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1.
Summary Using the patch-clamp technique we have identified a Ca2+-sensitive, voltage-dependent, maxi-K+ channel on the basolateral surface of rat pancreatic duct cells. The channel had a conductance of 200 pS in excised patches bathed in symmetrical 150mm K+, and was blocked by 1mm Ba2+. Channel openstate probability (P o ) on unstimulated cells was very low, but was markedly increased by exposing the cells to secretin, dibutyryl cyclic AMP, forskolin or isobutylmethylxanthine. Stimulation also shifted theP o /voltage relationship towards hyperpolarizing potentials, but channel conductance was unchanged. If patches were excised from stimulated cells into the inside-out configuration,P o remained high, and was not markedly reduced by lowering bath (cytoplasmic) Ca2+ concentration from 2mm to 0.1 m. However, activated channels were still blocked by 1mm Ba2+. ChannelP o was also increased by exposing the cytoplasmic face of excised patches to the purified catalytic subunit of cyclic AMP-dependent protein kinase., We conclude that cyclic AMP-dependent phosphorylation can activate maxi-K+ channels on pancreatic duct cells via a stable modification of the channel protein itself, or a closely associated regulatory subunit, and that phosphorylation alters the responsiveness of the channels to Ca2+. Physiologically, these K+ channels may contribute to the basolateral K+ conductance of the duct cell and, by providing a pathway for current flow across the basolateral membrane, play an important role in pancreatic bicarbonate secretion.  相似文献   

2.
Summary Potassium channels in membranes of isolatedNecturus enterocytes were studied using the patch-clamp technique. The most frequent channel observed had a conductance of 170 pS and reversal potential of 0 mV in symmetrical potassium-rich solutions. Channels were highly K+ selective. Channel activity was modulated by membrane potential and cytosolic Ca2+ concentration. Channel openings occurred in characteristic bursts separated by long closures. During bursts openings were interrupted by brief closures. Two gating modes controlled channel opening. The primary gate's sensitivity to intracellular Ca2+ concentration and membrane potential crucially determined long duration closures and bursting. In comparison, the second gate determining brief closures was largely insensitive to voltage and intracellular Ca2+ concentration. The channel was reversibly blocked by cytosolic barium exposure in a voltage-sensitive manner. Blockade reduced open-state probability without altering single-channel conductance and could be described, at relatively high Ca2+ concentration, by a three-state model where Ba2+ interacted with the open channel with a dissociation constant of about 10–4 m at 0 mV.  相似文献   

3.
Summary Four types of nonvoltage-activated potassium channels in the body-wall muscles ofDrosophila third instar larvae have been identified by the patch-clamp technique. Using the inside-out configuration, tetraethylammonium (TEA). Ba2+, and quinidine were applied to the cytoplasmic face of muscle membranes during steady-state channel activation. The four channels could be readily distinguished on the basis of their pharmacological sensitivities and physiological properties. The KST channel was the only type that was activated by stretch. It had a high unitary conductance (100 pS in symmetrical 130/130mm KCl solution), was blocked by TEA (K d 35mm), and was the most sensitive to Ba2+ (complete block at 10–4 m). A Ca2+-activated potassium channel. KCF 72pS (130/130mm KCl), was gated open at>10–8 m Ca2+, was the least sensitive to Ba2+ (K d of 3mm) and TEA (K d of 100mm), and was not affected by quinidine. K2 was a small conductance channel of 11 pS (130/2 KCl, pipette/bath), and was very sensitive to quinidine, being substantially blocked at 0.1mm. It also exhibited a half block at 0.3mm Ba2+ and 25mm TEA. A fourth channel type, K3, was the most sensitive to TEA (half block<1mm). It displayed a partial block to Ba2+ at 10mm, but no block by 0.1mm quinidine. The blocking effects of TEA, Ba2+ and quinidine were reversible in all channels studied. The actions of TEA and Ba2+ appeared qualitatively different: in all four channels. TEA reduced the apparent unitary conductance, whereas Ba2+ decreased channel open probability.  相似文献   

