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1.
Extracellular acidosis affects both permeation and gating of the expressed rat skeletal muscle Na+ channel (μ1). Reduction of the extracellular pH produced a progressive decrease in the maximal whole-cell conductance and a depolarizing shift in the whole-cell current-voltage relationship. A smaller depolarizing shift in the steady-state inactivation curve was observed. The pK of the reduction of maximal conductance was 6.1 over the pH range studied. An upper limit estimate of the pK of the shift of the half-activation voltage was 6.1. The relative reduction in the maximal whole-cell conductance did not change with higher [Na+] o . The conductance of single fenvalerate-modified Na+ channels was reduced by extracellular protons. Although the single-channel conductance increased with higher [Na+] o , the maximal conductances at pH 7.6, 7.0 and 6.0 did not converge at [Na+] o up to 280 mm, inconsistent with a simple electrostatic effect. A model incorporating both Na+ and H+ binding in the pore and cation binding to a Gouy-Chapman surface charge provided a robust fit to the single-channel conductance data with an estimated surface charge density of 1e/439?2. Neither surface charge nor proton block alone suffices to explain the effects of extracellular acidosis on Na+ channel permeation; both effects play major roles in mediating the response to extracellular pH. Received: 14 May 1996/Revised: 19 September 1996  相似文献   

2.
The gating cycle of CFTR (Cystic Fibrosis Transmembrane conductance Regulator) chloride channels requires ATP hydrolysis and can be interrupted by exposure to the nonhydrolyzable nucleotide AMP-PNP. To further characterize nucleotide interactions and channel gating, we have studied the effects of AMP-PNP, protein kinase C (PKC) phosphorylation, and temperature on gating kinetics. The rate of channel locking increased from 1.05 × 10−3 sec−1 to 58.7 × 10−3 sec−1 when AMP-PNP concentration was raised from 0.5 to 5 mm in the presence of 1 mm MgATP and 180 nm protein kinase A catalytic subunit (PKA). Although rapid locking precluded estimation of P o or opening rate immediately after the addition of AMP-PNP to wild-type channels, analysis of locking rates in the presence of high AMP-PNP concentrations revealed two components. The appearance of a distinct, slow component at high [AMP-PNP] is evidence for AMP-PNP interactions at a second site, where competition with ATP would reduce P o and thereby delay locking. All channels exhibited locking when they were strongly phosphorylated by PKA, but not when exposed to PKC alone. AMP-PNP increased P o at temperatures above 30°C but did not cause locking, evidence that the stabilizing interactions between domains, which have been proposed to maintain CFTR in the open burst state, are relatively weak. The temperature dependence of normal CFTR gating by ATP was strongly asymmetric, with the opening rate being much more temperature sensitive (Q 10= 9.6) than the closing rate (Q 10= 3.6). These results are consistent with a cyclic model for gating of phosphorylated CFTR. Received: 28 August 1997/Revised: 4 February 1998  相似文献   

3.
The gating of ryanodine receptor calcium release channels (RyRs) depends on myoplasmic Ca2+ and Mg2+ concentrations. RyRs from skeletal and cardiac muscle are activated by μm Ca2+ and inhibited by mm Ca2+ and Mg2+. 45Ca2+ release from skeletal SR vesicles suggests two mechanisms for Mg2+-inhibition (Meissner, Darling & Eveleth, 1986, Biochemistry 25:236–244). The present study investigates the nature of these mechanisms using measurements of single-channel activity from cardiac- and skeletal RyRs incorporated into planar lipid bilayers. Our measurements of Mg2+- and Ca2+-dependent gating kinetics confirm that there are two mechanisms for Mg2+ inhibition (Type I and II inhibition) in skeletal and cardiac RyRs. The mechanisms operate concurrently, are independent and are associated with different parts of the channel protein. Mg2+ reduces P o by competing with Ca2+ for the activation site (Type-I) or binding to more than one, and probably two low affinity inhibition sites which do not discriminate between Ca2+ and Mg2+ (Type-II). The relative contributions of the two inhibition mechanisms to the total Mg2+ effect depend on cytoplasmic [Ca2+] in such a way that Mg2+ inhibition has the properties of Types-I and II inhibition at low and high [Ca2+] respectively. Both mechanisms are equally important when [Ca2+] = 10 μm in cardiac RyRs or 1 μm in skeletal RyRs. We show that Type-I inhibition is not the sole mechanism responsible for Mg2+ inhibition, as is often assumed, and we discuss the physiological implications of this finding. Received: 1 January 1996/Revised: 14 November 1996  相似文献   

