observed channels are permeable for K+ ions in both directions and that these basolateral K+ channels in rabbit proximal straight tubule are not directly dependent on Ca2+ ions. 相似文献