4.
Summary K+ channels in cultured rat pancreatic islet cells have been studied using patch-clamp single-channel recording techniques in cell-attached and excised inside-out and outside-out membrane patches. Three different K+-selective channels have been found. Two inward rectifier K+ channels with slope conductances of about 4 and 17 pS recorded under quasi-physiological cation gradients (Na+ outside, K+ inside) and maximal conductances recorded in symmetrical K+-rich solutions of about 30 and 75 pS, respectively. A voltage- and calcium-activated K channel was recorded with a slope conductance of about 90 pS under the same conditions and a maximal conductance recorded in symmetrical K+-rich solutions of about 250 pS. Single-channel current recording in the cell-attached conformation revealed a continuous low level of activity in an apparently small number of both the inward rectifier K+ channels. But when membrane patches were excised from the intact cell a much larger number of inward rectifier K+ channels became transiently activated before showing an irreversible decline. In excised patches opening and closing of both the inward rectifier K+ channels were unaffected by voltage, internal Ca2+ or externally applied tetraethyl-ammonium (TEA) but the probability of opening of both inward rectifier K+ channels was reduced by internally applied 1–5mm adenosine-5-triphosphate (ATP). The large K+ channel was not operational in cell-attached membrane patches, but in excised patches it could be activated at negative membrane potentials by 10–7 to 10–6 m internal Ca2+ and blocked by 5–10mm external TEA.  相似文献   

5.
Summary Calcium-activated potassium channels were the channels most frequently observed in primary cultured normal mammary cell and in the established mammary tumor cell, MMT060562. In both cells, single-channel and whole-cell clamp recordings sometimes showed slow oscillations of the Ca2+-gated K+ current. The characteristics of the Ca2+-activated K+ channels in normal and cancerous mammary cells were quite similar. The slope conductances changed from 8 to 70 pS depending on the mode of recording and the ionic composition in the patch electrode. The open probability of this channel increased between 0.1 to 1 m of the intracellular Ca2+, but it was independent of the membrane potential.Charybdotoxin reduced the activity of the Ca2+-activated K+ channel and the oscillation of the membrane current, but apamin had no apparent effect. The application of tetraethylammonium (TEA) from outside and BaCl2 from inside of the cell diminished the activity of the channel. The properties of this channel were different from those of both the large conductance (BK or MAXI K) and small conductance (SK) type Ca2+-activated K+ channels.  相似文献   

6.
Summary The Ca2+-activated K+ channel in rat pancreatic islet cells has been studied using patch-clamp single-channel current recording in excised inside-out and outside-out membrane patches. In membrane patches exposed to quasi-physiological cation gradients (Na+ outside, K+ inside) large outward current steps were observed when the membrane was depolarized. The single-channel current voltage (I/V) relationship showed outward rectification and the null potential was more negative than –40 mV. In symmetrical K+-rich solutions the single-channelI/V relationship was linear, the null potential was 0 mV and the singlechannel conductance was about 250 pS. Membrane depolarization evoked channel opening also when the inside of the membrane was exposed to a Ca2+-free solution containing 2mm EGTA, but large positive membrane potentials (70 to 80 mV) were required in order to obtain open-state probabilities (P) above 0.1. Raising the free Ca2+ concentration in contact with the membrane inside ([Ca2+]i) to 1.5×10–7 m had little effect on the relationship between membrane potential andP. When [Ca2+]i was increased to 3×10–7 m and 6×10–7 m smaller potential changes were required to open the channels. Increasing [Ca2+]i further to 8×10–7 m again activated the channels, but the relationship between membrane potential andP was complex. Changing the membrane potential from –50 mV to +20 mV increasedP from near 0 to 0.6 but further polarization to +50 mV decreasedP to about 0.2. The pattern of voltage activation and inactivation was even more pronounced at [Ca2+]i=1 and 2 m. In this situation a membrane potential change from –70 to +20 mV increasedP from near 0 to about 0.7 but further polarization to +80 mV reducedP to less than 0.1. The high-conductance K+ channel in rat pancreatic islet cells is remarkably sensitive to changes in [Ca2+]i within the range 0.1 to 1 m which suggests a physiological role for this channel in regulating the membrane potential and Ca2+ influx through voltage-activated Ca2+ channels.  相似文献   