4.
A K+ channel with a main conductance of 29 pS was recorded after the incorporation of coronary artery membrane vesicles into lipid bilayers. This channel was identified as an ATP-sensitive K+ channel (KATP) because its activity was diminished by the internal application of 50–250 μm ATP-Na2. Moreover, it was opened when 10–50 μm pinacidil was externally applied. Single-channel records revealed the existence of several (sub)conductance states. At 0 mV and with a 5/250 KCl gradient, the main conductance of the KATP channel was 29 pS. The other (sub)conductance states were less frequent and had discrete values of 12, 17 and 22 pS. Pinacidil stabilized the channel open state primarily in the 29 pS conductance level; whereas ATP inhibited all the conductance levels. In general, KATP channels were characterized by brief openings followed by long closings (open probability, P o ≈ 0.02); only occasionally (3 out of 12 experiments) did the KATP channels have a high open probability (P o ≥ 0.7). Channel activity could be increased or rescued by adding 2.5–10 mm UDP-TRIS and 0.5–2 mm MgCl2 to the internal side of the channel. Received: 7 November 1995/Revised: 10 June 1996  相似文献   

5.
The modulation of the calmodulin-induced inhibition of the calcium release channel (ryanodine receptor) by two sulfhydryl oxidizing compounds, 4-(chloromercuri)phenyl–sulfonic acid (4-CMPS) and 4,4′-dithiodipyridine (4,4′-DTDP) was determined by single channel current recordings with the purified and reconstituted calcium release channel from rabbit skeletal muscle sarcoplasmic reticulum (HSR) and [3H]ryanodine binding to HSR vesicles. 0.1 μm CaM reduced the open probability (P o ) of the calcium release channel at maximally activating calcium concentrations (50–100 μm) from 0.502 ± 0.02 to 0.137 ± 0.022 (n= 28), with no effect on unitary conductance. 4-CMPS (10–40 μm) and 4,4′-DTDP (0.1–0.3 mm) induced a concentration dependent increase in P o (> 0.9) and caused the appearance of longer open states. CaM shifted the activation of the calcium release channel by 4-CMPS or 4,4′-DTDP to higher concentrations in single channel recordings and [3H]ryanodine binding. 40 μm 4-CMPS induced a near maximal (P o > 0.9) and 0.3 mm 4,4′-DTDP a submaximal (P o = 0.74) channel opening in the presence of CaM, which was reversed by the specific sulfhydryl reducing agent DTT. Neither 4-CMPS nor 4,4′-DTDP affected Ca-[125I]calmodulin binding to HSR. 1 mm MgCl2 reduced P o from 0.53 to 0.075 and 20–40 μm 4-CMPS induced a near maximal channel activation (P o > 0.9). These results demonstrate that the inhibitory effect of CaM or magnesium in a physiological concentration is diminished or abolished at high concentrations of 4-CMPS or 4,4′-DTDP through oxidation of activating sulfhydryls on cysteine residues of the calcium release channel. Received: 22 July 1999/Revised: 15 November 1999  相似文献   

