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1.
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
2.
J. Hirsch J. Leipziger U. Fröbe E. Schlatter 《Pflügers Archiv : European journal of physiology》1993,422(5):492-498
In the luminal membrane of rat cortical collecting duct (CCD) a big Ca2+-dependent and a small Ca2+-independent K+ channel have been described. Whereas the latter most likely is responsible for the K+ secretion in this nephron segment, the function of the large-conductance K+ channel is unknown. The regulation of this channel and its possible physiological role were examined with the conventional cell-free and the cell-attached nystatin patch-clamp techniques. Patch-clamp recordings were obtained from the luminal membrane of isolated perfused CCD segments and from freshly isolated CCD cells. Intracellular calcium was measured using the calcium-sensitive dye fura-2. The large-conductance K+ channel was strongly voltage- and calcium-dependent. At 3 mol/l cytosolic Ca2+ activity it was half-maximally activated. At 1 mmol/l it was neither regulated by cytosolic pH nor by ATP. At 1 mol/l Ca2+ activity the open probability (P
o) of this channel was pH-dependent. At pH 7.0 P
o was decreased to 4±2% (n=9) and at pH 8.5 it was increased to 425±52% (n=9) of the control. At this low Ca2+ activity the P
o of the channel was reduced by 1 mmol/l ATP to 8±4% (n=6). Cell swelling activated the large-conductance K+ channel (n=14) and hyperpolarized the membrane potential of the cells by 9±1 mV (n=23). Intracellular Ca2+ activity increased after hypotonic stress. This increase depended on the extracellular Ca2+ activity. A possible physiological function of the large-conductance K+ channel in rat CCD cells may be the reduction of the intracellular K+ concentration after cell swelling. Once this channel is activated by increases in the cytosolic Ca2+ activity it can be regulated by changes in cellular pH and ATP.Supported by DFG Schl 277/2-3 相似文献
3.
Takako Ohno-Shosaku Chosaburo Yamamoto 《Pflügers Archiv : European journal of physiology》1992,422(3):260-266
To determine whether membranes of mammalian central neurons contain an ATP-sensitive K+ (KATP) channel similar to that present in pancreatic cells, the patch-clamp technique was applied to cultured neurons prepared from the neonatal rat cerebral cortex and hippocampus. In whole-cell experiments with hippocampal neurons, extracellular application of 0.5 mM diazoxide (a KATP channel activator) elicited a hyperpolarization concomitant with an increase in membrane conductance, whereas application of 0.5 mM tolbutamide (a KATP channel blocker) induced a depolarization with a decrease in conductance. Similar results were obtained with cortical neurons. In outside-out patch experiments with cortical neurons, a K+ channel sensitive to these drugs was found. The channel was completely blocked by 0.5 mM tolbutamide and activated by 0.5 mM diazoxide. The single-channel conductance was 65 pS under symmetrical 145 mM K+ conditions and 24 pS in a physiological K+ gradient. In inside-out patch experiments, this channel was demonstrated to be inhibited by an application of 0.2–1 mM ATP to the cytoplasmic surface of the patch membrane. These results indicate that the membranes of rat cortical neurons contain a KATP channel that is quite similar to that found in pancreatic cells. It is also suggested that the same or a similar K+ channel may exist in membranes of hippocampal neurons. 相似文献
4.