7.
Using the two-microelectrode voltage clamp technique in Xenopus laevis oocytes, we estimated Na+-K+-ATPase activity from the dihydroouabain-sensitive current (I DHO) in the presence of increasing concentrations of tetraethylammonium (TEA+; 0, 5, 10, 20, 40 mm), a well-known blocker of K+ channels. The effects of TEA+ on the total oocyte currents could be separated into two distinct parts: generation of a nonsaturating inward current increasing with negative membrane potentials (V M) and a saturable inhibitory component affecting an outward current easily detectable at positive V M. The nonsaturating component appears to be a barium-sensitive electrodiffusion of TEA+ which can be described by the Goldman-Hodgkin-Katz equation, while the saturating component is consistent with the expected blocking effect of TEA+ on K+ channels. Interestingly, this latter component disappears when the Na+-K+-ATPase is inhibited by 10 m DHO. Conversely, TEA+ inhibits a component of I DHO with a k d of 25±4 mm at +50 mV. As the TEA+-sensitive current present in I DHO reversed at –75 mV, we hypothesized that it could come from an inhibition of K+ channels whose activity varies in parallel with the Na+-K+-ATPase activity. Supporting this hypothesis, the inward portion of this TEA+-sensitive current can be completely abolished by the addition of 1 mm Ba2+ to the bath. This study suggests that, in X. laevis oocytes, a close link exists between the Na-K-ATPase activity and TEA+-sensitive K+ currents and indicates that, in the absence of effective K+ channel inhibitors, I DHO does not exclusively represent the Na+-K+-ATPase-generated current.  相似文献   

8.
Summary Current noise power spectra of the voltage-clamped (V=0)Necturus gallbladder, exposed to NaCl-Ringer's on both sides contained a relaxation noise component, which overlapped with a 1/f noise component, with being about 2. Substitution of all Na+ by K+ on either the serosal or mucosal side increased the relaxation as well as the 1/f noise component considerably. InNecturus gallbladder both noise components are reduced by addition of 10mm 2,4,6-triaminopyrimidine (TAP) or 5mm of tetraethylammonium (TEA+) added to ification of the mucosal solution to pH 5 and lower. Fivemm of tetraethylammonium (TEA+) added to the mucosal solution, abolished K+ relaxation noise and decreased the 1/f noise component. Applying a Cs+ concentration gradient across the epithelium did not yield relaxation noise. However, if Rb+ was substituted for all Na+ on one side, a Lorentzian noise component appeared in the spectrum. Its plateau was smaller than with KCl-Ringer's on the respective side. These data confirm the existence of fluctuating K+ channels in the apical membrane of theNecturus gallbladder. Furthermore it can be concluded that these channels have the permeability sequence K+>Rb+>Sc+. The inhibition of the fluctuations by mucosal acidification indicates the existence of acidic sites in the channel. The single-channel conductance was estimated to be between 6.5 and 40 pS.  相似文献   

9.
Summary Human red cells were prepared with various cellular Na+ and K+ concentrations at a constant sum of 156mm. At maximal activation of the K+ conductance,g K(Ca), the net efflux of K+ was determined as a function of the cellular Na+ and K+ concentrations and the membrane potential,V m , at a fixed [K+]ex of 3.5mm.V m was only varied from (V m E K)25 mV and upwards, that is, outside the range of potentials with a steep inward rectifying voltage dependence (Stampe & Vestergaard-Bogind, 1988).g K(Ca) as a function of cellular Na+ and K+ concentrations atV m =–40, 0 and 40 mV indicated a competitive, voltage-dependent block of the outward current conductance by cellular Na+. Since the present Ca2+-activated K+ channels have been shown to be of the multi-ion type, the experimental data from each set of Na+ and K+ concentrations were fitted separately to a Boltzmann-type equation, assuming that the outward current conductance in the absence of cellular Na+ is independent of voltage. The equivalent valence determined in this way was a function of the cellular Na+ concentration increasing from 0.5 to 1.5 as this concentration increased from 11 to 101mm. Data from a previous study of voltage dependence as a function of the degree of Ca2+ activation of the channel could be accounted for in this way as well. It is therefore suggested that the voltage dependence ofg K(Ca) for outward currents at (V m E K)>25 25 mV reflects a voltage-dependent Na+ block of the Ca2+-activated K+ channels.  相似文献   