6.
Membrane fractions highly enriched in chicken lens MIP (MIP28) were found to form ion channels when incorporated into planar lipid bilayers. The channels displayed prominent unitary conductances of about 60 and 290 pS in symmetric 150 mm KCl solution and were slightly anion selective. For both depolarizing and hyperpolarizing voltages, voltage sensitivity of the MIP28-induced conductance could be fit by a Boltzmann relation, symmetric around zero mV, with V 0 = 18.5 mV, n= 4.5 and g min/g max= 0.17. Channel properties were not appreciably altered by pH in the range of 5.8 to 7, although channel incorporation was observed to occur more frequently at lower pH values. Calcium, at millimolar concentrations, decreased the channel mean open time. Partial proteolysis of MIP28 to yield MIP21 did not appreciably affect single-channel conductance or voltage sensitivity of the reconstituted channels. MIP28 was not phosphorylated by cAMP dependent protein kinase (PKA). Although unitary conductance and selectivity of the chicken MIP channel are similar to those reported for the bovine MIP (MIP26), the voltage sensitivity of MIP28 was higher than that of the bovine homologue, and voltage sensitivity of MIP28 was not modulated by treatments previously shown to affect MIP26 voltage gating (partial proteolysis and protein phosphorylation by PKA: (Ehring et al., 1990). The existence of such strikingly different functional properties in highly homologous channel isoforms may provide a useful system for exploration of the structure-function relations of MIP channels. Received: 27 March 1996/Revised: 5 August 1996  相似文献   

7.
The existence of invertebrate forms of the RyR has recently been confirmed (Takeshima et al., 1994, Puente et al., 2000). However, information on the functional properties of this insect RyR is still limited. We report the functional characterization of a RyR from the thoracic muscle of H. virescens (Scott-Ward et al., 1997). A simple purification protocol produced membranes from homogenized prefrozen H. virescens thoracic muscle with a [3H]-ryanodine binding activity of 1.19 ± 0.21 pmol/mg protein (mean ±se; n= 4). [3H]-Ryanodine binding to the H. virescens receptor was dependent on the ryanodine concentration in a hyperbolic fashion with a K D of 3.82 nm (n= 4). [3H]-ryanodine binding was dependent on [Ca2+] in a biphasic manner and was stimulated by 1 mm ATP. Millimolar caffeine did not stimulate [3H]-ryanodine binding to H. virescens membranes in the presence of either nanomolar or micromolar Ca2+. A protein of at least 400 KDa was recognized in H. virescens membrane proteins by a specific anti-H. virescens RyR antibody. Discontinuous density sucrose gradient fractionation of microsomal membranes produced vesicles suitable for single-channel studies. Ca2+-sensitive, Ca2+-permeable channels were successfully inserted into artificial lipid bilayers from H. virescens membrane vesicles. The H. virescens RyR-channel displayed a Ca2+ conductance of ∼110 pS and underwent a persistent and characteristic modification of ion handling and gating following addition of 100 nm ryanodine. The gating of H. virescens channels was sensitive to ATP and ruthenium red in a manner similar to mammalian RyR. This is the first report to describe the single channel and [3H]-ryanodine binding properties of a native insect RyR. Received: 3 July 2000/Revised: 17 October 2000  相似文献   

8.
We show that rabbit skeletal RyR channels in lipid bilayers can be activated or inhibited by NO, in a manner that depends on donor concentration, membrane potential and the presence of channel agonists. 10 μm S-nitroso-N-acetyl-penicillamine (SNAP) increased RyR activity at −40 mV within 15 sec of addition to the cis chamber, with a 2-fold increase in frequency of channel opening (F o ). 10 μm SNAP did not alter activity at +40 mV and did not further activate RyRs previously activated by 2 mm cis ATP at +40 or −40 mV. In contrast to the increase in F o with 10 μm SNAP, 1 mm SNAP caused a 2-fold reduction in F o but a 1.5-fold increase in mean open time (T o ) at −40 mV in the absence of ATP. 1 mm SNAP or 0.5 mm sodium nitroprusside (SNP) induced ∼3-fold reductions in F o and T o at +40 or −40 mV when channels were activated by 2 mm cis ATP or in channels activated by 6.5 μm peptide A at −40 mV (peptide A corresponds to part of the II–III loop of the skeletal dihydropyridine receptor). Both SNAP-induced activation and SNAP/SNP-induced inhibition were reversed by 2 mm dithiothreitol. The results suggest that S-Nitrosylation or oxidation of at least three classes of protein thiols by NO each produced characteristic changes in RyR activity. We propose that, in vivo, initial release of NO activates RyRs, but stronger release increases [NO] and inhibits RyR activity and contraction. Received: 27 August 1999/Revised: 25 October 1999  相似文献   