E. Schlatter S. Haxelmans J. Hirsch J. Leipziger 《Pflügers Archiv : European journal of physiology》1994,428(5-6):631-640
The K+ channels of the principal cells of rat cortical collecting duct (CCD) are pH sensitive in excised membranes. K+ secretion is decreased with increased H+ secretion during acidosis. We examined whether the pH sensitivity of these K+ channels is present also in the intact cell and thus could explain the coupling between K+ and H+ secretion. Membrane voltages (V
m), whole-cell conductances (g
c), and single-channel currents of K+ channels were recorded from freshly isolated CCD cells or isolated CCD segments with the patch-clamp method. Intracellular pH (pHi) was measured using the pH-sensitive fluorescent dye 2-7-bis(carboxyethyl)-5-6-carboxyfluorescein (BCECF). Acetate (20 mmol/l) had no effect on V
m, g
c, or the activity of the K+ channels in these cells. Acetate, however, acidified pHi slightly by 0.17±0.04 pH units (n=19). V
m depolarized by 12±3 mV (n=26) and by 23±2 mV (n=66) and g
c decreased by 26±5% (n=13) and by 55±5% (n=12) with 3–5 or 8–10% CO2, respectively. The same CO2 concentrations decreased pHi by 0.49±0.07 (n=15) and 0.73±0.11 pH units (n=12), respectively. Open probability (P
o) of all four K+ channels in the intact rat CCD cells was reversibly inhibited by 8–10% CO2. pHi increased with the addition of 20 mmol/l NH4
+/NH3 by a maximum of 0.64±0.08 pH units (n=33) and acidified transiently by 0.37±0.05 pH units (n=33) upon NH4
+/NH3 removal. In the presence of NH4
+/NH3
V
m depolarized by 16±2 mV (n=66) and g
c decreased by 26±7% (n=16). The activity of all four K+ channels was also strongly inhibited in the presence of NH4
+/NH3. The effect of NH4
+/NH3 on V
m and g
c was markedly increased when the pH of the NH4
+/NH3-containing solution was set to 8.5 or 9.2. From these data we conclude that cellular acidification in rat CCD principal cells down-regulates K+ conductances, thus reduces K+ secretion by direct inhibition of K+ channel activity. This pH dependence is present in all four K+ channels of the rat CCD. The inhibition of K+ channels by NH4
+/NH3 is independent of changes in pHi and rather involves an effect of NH3. 相似文献
5.
T. Leinders H. P. M. Vijverberg 《Pflügers Archiv : European journal of physiology》1992,422(3):223-232
Single-channel properties of Ca2+-activated K+ channels have been investigated in excised membrane patches of N1E-115 mouse neuroblastoma cells under asymmetric K+ concentrations at 0 mV. The SK channels are blocked by 3 nM external apamin, are unaffected by 20 mM external tetraethylammonium (TEA) and have a single-channel conductance of 5.4 pS. The half-maximum open probability and opening frequency of SK channels are observed at 1 M internal Ca2+. Concentration/effect curves of these parameters are very steep with exponential slope factors between 7 and 13. Opentime distributions demonstrate the existence of at least two open states. The mean short open time increases with [Ca2+]i, whereas the mean long open time is independent of [Ca2+]i. At low [Ca2+]i the short-lived open state predominates. At saturating [Ca2+]i the number of longlived openings is more enhanced than the number of short-lived openings and both open states occur equally frequently. The opening frequency as well as the open times of SK channels are independent of the membrane potential in the range of –16 to +40 mV. The results indicate that activation of K+ current through SK channels is mainly determined by the Ca2+-dependent single-channel opening frequency. BK channels in N1E-115 cells are insensitive to 100 nM external apamin, are sensitive to external TEA in the millimolar range and have a single-channel conductance of 98 pS. Half-maximum open probability and opening frequency of the BK channel are observed at 7.5–21 M internal Ca2+. The slope factors of concentration/effect curves range between 1.7 and 2.9. As the BK channel open time is markedly enhanced at raised [Ca2+]i, the Ca2+ dependence of the current through BK channels is determined by the single-channel opening frequency as well as the open time. SK as well as BK channels appear to be clustered and interact in a negative cooperative manner in multiple channel patches. The differences in Ca2+ dependence suggest that BK channels are activated by a local high [Ca2+]i associated with Ca2+ influx, whereas SK channels may be activated by Ca2+ released from internal stores as well. 相似文献
6.