10.
Summary The tight-seal whole-cell recording method has been used to studyNecturus choroid plexus epithelium. A cell potential of –59±2 mV and a whole cell resistance of 56±6 M were measured using this technique. Application of depolarizing step potentials activated voltage-dependent outward currents that developed with time. For example, when the cell was bathed in 110mm NaCl Ringer solution and the interior of the cell contained a solution of 110mm KCl and 5nm Ca2+, stepping the membrane potential from a holding value of –50 to –10 mV evoked outward currents which, after a delay of greater than 50 msec, increased to a steady state in 500 msec. The voltage dependence of the delayed currents suggests that they may be currents through Ca2+-activated K_ channels. Based on the voltage dependence of the activation of Ca2+-activated K+ channels, we have devised a general method to isolate the delayed currents. The delayed currents were highly selective for K+ as their reversal potential at different K+ concentration gradients followed the Nernst potential for K+. These currents were reduced by the addition of TEA+ to the bath solution and were eliminated when Cs+ or Na+ replaced intracellular K+. Increasing the membrane potential to more positive values decreased both the delay and the half-times (t 1/2) to the steady value. Increasing the pipette Ca2+ also decreased the delay and decreasedt 1/2. For instance, when pipette Ca2+ was increased from 5 to 500nm, the delay andt 1/2 decreased from values greater than 50 and 150 msec to values less than 10 and 50 msec. We conclude that the delayed currents are K+ currents through Ca2+-activated K+ channels.At the resting membrane potential of –60 mV, Ca2+-activated K+ channels contribute between 13 to 25% of the total conductance of the cell. The contribution of these channels to cell conductance nearly doubles with membrane depolarization of 20–30 mV. Such depolarizations have been observed when cerebrospinal fluid (CSF) secretion is stimulated by cAMP and with intracellular Ca2+. Thus the Ca2+-activated K+ channels may play a specific role in maintaining intracellular K+ concentrations during CSF secretion.  相似文献   

11.
Summary Patch-clamp methods were used to study single-channel events in isolated oxyntic cells and gastric glands fromNecturus maculosa. Cell-attached, excised inside-out and outside-out patches from the basolateral membrane frequently contained channels which had conductances of 67±21 pS in 24% of the patches and channels of smaller conductance, 33±6 pS in 56% of the patches. Channels in both classes were highly selective for K+ over Na+ and Cl, and shared linear current-voltage relations. The 67-pS channel was activated by membrane depolarization, whereas the activity of the 33-pS channel was relatively voltage independent. The larger conductance channels were activated by intracellular Ca2+ in the range between 5 and 500nm, but unaffected by cAMP. The smaller conductance channels were activated by cAMP, but not Ca2+. The presence of K+ channels in the basolateral membrane which are regulated by these known second messengers can account for the increase in conductance and the hyperpolarization of the membrane observed upon secretagogue stimulation.  相似文献   

12.
Xylem parenchyma cells are situated around the (apoplastic) xylem vessels and are involved in the control of the composition of the xylem sap by exporting and resorbing solutes. We investigated properties of the K+ inward rectifier in the plasma membrane of these cells by performing patch clamp experiments on protoplasts in the whole-cell configuration. Inward currents were sensitive to the K+ channel blocker TEA+ at a high concentration (20 mm). Barium, another classical K+ channel blocker, inhibited K+ currents with a K i of about 1.3 mm. In contrast to guard cells, the cytosolic Ca2+ level proved to be ineffective in regulating the K+ conductance at hyperpolarization. External Ca2+ blocked currents weakly in a voltage-dependent manner. From instantaneous current-voltage curves, we identified a binding site in the channel pore with an electrical distance of about 0.2 to 0.5. Lanthanum ions reduced the inward current in a voltage-dependent manner and simultaneously displaced the voltage at which half of the channels are in the open state to more positive values. This finding was interpreted as resulting from a sum of two molecular effects, an interaction with the mouth of the channel that causes a reduction of current, and a binding to the voltage sensor, leading to a shielding of surface charges and, subsequently, a modulation of channel gating.A comparison between the K+ inward rectifier in xylem parenchyma cells, guard cells and KAT1 from Arabidopsis leads to the conclusion that these rectifiers form subtypes within one class of ion channels. The ineffectiveness of Ca2+ to control K+ influx in xylem parenchyma cells is interpreted in physiological terms.  相似文献   