9.
Properties of large conductance Ca2+-activated K+ channels were studied in the soma of motoneurones visually identified in thin slices of neonatal rat spinal cord. The channels had a conductance of 82 ± 5 pS in external Ringer solution (5.6 mm K+ o //155 mm K+ i ) and 231 ± 4 pS in external high-K o solution (155 mm K+ o //155 mm K+ i ). The channels were activated by depolarization and by an increase in internal Ca2+ concentration. Potentials of half-maximum channel activation (E50) were −13, −34, −64 and −85 mV in the presence of 10−6, 10−5, 10−4 and 10−3 m internal Ca2+, respectively. Using an internal solution containing 10−4 m Ca2+, averaged KCa currents showed fast activation within 2–3 msec after a voltage step to +50 mV. Averaged KCa currents did not inactivate during 400 msec voltage pulses. External TEA reduced the apparent single-channel amplitude with a 50% blocking concentration (IC50) of 0.17 ± 0.02 mm. KCa channels were completely suppressed by externally applied 100 mm charybdotoxin. It is concluded that KCa channels activated by Ca2+ entry during the action potential play an important role in the excitability of motoneurones. Received: 7 November 1996/Revised: 29 October 1997  相似文献   

10.
Large Conductance Ca2+-Activated K+ Channels in Human Meningioma Cells   总被引:2,自引:0,他引:2  
Cells from ten human meningiomas were electrophysiologically characterized in both living tissue slices and primary cultures. In whole cells, depolarization to voltages higher than +80 mV evoked a large K+ outward current, which could be blocked by iberiotoxin (100 nm) and TEA (half blocking concentration IC50= 5.3 mm). Raising the internal Ca2+ from 10 nm to 2 mm shifted the voltage of half-maximum activation (V 1/2) of the K+ current from +106 to +4 mV. Respective inside-out patch recordings showed a voltage- and Ca2+-activated (BK Ca ) K+ channel with a conductance of 296 pS (130 mm K+ at both sides of the patch). V 1/2 of single-channel currents was +6, −12, −46, and −68 mV in the presence of 1, 10, 100, and 1000 μm Ca2+, respectively, at the internal face of the patch. In cell-attached patches the open probability (P o ) of BK Ca channels was nearly zero at potentials below +80 mV, matching the activation threshold for whole-cell K+ currents with 10 nm Ca2+ in the pipette. Application of 20 μm cytochalasin D increased P o of BK Ca channels in cell-attached patches within minutes. These data suggest that the activation of BK Ca channels in meningioma cells does not only depend on voltage and internal Ca2+ but is also controlled by the cytoskeleton. Received 18 June 1999/Revised: 18 January 2000  相似文献   

11.
Opening of ATP-sensitive K+ (KATP) channels by the uncoupler of oxidative phosphorylation, 2,4 dinitrophenol (DNP), has been assumed to be secondary to metabolic inhibition and reduced intracellular ATP levels. Herein, we present data which show that DNP (200 μm) can induce opening of cardiac KATP channels, under whole-cell and inside-out conditions, despite millimolar concentrations of ATP (1–2.5 mm). DNP-induced currents had a single channel conductance (71 pS), inward rectification, reversal potential, and intraburst kinetic properties (open time constant, τopen: 4.8 msec; fast closed time constant, τclosed(f): 0.33 msec) characteristic of KATP channels suggesting that DNP did not affect the pore region of the channel, but may have altered the functional coupling of the ATP-dependent channel gating. A DNP analogue, with the pH-titrable hydroxyl replaced by a methyl group, could not open KATP channels. The pH-dependence of the effect of DNP on channel opening under whole-cell, cell-attached, and inside-out conditions suggested that transfer of protonated DNP across the sarcolemma is essential for activation of KATP channels in the presence of ATP. We conclude that the use of DNP for metabolic stress-induced KATP channel opening should be reevaluated. Received: 10 September 1996/Revised: 27 December 1996  相似文献   