Jos A. H. Verheugen Regina G. D. M. van Kleef Marga Oortgiesen Henk P. M. Vijverberg 《Pflügers Archiv : European journal of physiology》1994,426(6):465-471
Ca2+-activated K+ [K(Ca)] channels were studied in excised patches of resting and activated human peripheral blood T lymphocytes. The K(Ca) channel had a single-channel conductance of 50±6 pS in symmetrical high-K+ solutions in the potential range of –100 to –10 mV and was inwardly rectifying at more depolarized potentials. The channel was sensitive to block by charybdotoxin (10 nM) and insensitive to apamin (3 nM). Half-maximum activation occurred at an internal free Ca2+ concentration of 360±110 nM. The concentration-effect curve had a slope factor of 0.83±0.12, suggesting a 11 interaction of Ca2+ ions with the channel. Ca2+ affects the open time probability of the K(Ca) channels, mainly by modulating the frequency of channel opening. The open probability did not show voltage dependence. The kinetics of the channel could be described assuming one open state and two closed states. The time constant of the exponential describing the open time distribution amounted to 2.8±1.2 ms, whereas the closed time distribution could be described with two exponentials with time constants of 0.2±0.05 ms and 8.0±2.1 ms, respectively. Resting T lymphocytes expressed a low number of channels but the density of channels increased dramatically during chronic phytohaemagglutinin stimulation. 相似文献
7.
Alfred H. Gitter Klaus W. Beyenbach Chadwick W. Christine Peter Gross Will W. Minuth Ebrhard Frömter 《Pflügers Archiv : European journal of physiology》1987,408(3):282-290
Using the patch clamp technique, one type of K+ channel was identified in the apical cell membrane of cultured principal cells of rabbit renal collecting ducts in the cell-attached or excised-patch configuration. The channel was highly selective for K+ over Na+ (typically 30-70-fold) and had a conductance of 180, SD±39 pS (n=6), referred to a situation of 140 mmolar K+-Ringer solution present on either surface of the patch membrane. Channel activity was completely blocked by Ba2+ (5 mmol/l) and partially inhibited by Na+. The latter was evidenced by a deviation from Goldman rectification at high cytoplasm-positive membrane potentials, which was observed when Na+ competed with K+ for channel entrace from the cytoplasmic surface. Channel open probability depended strongly on membrane voltage and cytoplasmic Ca2+ concentration. Open-close kinetics exhibited double exponential behaviour, with a strong voltage dependence of the slow open time constant. Infrequently also a substate conductance level was identified. The voltage and calcium dependence suggest that the channel plays a role in adjusting K+ secretion to Na+ absorption in the fine regulation of cation excretion in renal collecting ducts. 相似文献
8.
Luis Vaca William P. Schilling Diana L. Kunze 《Pflügers Archiv : European journal of physiology》1992,422(1):66-74
The purpose of the present study was to determine the mechanism by which bradykinin activates the small conductance, inwardly rectifying, Ca2+-activated K+ channel (KCa) found in cultured bovine aortic endothelial cells. Channel activity was studied using the patch-clamp technique in whole-cell, cell-attached, inside-out and outside-out configurations. Channel conductance at potentials positive to 0 mV was 10±2 pS and at potentials negative to 0 mV 30±3 pS (n=7) when examined in symmetrical K+ (150 mmol/l) solutions. The channel open probability (P
o) was only weakly voltage dependent changing approximately 0.2 units over 160 mV. In contrast, raising the intracellular Ca2+ concentration from 100 nmol/l to 10 mol/l at –60 mV produced a graded increase in channel P
o from 0.15 to 0.96; the concentration required for half-maximum response (apparent K0.5) was 719 nmol/l. At a constant Ca2+ concentration, application of guanosine triphosphate (GTP) to the cytoplasmic surface of the patch increased channel P
o. This effect was dependent upon the simultaneous presence of both GTP and Mg2+, and was reversed by the subsequent application of the guanosine diphosphate (GDP) analogue, guanosine-5-O-(2-thiodiphosphate) (GDPS). The hydrolysis-resistant GTP analogue, guanosine-5-O-(3-thiotriphosphate) (GTPS), induced a long-lasting increase in channel P
o. In the presence of Mg2+-GTP, the apparent K0.5 for Ca2+ decreased from a control value of 722 nmol/l to 231 nmol/l. Addition of bradykinin to outside-out patches previously exposed to intracellular Mg2+-GTP further enhanced KCa activity, shifting the apparent K0.5 for Ca2+ from 228 nmol/l to 107 nmol/l. This activation by bradykinin was not observed in patches following prior exposure to GDPS. These results suggest that bradykinin can activate the KCa channel of vascular endothelial cells via a G-protein-mediated change in the sensitivity of the channel for Ca2+. We postulate that vasoactive agonists may use this mechanism to maintain an elevated K+ permeability as the intracellular Ca2+ concentration returns towards normal resting levels. 相似文献
9.