13.
14.
Summary Using patch-clamp techniques, we have studied Ca2+-activated K+ channels in the basolateral membrane of freshly isolated epithelial cells from rabbit distal colon. Epithelial cell clusters were obtained from distal colon by gentle mechanical disruption of isolated crypts. Gigaohm seals were obtained on the basolateral surface of the cell clusters. At the resting potential (approximately –45 mV), with NaCl Ringer's bathing the cell, the predominant channels had a conductance of 131±25 pS. Channel activity depended on voltage as depolarization of the membrane increased the open probability. In excised inside-out patches, channels were found to be selective for K+ over Na+. Channel activity correlated directly with bath Ca2+ concentration in the excised patches. Channel currents were blocked by 5mm TEA+ and 1mm Ba2+. In cell-attached patches, after addition of the Ca2+ ionophore A23187, which increases intracellular Ca2+, open probability was markedly increased. Channel activity was also regulated by cAMP as addition of 1mm dibutyryl-cAMP in the bath solution in cell-attached patches increased channel open probability over 20-fold. Channels that had been activated by cAMP were further activated by Ca2+. We conclude that the basolateral membrane of epithelial cells from descending colon contains a class of potassium channels, which are regulated by intracellular Ca2+ and cAMP.  相似文献   

15.
Summary A simple procedure was developed for the isolation of a sarcolemma-enriched membrane preparation from homogenates of bullfrog (Rana catesbeiana) heart. Crude microsomes obtained by differential centrifugation were fractionated in Hypaque density gradients. The fraction enriched in surface membrane markers consisted of 87% tightly sealed vesicles. The uptake of86Rb+ by the preparation was measured in the presence of an opposing K+ gradient using a rapid ion exchange technique. At low extravesicular Rb+ concentrations, at least 50% of the uptake was blocked by addition of 1mm ouabain to the assay medium. Orthovanadate (50 m), ADP (2.5mm), or Mg (1mm) were also partial inhibitors of Rb+ uptake under these conditions, and produced a complete block of Rb+ influx in the presence of 1mm ouabain. When86Rb+ was used as a tracer of extravesicular K+ (Rb 0 + 40 m K 0 + =0.1–5mm) a distinct uptake pathway emerged, as detected by its inhibition by 1mm Ba2+ (K 0.5=20 m). At a constant internal K+ concentration (K in + =50mm) the magnitude of the Ba2+-sensitive K+ uptake was found to depend on K 0 + in a manner that closely resembles the K+ concentration dependence of the background K+ conductance (I Kl) observed electrophysiologically in intact cardiac cells. We conclude that K+ permeates passively this preparation through two distinct pathways, the sodium pump and a system identifiable as the background potassium channel.  相似文献   

16.
Summary Ca2+-activated K+ channels were studied in cultured medullary thick ascending limb (MTAL) cells using the patch-clamp technique in the inside-out configuration. The Ca2+ activation site was modified using N-bromoacetamide (NBA). 1mm NBA in the bath solution, at 2.5 m Ca2+ reduces the open probability,P o , of the channel to <0.01, without an effect on single-channel conductance. NBA-modified channels are still Ca2+-sensitive, requiring 25mm Ca2+ to raiseP o to 0.2. Both before and after NBA modification channel openings display at least two distributions, indicative of more than one open state. High Ca2+ (1mm) protects the channels from modification. Also presented is a second class of Ca2+-activated K+ channels which are normally present in MTAL cells which open infrequently at 10 m Ca2+ (P o =0.01) but have aP o of 0.08 at 1mm Ca2+. We can conclude (i) that NBA modifies the channel by shifting Ca2+-sensitivity to very high Ca2+, (ii) that NBA acts on a site involved in Ca2+ gating, and (iii) that a low affinity channel is present in the apical cell membrane with characteristics similar to those of normal channels modified with NBA.  相似文献   

