共查询到20条相似文献,搜索用时 15 毫秒
1.
Extracellular protons have been shown to modulate voltage-activated ionic channels. It has been proposed that synaptic modulation
by exocytosed vesicular protons would be a characteristic feature of ribbon-type synapses. Type-I hair cells have a calyceal
afferent junction with a diffusionally restricted synaptic cleft. These led us to study the action of extracellular pH changes
on the voltage-activated Ca2+ and K+ currents evaluated using a whole-cell patch clamp in isolated cells. The amplitude of the Ca2+ and the K+ current were reduced by extracellular acidification, but without significant changes with extracellular alkalization. A shift
in the voltage dependence to a more positive membrane potential was achieved at pH < 6.8. Our results shows that the presynaptic
K+ and Ca2+ currents are modulated by protons, indicating that protons released along with an afferent neurotransmitter would participate
as a feedback mechanism in type-I hair cells.
Special issue article in honor of Dr. Ricardo Tapia. 相似文献
2.
Human capillary endothelial cells (HCEC) in normal media contain noninactivating outwardly rectifying chloride currents,
TEA-sensitive delayed rectifier K+ currents and an inward rectifier K+ current. Two additional ionic currents are induced in HCEC when the media are allowed to become conditioned: A Ca2+-activated K+ current (BKCA) that is sensitive to iberiotoxin is induced in 23.5% of the cells, a transient 4-AP-sensitive K+ current (A current) is induced in 24.7% of the cells, and in 22.3% of the cells both the transient and BKCA currents are
coinduced. The EC50 for Ca2+ activation of the BKCA current in HCEC from conditioned media is 213 nM. RNA message for BKCA (hSlo clone) is undetecable
after PCR amplification in control cells but is seen in those from conditioned cells. The induction of BKCA current is not
blocked by conditioning with inhibitors of nitric oxide synthase, cyclo-oxgenase or lypo-oxygenase pathways. Apparently the
characteristics of human endothelial cells are highly malleable and can be easily modified by their local environment.
Received: 21 May 1998/Revised: 23 September 1998 相似文献
3.
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
4.
D. R. Laver 《The Journal of membrane biology》1990,118(1):55-67
Summary The patch-clamp technique is used here to investigate the kinetics of Ca2+ block in single high-conductance Ca2+-activated K+ channels. These channels are detected in the membrane surounding cytoplasmic drops fromChara australis, a membrane which originates from the tonoplast of the parent cell. The amplitudes and durations of single channel events are measured over a wide range of membrane potential (–300 to 200 mV). Ca2+ on either side of the channel reduces its K+ conductance and alters its ion-gating characteristics in a voltage-dependent manner. This Ca2+-induced attenuation of conductance is analyzed using the theory of diffusion-limited ion flow through pores. Interaction of external Ca2+ with the channel's ion-gating mechanism is examined in terms of a kinetic model for ion-gating that includes two voltage-dependent gating mechanisms. The kinetics of channel block by external Ca2+ indicates that (i) external Ca2+ binds at two sites, a superficial site and a deep site, located at 8 and 40% along the trans-pore potential difference, (ii) the external vestibule cannot be occupied by more than one Ca2+ or K+, and (iii) the kinetics of Ca2+ binding at the deep site is coupled with that of a voltage-dependent gate on the external side of the channel. Kinetics of channel block by internal Ca2+ indicates that more than one Ca2+ is involved. 相似文献
5.
《Molecular membrane biology》2013,30(2):83-110
Freshly dissociated cells from the stomach muscularis of the toad Bufo marinus have been employed to carry out a systematic set of electrophysiological studies on the membrane properties of smooth muscle. The existence of Ca2+-activated K+ channels became apparent during the first studies under current clamp. In subsequent studies under voltage clamp, a Ca2+-activated, TEA-sensitive outward current was evident, and it was more than an order of magnitude larger than any other current observed in the cells. The channel responsible, at least in part, for this large outward current has been identified on the basis of single-channel records, and some of its main characteristics have been studied. It is similar in many respects to the large-conductance, Ca2+-activated K+ channel seen in other preparations. This channel has now been found in a considerable diversity of smooth muscle types. 相似文献
6.
Ca2+-activated K+ channels were studied in C6-glioma cells in an attempt to correlate changes in expression with cell proliferation and differentiation. In this study, we treated C6-glioma cells with thapsigargin for 48 h. Cell proliferation was markedly inhibited, and cell morphology changed from round to a spindle differentiated shape. Furthermore, intracellular calcium concentration was initially increased during acute treatment with thapsigargin. The internal [Ca2+]i pool was eventually depleted after a 48-h thapsigargin treatment. We have characterized Ca2+-activated K+ currents in less differentiated C6 cells. After differentiation of C6 cells induced by thapsigargin, Ca2+-activated K+ currents were selectively suppressed. These data lend further support to the notion that the expression of Ca2+-activated K+ channels is intimately associated with the proliferation of C6-glioma cells, and the suppression of Ca2+-activated K+ channels coincides with the inhibition of proliferation and subsequent induction of cell differentiation. 相似文献
7.