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
10.
Debebe Gebremedhin Pierre Bonnet Andrew S. Greene Sarah K. England Nancy J. Rusch Julian H. Lombard David R. Harder 《Pflügers Archiv : European journal of physiology》1994,428(5-6):621-630
The cellular mechanisms mediating hypoxia-induced dilation of cerebral arteries have remained unknown, but may involve modulation of membrane ionic channels. The present study was designed to determine the effect of reduced partial pressure of O2, PO
2, on the predominant K+ channel type recorded in cat cerebral arterial muscle cells, and on the diameter of pressurized cat cerebral arteries. A K+-selective single-channel current with a unitary slope conductance of 215 pS was recorded from excised inside-out patches of cat cerebral arterial muscle cells using symmetrical KCl (145 mM) solution. The open state probability (NP
o) of this channel displayed a strong voltage dependence, was not affected by varying intracellular ATP concentration [(ATP]i) between 0 and 100 M, but was significantly increased upon elevation of intracellular free Ca2+ concentration ([Ca2+]i). Low concentrations of external tetraethylammonium (0.1–3 mM) produced a concentration-dependent reduction of the unitary current amplitude of this channel. In cell-attached patches, where the resting membrane potential was set to zero with a high KCl solution, reduction of O2 from 21% to < 2% reversibly increased the NP
o, mean open time, and event frequency of the Ca2+-sensitive, high-conductance single-channel K+ current recorded at a patch potential of + 20 mV. A similar reduction in PO2 also produced a transient increase in the activity of the 215-pS K+ channel measured in excised inside-out patches bathed in symmetrical 145 mM KCl, an effect which was diminished, or not seen, during a second application of hypoxic superfusion. Hypoxia had no effect on [Ca2+]i or intracellular pH (pHi) of cat cerebral arterial muscle cells, as measured using Ca2+- or pH-sensitive fluorescent probes. Reduced PO2 caused a significant dilation of pressurized cerebral arterial segments, which was attenuated by pre-treatment with 1 mM tetraethylammonium. These results suggest that reduced PO2 increases the activity of a high-conductance, Ca2+-sensitive K+ channel in cat cerebral arterial muscle cells, and that these effects are mediated by cytosolic events independent of changes in [Ca2+]i and pHi. 相似文献
11.
Eberhard Schlatter Emanuel Lohrmann Rainer Greger 《Pflügers Archiv : European journal of physiology》1992,420(1):39-45
In this study we examined by impalement techniques properties of the macroscopic K+ conductances in the luminal and basolateral membrane of principal cells from isolated perfused cortical collecting ducts (CCD) of the rat. Both membranes possess a dominating K+ conductance. Compared to their behaviour with K+, both membranes appear much less permeable to NH
4
+
and Rb+, and the K+ conductances of both membranes are inhibited by these cations. In light of these findings, it is very unlikely that significant amounts of NH
4
+
, which is secreted in the CCD, cross the principal cells as NH
4
+
. Several inhibitors with known effects on K+ channels in patch-clamp studies have been examined. Tetraethylammonium, which inhibits the excised K+ channels of these cells, has no effect on the macroscopic K+ conductances of either membrane. Verapamil, which inhibits the K+ channels in the luminal membrane, acts predominantly on the basolateral membrane K+ conductance in the intact tubule. Therefore, some of the macroscopic properties of the K+ conductances are distinct from those of single channels thus far observed in patchclamp studies.Supported by DFG Schl 277/2-1 and Gr 480/10 相似文献
12.