17.
Summary The effects of short (1 sec) and long (1 min) transepithelial current clamps on membrane voltages and resistances ofNecturus gallbladder were investigated. Transepithelial and cell membrane current-voltage relationships determined from 1-sec clamps revealed that: a) depolarization of the apical membrane voltage (V mc) results in a marked decrease in apical membrane fractional resistance (fR a), whereas hyperpolarization ofV mc results in either no change infR a or a small increase, and b) the voltage-dependent changes infR a are essentially complete within 500 msec. Exposure of the tissue to 5mm TEA+ on the mucosal side caused no significant change in baselineV mc (–69±2 mV) and yet virtually abolished the voltage dependence offR a. A possible interpretation of these results is that two types of K+ channels exist in the apical membrane, with different voltage dependencies and TEA+ sensitivities. Acidification or Ba2+ addition to the mucosal solution also reduced the voltage-dependent changes infR a. The time courses of the changes infR a and in the cable properties of the epithelium were assessed during 1-min transepithelial current clamps (±200 A/cm2). No secondary change infR a was observed with mucosa-to-serosa currents, but a slow TEA+-sensitive decrease infR a (half-time of seconds) was evident with serosa-to-mucosa currents. Cable analysis experiments demonstrated that the initial (<500 msec) voltage-dependent decrease infR a is due to a fall in apical membrane resistance. The later decrease infR a is due to changes in both cell membrane resistances attributable to the increase in transcellular current flow resulting from a fall in paracellular conductance. The voltage dependence of the apical membrane conductance is a more significant problem in estimatingfR a than the current-induced effects on the lateral intercellular spaces. In principle, TEA+ can be used to prevent the nonlinear behavior ofR a during measurements of the voltage divider or membrane resistance ratio.  相似文献   

18.
Summary Using Ca2+- and K+-selective microelectrodes, the cytosolic free Ca2+ and K+ concentrations were measured in mouse fibroblastic L cells. When the extracellular Ca2+ concentration exceeded several micromoles, spontaneous oscillations of the intracellular free Ca2+ concentration were observed in the submicromolar ranges. During the Ca2+ oscillations, the membrane potential was found to oscillate concomitantly. The peak of cyclic increases in the free Ca2+ level coincided in time with the peak of periodic hyperpolarizations. Both oscillations were abolished by reducing the extracellular Ca2+ concentration down to 10–7 m or by applying a Ca2+ channel blocker, nifedipine (50 m). In the presence of 0.5mm quinine, an inhibitor of Ca2+-activated K+ channel, sizable Ca2+ oscillations still persisted, while the potential oscillations were markedly suppressed. Oscillations of the intracellular K+ concentration between about 145 and 140mm were often associated with the potential oscillations. The minimum phase of the K+ concentration was always 5 to 6 sec behind the peak hyperpolarization. Thus, it is concluded that the oscillation of membrane potential results from oscillatory increases in the intracellular Ca2+ level, which, in turn, periodically stimulate Ca2+-activated K+ channels.  相似文献   

19.
Summary The apical membrane K+ permeability of the newt proximal tubular cells was examined in the doubly perfused isolated kidney by measuring the apical membrane potential change (V a change) during alteration of luminal K+ concentration and resultant voltage deflections caused by current pulse injection into the lumen.V a change/decade for K+ was 50 mV at K+ concentration higher than 25mm, and the resistance of the apical membrane decreased bt 58% of control when luminal K+ concentration was increased from 2.5 to 25mm. Ba2+ (1mm in the lumen) reducedV a change/decade to 24 mV and increased the apical membrane resistance by 70%. These data support the view that Ba2+-sensitive K+ conductance exists in the apical membrane of the newt proximal tubule. Furthermore, intracellular K+ activity measured by K+-selective electrode was 82.4 ± 3.6 meq/liter, which was higher than that predicted from the Nernst equation for K+ across both cell membranes. Thus, it is concluded that cell K+ passively diffuses, at least in part, through the K+ conductive pathway of the apical membrane.  相似文献   

20.
Elevated levels of intracellular Ca2+ activate a K+-selective permeability in the membrane of human erythrocytes. Currents through single channels were analysed in excised inside-out membrane patches. The effects of several ions that are known to inhibit K+ fluxes are described with respect to the single-channel events. The results suggest that the blocking ions can partly move into the channels (but cannot penetrate) and interact with other ions inside the pore. The reduction of single-channel conductance by Cs+, tetraethylammonium and Ba2+ and of single-channel activity by quinine and Ba2+ is referred to different rates of access to the channel. The concentration- and voltage-dependent inhibition by ions with measurable permeability (Na+ and Rb+) can be explained by their lower permeability, with single-file movement and ionic interactions inside the pore.  相似文献   

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