Large conductance Ca2+-activated potassium channels (BK) are targets for research that explores therapeutic means to various diseases, owing to the roles of the channels in mediating multiple physiological processes in various cells and tissues. We investigated the pharmacological effects of curcumin, a compound isolated from the herb Curcuma longa, on BK channels. As recorded by whole-cell patch-clamp, curcumin increased BK (α) and BK (α+β1) currents in transfected HEK293 cells as well as the current density of BK in A7r5 smooth muscle cells in a dose-dependent manner. By incubating with curcumin for 24 hours, the current density of exogenous BK (α) in HEK293 cells and the endogenous BK in A7r5 cells were both enhanced notably, though the steady-state activation of the channels did not shift significantly, except for BK (α+β1). Curcumin up-regulated the BK protein expression without changing its mRNA level in A7r5 cells. The surface expression and the half-life of BK channels were also increased by curcumin in HEK293 cells. These effects of curcumin were abolished by MG-132, a proteasome inhibitor. Curcumin also increased ERK 1/2 phosphorylation, while inhibiting ERK by U0126 attenuated the curcumin-induced up-regulation of BK protein expression. We also observed that the curcumin-induced relaxation in the isolated rat aortic rings was significantly attenuated by paxilline, a BK channel specific blocker. These results show that curcumin enhances the activity of the BK channels by interacting with BK directly as well as enhancing BK protein expression through inhibiting proteasomal degradation and activating ERK signaling pathway. The findings suggest that curcumin is a potential BK channel activator and provide novel insight into its complicated pharmacological effects and the underlying mechanisms. 相似文献
8.
Stimulation of airway myocytes by contractile agents such as acetylcholine (ACh) activates a Ca2+-activated Cl– current (IClCa) which may play a key role in calcium homeostasis of airway myocytes and hence in airway reactivity. The aim of the present study was to model IClCa in airway smooth muscle cells using a computerised model previously designed for simulation of cardiac myocyte functioning. Modelling was based on a simple resistor-battery permeation model combined with multiple binding site activation by calcium. In order to validate the model, a combination of equations, used to mimic [Ca2+]i response to ACh stimulation, were incorporated into the model. The results indicate that the model developed in this article accounts for experimental recordings and electrophysiological characteristics of this current in airway smooth muscle cells, with parameter values consistent with those calculated from experimental data. Such a model may thus be used to predict IClCa functioning, though additional experimental data from airway myocytes would be useful to more accurately determine some parameter values of the model. 相似文献
9.
Hanaoka K Wright JM Cheglakov IB Morita T Guggino WB 《The Journal of membrane biology》1999,172(3):193-201
We previously cloned a MaxiK channel α-subunit isoform, rbslo1, from rabbit kidney with an amino acid sequence highly homologous to mslo but with a 59 amino acid insertion between S8 and S9 (Morita et al., 1997. Am. J. Physiol.
273:F615–F624). rbslo1 activation properties differed substantially from mslo with much greater Ca2+ sensitivity, half-activation potential of −49 mV in 1 μm Ca2+. We now report single-channel analysis of rbslo1 and delA, a construct produced by removal of the 59 amino acid insertion at site A. delA is identical to mslo from upstream of S1 to downstream of S10 with the exception of 8 amino acids. Slope of the steady-state Boltzmann voltage
activation curve was 8.1 mV per e-fold change in probability of opening for both rbslo1 and delA. The apparent [Ca2+]
i
properties in delA were more like mslo but the voltage-activation properties remained distinctly rbslo1. Ca2+ affinity decreased and transmembrane voltage effects on apparent Ca2+ affinity increased in delA. The differences between rbslo1 and other cloned channels appear to be localized at insertion site A with both the insertion sequence and amino acid substitutions
near site A being important. The steeper activation slope makes the channel more responsive to small changes in transmembrane
voltage while the insertion sequence makes the channel functional at physiological low levels of [Ca2+]
i
.
Received: 23 August 1999 相似文献
10.