Takako Ohno-Shosaku Takahiro Kubota Jun Yamaguchi Masaaki Fukase Takuo Fujita Mamoru Fujimoto 《Pflügers Archiv : European journal of physiology》1989,413(5):562-564
Using the patch clamp technique, we identified an inwardly rectifying K+ channel in the membrane of opossum kidney cells. The single channel conductance was about 90 pS for inward currents and 30 pS for outward currents under a symmetrical high-K+ condition. The activity of the channel was found to decrease with time during recording from inside-out patches. In the solution with submicromolar Ca2+, the activity disappeared within 4–20 min. Intracellular Ca2+ promoted the run-down of the channel activity at 0.1–1 mM, whereas millimolar Mg-ATP restored the activity after run-down. The run-down channels could never be reactivated by ATP in the absence of Mg2+, or by a nonhydrolyzable ATP analog, AMPPNP, even in the presence of Mg2+. 相似文献
13.
M. Bleich E. Schlatter R. Greger 《Pflügers Archiv : European journal of physiology》1990,415(4):449-460
In vitro perfused rat thick ascending limbs of Henle's loop (TAL) were used (n=260) to analyse the conductance properties of the luminal membrane applying the patch-clamp technique. Medullary (mTAL) and cortical (cTAL) tubule segments were dissected and perfused in vitro. The free end of the tubule was held and immobilized at one edge by a holding pipette kept under continuous suction. A micropositioner was used to insert a patch pipette into the lumen, and a gigaohm seal with the luminal membrane was achieved in 455 instances out of considerably more trials. In approximately 20% of all gigaohm seals recordings of single ionic channels were obtained. We have identified only one single type of K+ channel in these cell-attached and cell-excised recordings. In the cell-attached configuration with KCl or NaCl in the pipette, the channel had a conductance of 60±6 pS (n=24) and 31±7 pS (n=4) respectively. In cell-free patches with KCl either in the patch pipette or in the bath and with a Ringer-type solution (NaCl) on the opposite side the conductance was 72±4 pS (n=37) at a clamp voltage of 0 mV. The permeability was 0.33±0.02 · 10±12 cm3/s. The selectivity sequence für this channel was: K+=Rb+=NH
4
+
=Cs+>Li+Na+=0; the conductance sequence was K+Li+Rb+=Cs+= NH
4
+
=Na+=0. In excised patches Rb+, Cs+ and NH
4
+
when present in the bath at 145 mmol/l all inhibited K+ currents out of the pipette. The channel kinetics were described by one open (9.5±1.5 ms, n=18) and by two closed (1.4±0.1 and 14±2 ms) time constants. The open probability of this channel was increased by depolarization. The channel open probability was reduced voltage dependently by Ba2+ (half maximal inhibition at 0 mV: 0.07 mmol/l) from the cytosolic side. Verapamil, diltiazem, quinine and quinidine inhibited at approximately 1 mol/l ±0.1 mmol/l from either side. Similarly, the amino cations lidocaine, tetraethylammonium and choline inhibited at 10–100 mmol/l. The channel was downregulated in its open probability by cytosolic Ca2+ activities > 10±7 mol/l and by adenosine triphosphate 10±4 mol/l. The open probability was downregulated by decreasing cytosolic pH (2-fold by a decrease in pH by 0.2 units). The described channel differs in several properties from the K+ channels of other epithelia and of renal cells and TAL cells in culture. It appears to be responsible for K+ recycling in the TAL segment.Preliminary reports of the present study have been given at the following conferences: Tagung der Deutschen Physiologischen Gesellschaft, Würzburg, October 1988; Membranforum, Frankfurt, April 1989; 3rd Int. Conf. Diur., Mexico City, April 1989; 3rd Nephrology Forefront Symposium, Arrola, July, 1989; IUPS meeting, Helsinki, July 1989. This study has been supported by Deutsche Forschungsgemeinschaft Grant No. Gr 480/9 相似文献
14.