12.
Single inward rectifier K+ channels were studied in Xenopus laevis embryonic myocytes. We have characterized in detail the channel which is most frequently observed (Kir) although we routinely observe three other smaller current levels with the properties of inward rectifier K+ channels (Kir(0.3), Kir(0.5) and Kir(0.7)). For Kir, slope conductances of inward currents were 10.3, 20.3, and 27.9 pS, in 60, 120 and 200 mM [K+] o respectively. Extracellular Ba2+ blocked the normally high channel activity in a concentration-dependent manner (K A = 7.8 μm, −90 mV). In whole-cell recordings of inward rectifier K+ current, marked voltage dependence of Ba2+ block over the physiological range of potentials was observed. We also examined current rectification. Following step depolarizations to voltages positive to E K , outward currents through Kir channels were not observed even when the cytoplasmic face of excised patches were exposed to Mg2+-free solution at pH 9.1. This was probably also true for Kir(0.3), Kir(0.5) and Kir(0.7) channels. We then examined the possibility of modulation of Kir channel activity and found neither ATP nor GTP-γS had any effect on Kir channel activity when added to the solution perfusing the cytoplasmic face of a patch. Kinetic analysis revealed Kir channels with a single open state (mean dwell time 72 msec) and two closed states (time constants 1.4, 79 msec). These results suggest that the native Kir channels of Xenopus myocytes have similar properties to the cloned strong inward rectifier K+ channels, in terms of conductance, kinetics and barium block but does show some differences in the effects of modulators of channel activity. Furthermore, skeletal muscle may contain either different inward rectifier channels or a single-channel type which can exist in stable subconductance states. Received: 16 September 1996/Revised: 14 March 1997  相似文献   

13.
Data obtained with the lipid bilayer technique indicate that cis (cytoplasmic) concentration of 4.4–22 mm hydrogen peroxide (H2O2), is a water-soluble oxidant. [H2O2] cis (n= 26) reversibly inhibits the multisubconductance SCl channel of the sarcoplasmic reticulum vesicles from rabbit skeletal muscle. At −40 mV, the mean values of the current amplitude (I) and the probability of the SCl channel being open (P o ) were reduced significantly (n= 8) from −6.14 ± 0.42 pA and 0.69 ± 0.06 (for all conductance levels) in control 0.0 mm [H2O2] cis to −1.10 ± 0.51 pA and 0.13 ± 0.04 (for the intermediate subconductance states) in 8.8 mm [H2O2] cis , respectively. The [H2O2] cis -induced decrease in P o is mainly due to a decrease in the mean open time T o . The mechanism of [H2O2] cis effects on the multiconductance SCl channel is characterized by a mode shift in the channel state from the main conductance state to the low subconductance states. The estimated concentration of the [H2O2] cis for the half inhibitory constant, K i , was 11.78 mm, higher than the estimated 8.0 and 8.1 mm for the parameters P o and T o , respectively, indicating that the conductance of the SCl channel is less sensitive than the gating kinetics of the channel. After a lag period of between 30 to 60 sec, the lipophilic SH-oxidizing agent 4,4′-dithiodipyridine (4,4′-DTDP) added to the cis side at 1.0 mm removed the inhibitory effects of 8.8 mm [H2O2] cis . The 4,4′-DTDP-enhanced SCl channel activity was blocked after the addition of 0.5 mm ATP to the cis side of the channel. The addition of 1.0 mm 4,4′-DTDP to the cis or trans solutions facing an SCl channel already subjected to 0.5 mm [ATP] cis or [ATP] trans failed to activate the ATP-inhibited SCl channel. These findings suggest that 4,4′-DTDP is not preventing the binding of ATP to its binding site on the channel protein. The interaction of H2O2 with the SCl channel proteins is consistent with a thiol-disulfide redox state model for regulating ion transport, where SH groups can directly modify the function of the channel and/or the availability of regulatory sites on the channel proteins. The H2O2 effects on the Ca2+ countercurrent through the SCl channel are also consistent with H2O2-modification of the mechanisms involved in the Ca2+ regulation, which underlies excitation-contraction coupling in skeletal muscle. Received: 27 April 1999/Revised: 1 July 1999  相似文献   