Hepatocyte growth factor/scatter factor (HGF/SF) stimulates migration of various cells and has been linked via Met tyrosine kinase-signaling to transformation and the metastatic phenotype. Migration of transformed MDCK-F cells depends on activation of a charybdotoxin-sensitive, volume-activated membrane K+ current. Thus, we used patch-clamp electrophysiology and transwell migration assays to determine whether HGF/SF stimulation of MDCK II cell migration depends on the activation of membrane K+ currents. HGF/SF activated a membrane K+ current that increased over 24 hr, and which could be modulated by increasing intracellular calcium concentration, [Ca2+]i. Charybdotoxin (ChTX, 50 nM), iberiotoxin (IbTX, 100 nM), stichodactyla toxin (Stk, 100 nM) and clotrimazole (CLT, 1 mM) all inhibited this current. HGF/SF (100 scatter units/ml) significantly increased MDCK II cell migration over 8 hr compared to control cells. Addition of ChTX (50 nM), IbTX (100 nM), Stk (100 nM) or CLT (1 microM) inhibited the HGF/SF-stimulated MDCK II cell migration. We conclude that the activation of membrane Ca2+-activated K+current is necessary for HGF/SF stimulation of MDCK II cell. 相似文献
11.
The Ach-evoked Ca2+-activated K+ Current in Mouse Mandibular Secretory Cells. Single Channel Studies
Although acetylcholine (ACh) is able to activate voltage- and Ca2+-sensitive K+ (BK) channels in mouse mandibular secretory cells, our recent whole cell studies have suggested that these channels, like
those in sheep parotid secretory cells, do not contribute appreciably to the conductance that carries the ACh-evoked whole
cell K+ current. In the present study, we have used cell-attached patch clamp methods to identify and characterize the K+ channel type responsible for carrying the bulk of this current. When the cells were bathed in a NaCl-rich solution the predominant
channel type activated by ACh (1 μmol/l or 50 nmol/l) had a conductance only of 40 pS; it was not blocked by TEA but it was
sensitive to quinine and it conducted Rb+ to an appreciable extent. BK channels, which could be seen in some but not all patches from resting cells, also showed increased
activity when ACh was added to the bath, but they were much less conspicuous during ACh stimulation than the 40-pS channels.
When the cells were bathed in a KCl-rich rather than a NaCl-rich solution, a small-conductance K+ channel, sensitive to quinine but not to TEA, was still the most conspicuous channel to be activated by ACh although its
conductance was reduced to 25 pS. Our studies confirm that the ACh-evoked whole-cell K+ current is not carried substantially by BK channels and show that it is carried by a small-conductance K+ channel with quite different properties.
Received: 28 September 1995/Revised: 26 December 1995 相似文献
12.
Background
Apamin is commonly used as a small-conductance Ca2+-activated K+ (SK) current inhibitor. However, the specificity of apamin in cardiac tissues remains unclear.Objective
To test the hypothesis that apamin does not inhibit any major cardiac ion currents.Methods
We studied human embryonic kidney (HEK) 293 cells that expressed human voltage-gated Na+, K+ and Ca2+ currents and isolated rabbit ventricular myocytes. Whole-cell patch clamp techniques were used to determine ionic current densities before and after apamin administration.Results
Ca2+ currents (CACNA1c+CACNB2b) were not affected by apamin (500 nM) (data are presented as median [25th percentile;75th percentile] (from –16 [–20;–10] to –17 [–19;–13] pA/pF, P = NS), but were reduced by nifedipine to –1.6 [–3.2;–1.3] pA/pF (p = 0.008). Na+ currents (SCN5A) were not affected by apamin (from –261 [–282;–145] to –268 [–379;–132] pA/pF, P = NS), but were reduced by flecainide to –57 [–70;–47] pA/pF (p = 0.018). None of the major K+ currents (I Ks, I Kr, I K1 and I to) were inhibited by 500 nM of apamin (KCNQ1+KCNE1, from 28 [20]; [37] to 23 [18]; [32] pA/pF; KCNH2+KCNE2, from 28 [24]; [30] to 27 [24]; [29] pA/pF; KCNJ2, from –46 [–48;–40] to –46 [–51;–35] pA/pF; KCND3, from 608 [505;748] to 606 [454;684]). Apamin did not inhibit the I Na or I CaL in isolated rabbit ventricular myocytes (I Na, from –67 [–75;–59] to –68 [–71;–59] pA/pF; I CaL, from –16 [–17;–14] to –14 [–15;–13] pA/pF, P = NS for both).Conclusions
Apamin does not inhibit human cardiac Na+ currents, L-type Ca2+ currents or other major K+ currents. These findings indicate that apamin is a specific SK current inhibitor in hearts as well as in other organs. 相似文献13.
14.