Variations of membrane cholesterol alter the kinetics of Ca2+-dependent K+ channels and membrane fluidity in vascular smooth muscle cells 总被引:9,自引:0,他引:9
V. Bolotina V. Omelyanenko B. Heyes U. Ryan P. Bregestovski 《Pflügers Archiv : European journal of physiology》1989,415(3):262-268
The patch-clamp technique and fluorescence polarization analysis were used to study the dependence of Ca2+-dependent K+ channel kinetics and membrane fluidity on cholesterol (CHS) levels in the plasma membranes of cultured smooth muscle rabbit aortic cells. Mevinolin (MEV), a potent inhibitor of endogenous CHS biosynthesis was used to deplete the CHS content. Elevation of CHS concentration in the membrane was achieved using a CHS-enriching medium. Treatment of smooth muscle cells with MEV led to a nearly twofold increase in the rotational diffusion coefficient of DPH (D) and to about a ninefold elevation of probability of the channels being open (P
o). The addition of CHS to the cells membrane resulted in a nearly twofold decrease in D and about a twofold decrease in P
o. Elementary conductance of the channels did not change under these conditions. These data suggest that variations of the CHS content in the plasma membrane of smooth muscle cells affect the kinetic properties of Ca2+-dependent K+ channels presumably due to changes in plasma membrane fluidity. Our results give a possible explanation for the reported variability of Ca2+-dependent K+ channels kinetics in different preparations. 相似文献
15.
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
16.
Single channel Ca2+ currents inHelix pomatia neurons 总被引:4,自引:0,他引:4
Unitary Ca2+ currents of TEA injected Helix neurons were recorded in the Giga seal situation (6, 7) from microscopic membrane patches exposed to 50 mM [Ca2+]o, O [Na+]o, 20 mM [TEA+]o and 2.5 M [TTX]o. Constant field assumptions yield a channel permeability of 2.9±1.0×10–14 cm3s–1 corresponding to slope conductances of 5 to 15 pS between 0 and –30 mV. Frequency of occurrence of the units strongly increased with depolarization. Mean open time of the Ca2+ channels was about 3 ms without obvious dependence on voltage. A similar open time was seen with [Ba2+]o, yielding about double the current strength when compared with [Ca2+]o. 相似文献
17.
E. Schlatter U. Fröbe R. Greger 《Pflügers Archiv : European journal of physiology》1992,421(4):381-387
The study of ion conductances in the intact cortical collecting duct (CCD) with the patch-clamp method is rather difficult. An optimized method to isolate CCD cells from rat kidneys using an in vivo followed by an in vitro enzyme digestion is described. Individual CCD segments were collected after this digestion and incubated in EGTA-buffered medium. This procedure resulted in single cells or cell clusters. These freshly isolated CCD cells were studied with different modifications of the patch-clamp method. Membrane voltages measured in the cell-attached-nystatin configuration were –74 ±1mV (n=13) and –68±3 mV (n=22) in cells isolated from normal and mineralocorticoid-treated rats respectively. These values and those measured with the nystatin-perforated slow-whole-cell configuration (–79 ±1mV, n=23) are comparable to those measured in principal cells of isolated CCD segments. The cells hyperpolarized after the addition of amiloride and depolarized with the addition of adiuretin to the bath. The amiloride effect was enhanced when cells were isolated from deoxycorticosterone-acetate-treated rats. The cells were strongly depolarized upon elevation of the extracellular K+-concentration and did not demonstrate a measurable Cl– conductance. A large-conductance K+ channel (174 pS, n=5, cell-attached, 145 mmol/l K+ in the pipette; 140 pS, n=12, cell-free, 3.6 mmol/l K+ in the bath) was seen. It had a very low activity on the cell, but a high open probability when excised into a solution with 1 mmol/l Ca2+ on the cytosolic side. More often a small-conductance K+ channel (36–52 pS, n=19, cell-attached; 30 pS, n=5, cell-free) with a high open probability was found on the cell. These freshly isolated cells seem to be a powerful preparation to study the properties and regulation of ion conductances of rat CCD with several electrophysiological methods. These freshly isolated CCD cells maintain the conductance properties known from principal cells of the intact CCD. 相似文献
18.