14.
Nucleotide-dependent gating of ΔF508-CFTR was evaluated in membrane patches excised from HEK 293 and mouse L-cells and compared to observations on wt-CFTR channels recorded in the same expression systems. ΔF508-CFTR exhibited PKA activated, ATP-dependent channel gating. When compared to wt-CFTR, the K m for ATP was increased by ninefold (260 μm vs. 28 μm) and maximal open probability (P o ) was reduced by 49% (0.21 ± 0.06 vs. 0.41 ± 0.02). Additionally, in the absence of PKA, ΔF508-CFTR inactivated over a 1 to 5 min period whereas wt-CFTR remained active. Time-dependent inactivation could be mimicked in wt-CFTR by the intermittent absence of ATP in the cytosolic solution. The effects of 3-isobutyl-1-methyl xanthine (IBMX), a compound reported to stimulate ΔF508-CFTR, were evaluated on wt- and ΔF508-CFTR channels. At concentrations up to 5 mm, IBMX caused a concentration dependent reduction in the observed single channel amplitude (i) of wt-CFTR (maximal observed reduction 35 ± 3%). However, IBMX failed to significantly alter total patch current because of a concomitant 30% increase in P o . The effects of IBMX on ΔF508-CFTR were similar to effects on wt-CFTR in that i was reduced and P o was increased by similar magnitudes. Additionally, ΔF508-CFTR channel inactivation was dramatically slowed by IBMX. These results suggest that IBMX interacts with the ATP-bound open state of CFTR to introduce a short-lived nonconducting state which prolongs burst duration and reduces apparent single channel amplitude. A secondary effect observed in ΔF508-CFTR, which may result from this interaction, is a prolongation of the activated state. In light of previously proposed linear kinetic models of CFTR gating, these results suggest that IBMX traps CFTR in an ATP-bound state which may preclude inactivation of ΔF508-CFTR. Received: 5 February 1999/Revised: 25 March 1999  相似文献   

15.
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated, ATP-dependent chloride channel which may have additional functions. Recent reports that CFTR mediates substantial electrodiffusion of ATP from epithelial cells have led to the proposal that CFTR regulates other ion channels through an autocrine mechanism involving ATP. The aim of this study was to determine the ATP conductance of wild-type CFTR channels stably expressed in Chinese hamster ovary cells using patch clamp techniques. In the cell-attached configuration with 100 mm Mg · ATP or Tris · ATP solution in the pipette and 140 mm NaCl in the bath, exposing cells to forskolin caused the activation of a low-conductance channel having kinetics resembling those of CFTR. Single channel currents were negative at the resting membrane potential (V m ), consistent with net diffusion of Cl from the cell into the pipette. The transitions decreased in amplitude, but did not reverse direction, as V m was clamped at increasingly positive potentials to enhance the driving force for inward ATP flow (>+80 mV). In excised patches, single channel currents did not reverse under essentially biionic conditions (Clin/ATPout or ATPin/Clout), although PKA-activated currents were clearly visible in the same patches at voltages where they would be carried by chloride ions. Moreover, with NaCl solution in the bath and a mixture of ATP and Cl in the pipette, the single channel I/V curve reversed at the predicted equilibrium potential for chloride. CFTR channel currents disappeared when patches were exposed to symmetrical ATP solutions and were restored by reexposure to Cl solution. Finally, in the whole-cell configuration with NaCl in the bath and 100 mm MgATP or TrisATP in the pipette, cAMP-stimulated cells had time-independent, outwardly rectifying currents consistent with CFTR selectivity for external Cl over internal ATP. Whole-cell currents reversed near V m =−55 mV under these conditions, however the whole cell resistance measured at −100 mV was comparable to that of the gigaohm seal between the plasma membrane and glass pipette (7 GΩ). We conclude that CFTR does not mediate detectable electrodiffusion of ATP. Received: 8 November 1995/Revised: 23 January 1996  相似文献   