Alfredo Villarroel Osvaldo Alvarez Andres Oberhauser Ramon Latorre 《Pflügers Archiv : European journal of physiology》1988,413(2):118-126
A series of quaternary amonium (QA) ions were used to probe the gross architecture of the ion conduction pathway in a Ca2+-activated K+ channel from rat muscle membrane. The channels were inserted into planar phospholipid membranes and the single channel currents were measured in the presence of the different QA ions. Internally applied monovalent QA ions (e.g. tetramethylammonium and analogues) induced a voltage-dependent blockade with a unique effective valence of the block equal to 0.30, and blocking potency increases as the compound is made more hydrophobic. Blockade is relieved by increasing the K+ concentration of the internal or external side of the channel. The effective valence of block is independent of K+ concentration. These results suggest that, from the internal side, all monovalent QA ions interact with a site located in the channel conduction system. Divalent QA ions of the type n-alkylbis-,-trimethylammonium (bisQn) applied internally also block the channel in a voltage dependent fashion. For short chains (bisQ2-bisQ5), the effective valence decreases with chain length from 0.41 to 0.27, it remains constant for bisQ5 to bisQ6 and increases up to 0.54 for bisQ10. This dependence of block with chain length implies that 27% of the voltage drop within the channel occurs over a distance of 1 nm. Externally applied monovalent QA ions also block the channel. The site is specific for tetraethylammonium; increasing or decreasing the side chains in one methylene group decrease potency by about 400-fold. It is concluded that the Ca2+-activated K+ channel has wide mouths located at each end and that they are different in molecular nature. 相似文献
19.
Large-conductance Ca2+-activated K+ channels were studied in excised inside-out membrane patches from adult mouse skeletal muscle. The channels had a conductance
of about 250 pS in symmetrical 155 mM KCl solutions. They showed gating characteristics similar to those described for this
type of channel in rat and rabbit skeletal muscle. Polymyxin B, a cyclic polypeptide antibiotic, produced a voltage-dependent
block, whereas polymyxin E was only slightly effective. The concentration at which half-maximal blockage occurred was very
iow: 0.5 μg/ml at a voltage of + 30 mV. The blockage occurred with a Hill coefficient ofh=1.2. At negative membrane potentials, polymyxin B caused the appearance of a substate with a conductance of about 10% of
the fully open state. The mode of blockage is discussed and compared to the effect of polymyxin B on glucose uptake into the
muscle cell. 相似文献
20.
H. Lerche Ch. Fahlke P. A. Laizzo F. Lehmann-Horn 《Pflügers Archiv : European journal of physiology》1995,429(5):738-747
Ca2+-activated K+ channels of a large conductance (BKCa) in human skeletal muscle were studied by patch clamping membrane blebs and by using the three microelectrode voltage-clamp recording technique on resealed fibre segments. Single-channel recordings in bleb-attached and inside-out modes revealed BKCa conductances of 230 pS for symmetrical and 130 pS for physiological K+ distributions. Open probability increased with membrane depolarization and increasing internal [Ca2+]. The Hill coefficient was 2.0, indicating that at least two Ca2+ ions are required for full activation. Kinetic analysis revealed at least two open and three closed states. An additional long-lived inactivated state, lasting about 0.5–20 s, was observed following large depolarizations, when extracellular K+ was lowered to physiological values. BKCa were blocked by three means: (1) externally by tetraethylammonium which reduced single-channel amplitude (IC50 approx. 0.3 mM); (2) internally by polymyxin B which decreased the open probability (IC50 approx. 5 g/ml); and (3) externally by charybdotoxin which caused long-lasting periods of inactivation (IC50 <10 nM). Measurements on resealed fibre segments at physiological [K+] were in accordance with the single-channel data: only when intracellular [Ca2+] was elevated did charybdotoxin (50 nM) reduce the macroscopic membrane K+ conductance with depolarizing voltage steps. 相似文献