16.
A large conductance, Ca2+-activated K+ channel of the BK type was examined in cultured pituitary melanotrophs obtained from adult male rats. In cell-attached recordings the slope conductance for the BK channel was ≈190 pS and the probability (P o ) of finding the channel in the open state at the resting membrane potential was low (<<0.1). Channels in inside-out patches and in symmetrical 150 mm K+ had a conductance of ≈260 pS. The lower conductance in the cell-attached recordings is provisionally attributed to an intracellular K+ concentration of ≈113 mm. The permeability sequence, relative to K+, was K+ > Rb+ (0.87) > NH+ 4 (0.17) > Cs+≥ Na+ (≤0.02). The slope conductance for Rb+ was much less than for K+. Neither Na+ nor Cs+ carried measurable currents and 150 mm internal Cs+ caused a flickery block of the channel. Internal tetraethylammonium ions (TEA+) produced a fast block for which the dissociation constant at 0 mV (K D (0 mV)) was 50 mm. The K D (0 mV) for external TEA+ was much lower, 0.25 mm, and the blocking reaction was slower as evidenced by flickery open channel currents. With both internal and external TEA+ the blocking reaction was bimolecular and weakly voltage dependent. External charybdotoxin (40 nm) caused a large and reversible decrease of P o . The P o was increased by depolarization and/or by increasing the concentration of internal Ca2+. In 0.1 μm Ca2+ the half-maximal P o occurred at ≈100 mV; increasing Ca2+ to 1 μm shifted the voltage for the half-maximal P o to −75 mV. The Ca2+ dependence of the gating was approximated by a fourth power relationship suggesting the presence of four Ca2+ binding sites on the BK channel. Received: 23 October/Revised: 15 December 1995  相似文献   

17.
The lipid bilayer technique was used to examine the effects of the ATP-sensitive K+ channel inhibitor (glibenclamide) and openers (diazoxide, minoxidil and cromakalim) and Cl channel activators (GABA and diazepam) on two types of chloride channels in the sarcoplasmic reticulum (SR) from rabbit skeletal muscle. Neither diazepam at 100 μm nor GABA at 150 μm had any significant effect on the conductance and kinetics of the 75 pS small chloride (SCl) channel. Unlike the 150 pS channel, the SCl channel is sensitive to cytoplasmic glibenclamide with K i ∼ 30 μm. Glibenclamide induced reversible decline in the values of current (maximal current amplitude, I max and average mean current, I′) and kinetic parameters (frequency of opening F o , probability of the channel being open P o and mean open time, T o , of the SCl channel. Glibenclamide increased mean closed time, T c , and was a more potent blocker from the cytoplasmic side (cis) than from the luminal side (trans) of the channel. Diazoxide increased I′, P o , and T o in the absence of ATP and Mg2+ but it had no effect on I max and also failed to activate or remove the glibenclamide- and ATP-induced inhibition of the SCl channel. Minoxidil induced a transient increase in I′ followed by an inhibition of I max, whereas cromakalim reduced P o and I′ by increasing channel transitions to the closed state and reducing T o without affecting I max. The presence of diazoxide, minoxidil or cromakalim on the cytoplasmic side of the channel did not prevent [ATP] cis or [glibenclamide] cis from blocking the channel. The data suggest that the action(s) of these drugs are not due to their effects on the phosphorylation of the channel protein. The glibenclamide- and cromakalim-induced effects on the SCl channel are mediated via a ``flicker' type block mechanism. Modulation of the SCl channel by [diazoxide] cis and [glibenclamide] cis highlights the therapeutic potential of these drugs in regulating the Ca2+-counter current through this channel. Received: 2 September 1997/Revised: 20 March 1998  相似文献   

18.
The reactive disulfide 4,4′-dithiodipyridine (4,4′DTDP) was added to single cardiac ryanodine receptors (RyRs) in lipid bilayers. The activity of native RyRs, with cytoplasmic (cis) [Ca2+] of 10−7 m (in the absence of Mg2+ and ATP), increased within ∼1 min of addition of 1 mm 4,4′-DTDP, and then irreversibly ceased 5 to 6 min after the addition. Channels, inhibited by either 1 mm cis Mg2+ (10−7 m cis Ca2+) or by 10 mm cis Mg2+ (10−3 m cis Ca2+), or activated by 4 mm ATP (10−7 m cis Ca2+), also responded to 1 mm cis 4,4′-DTDP with activation and then loss of activity. P o and mean open time (T o ) of the maximally activated channels were lower in the presence of Mg2+ than in its absence, and the number of openings within the long time constant components of the open time distribution was reduced. In contrast to the reduced activation by 1 mm 4,4′-DTDP in channels inhibited by Mg2+, and the previously reported enhanced activation by 4,4′-DTDP in channels activated by Ca2+ or caffeine (Eager et al., 1997), the activation produced by 1 mm cis 4,4′-DTDP was the same in the presence and absence of ATP. These results suggest that there is a physical interaction between the ATP binding domain of the cardiac RyR and the SH groups whose oxidation leads to channel activation. Received: 8 September 1997/Revised: 20 January 1998  相似文献   

19.
The dependence of currents through the cyclic nucleotide-gated (CNG) channels of mammalian olfactory receptor neurons (ORNs) on the concentration of NaCl was studied in excised inside-out patches from their dendritic knobs using the patch-clamp technique. With a saturating concentration (100 μm) of adenosine 3′, 5′-cyclic monophosphate (cAMP), the changes in the reversal potential of macroscopic currents were studied at NaCl concentrations from 25 to 300 mm. In symmetrical NaCl solutions without the addition of divalent cations, the current-voltage relations were almost linear, reversing close to 0 mV. When the external NaCl concentration was maintained at 150 mm and the internal concentrations were varied, the reversal potentials of the cAMP-activated currents closely followed the Na+ equilibrium potential indicating that P Cl/P Na≈ 0. However, at low external NaCl concentrations (≤100 mm) there was some significant chloride permeability. Our results further indicated that Na+ currents through these channels: (i) did not obey the independence principle; (ii) showed saturation kinetics with K ms in the range of 100–150 mm and (iii) displayed a lack of voltage dependence of conductance in asymmetric solutions that suggested that ion-binding sites were situated midway along the channel. Together, these characteristics indicate that the permeation properties of the olfactory CNG channels are significantly different from those of photoreceptor CNG channels. Received: 7 November 1996/Revised: 24 March 1997  相似文献   

20.
The apical brush border membrane, the main target site of Bacillus thuringiensis toxins, was isolated from gypsy moth (Lymantria dispar) larval midguts and fused to artificial planar lipid bilayer membranes. Under asymmetrical N-methyl-d-glucamine-HCl conditions (450 mm cis/150 mm trans, pH 9.0), which significantly reduce endogenous channel activity, trypsin-activated Cry1Aa, a B. thuringiensis insecticidal protein active against the gypsy moth in vivo, induced a large increase in bilayer membrane conductance at much lower concentrations (1.1–2.15 nm) than in receptor-free bilayer membranes. At least 5 main single-channel transitions with conductances ranging from 85 to 420 pS were resolved. These Cry1Aa channels share similar ionic selectivity with P Cl/P NMDG permeability ratios ranging from 4 to 8. They show no evidence of current rectification. Analysis of the macroscopic current flowing through the composite bilayer suggested voltage-dependence of several channels. In comparison, the conductance of the pores formed by 100–500 nm Cry1Aa in receptor-free bilayer membranes was significantly smaller (about 8-fold) and their P Cl/P NMDG permeability ratios were also reduced (2- to 4-fold). This study provides a detailed demonstration that the target insect midgut brush border membrane material promotes considerably pore formation by a B. thuringiensis Cry toxin and that this interaction results in altered channel properties. Received: 23 February 2001/Revised: 15 June 2001  相似文献   

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