José A. Ramírez-Latorre 《PloS one》2012,7(12)
Many connections in the basal ganglia are made around birth when animals are exposed to a host of new affective, cognitive, and sensori-motor stimuli. It is thought that dopamine modulates cortico-striatal synapses that result in the strengthening of those connections that lead to desired outcomes. We propose that there must be a time before which stimuli cannot be processed into functional connections, otherwise it would imply an effective link between stimulus, response, and reward in uterus. Consistent with these ideas, we present evidence that early in development dopamine neurons are electrically immature and do not produce high-frequency firing in response to salient stimuli. We ask first, what makes dopamine neurons immature? and second, what are the implications of this immaturity for the basal ganglia? As an answer to the first question, we find that at birth the outward current is small (3nS-V), insensitive to , TEA, BK, and SK blockers. Rapidly after birth, the outward current increases to 15nS-V and becomes sensitive to , TEA, BK, and SK blockers. We make a detailed analysis of the kinetics of the components of the outward currents and produce a model for BK and SK channels that we use to reproduce the outward current, and to infer the geometrical arrangement of BK and channels in clusters. In the first cluster, T-type and BK channels are coupled within distances of 20 nm (200 Å). The second cluster consists of L-type and BK channels that are spread over distances of at least 60 nm. As for the second question, we propose that early in development, the mechanism of action selection is in a “locked-in” state that would prevent dopamine neurons from reinforcing cortico-striatal synapses that do not have a functional experiential-based value. 相似文献
15.
Palle Christophersen 《The Journal of membrane biology》1991,119(1):75-83
Summary The Ca2+-activated K+ channel of the human red cell membranes was characterized with respect to rectification and selectivity using the patch-clamp technique. In inside-out patches exposed to symmetric solutions of K+, Rb+, and NH
4
+
, respectively, inward rectifyingi-V curves were obtained. The zero current conductances were: K+ (23.5 pS±3.2)>NH
4
+
(14.2 pS±1.2)>Rb+ (11.4 pS±1.8). With low extracellular K+ concentrations (substitution with Na+) the current fluctuations reversed close to the Nernst potential for the K ion and the rectification as well as thei-V slopes decreased. With mixed intracellular solutions of K+ and Na+ enhanced rectification were observed due to a Na+ block of outward currents. From bi-ionic reversal potentials the following permeability sequence (P
K/P
X) was calculated: K+ (1.0)>Rb+ (1.4±0.1)>NH
4
+
(8.5±1.3)>Li+(>50); Na+ (>110); Cs+ (5). Li+, Na+, and Cs+ were not found to carry any current, and only minimum values of the permeability ratios were estimated. Tl+ was permeant, but the permeability and conductance were difficult to quantify, since with this ion the single channel activity was extremely low and the channels seemed to inactivate. The inward rectification in symmetric solutions indicate an asymmetric open channel structure, and the different selectivity sequences based on conductances and permeabilities reflect interionic interactions in the permeation process. 相似文献
16.
Ming-Yu Wei Lu Xue Li Tan Wen-Bo Sai Xiao-Cao Liu Qiu-Ju Jiang Jinhua Shen Yong-Bo Peng Ping Zhao Meng-Fei Yu Weiwei Chen Li-Qun Ma Kui Zhai Chunbin Zou Donglin Guo Gangjian Qin Yun-Min Zheng Yong-Xiao Wang Guangju Ji Qing-Hua Liu 《PloS one》2015,10(3)
The participation of large-conductance Ca2+ activated K+ channels (BKs) in chloroquine (chloro)-induced relaxation of precontracted airway smooth muscle (ASM) is currently undefined. In this study we found that iberiotoxin (IbTx, a selective inhibitor of BKs) and chloro both completely blocked spontaneous transient outward currents (STOCs) in single mouse tracheal smooth muscle cells, which suggests that chloro might block BKs. We further found that chloro inhibited Ca2+ sparks and caffeine-induced global Ca2+ increases. Moreover, chloro can directly block single BK currents completely from the intracellular side and partially from the extracellular side. All these data indicate that the chloro-induced inhibition of STOCs is due to the blockade of chloro on both BKs and ryanodine receptors (RyRs). We also found that low concentrations of chloro resulted in additional contractions in tracheal rings that were precontracted by acetylcholine (ACH). Increases in chloro concentration reversed the contractile actions to relaxations. In the presence of IbTx or paxilline (pax), BK blockers, chloro-induced contractions were inhibited, although the high concentrations of chloro-induced relaxations were not affected. Taken together, our results indicate that chloro blocks BKs and RyRs, resulting in abolishment of STOCs and occurrence of contraction, the latter will counteract the relaxations induced by high concentrations of chloro. 相似文献
17.
K.V. Kochetkov V.N. Kazachenko O.V. Aslanidi N.K. Chemeris A.B. Gapeyev 《Journal of biological physics》1999,25(2-3):211-222
Using the patch-voltage clamp technique and the rescaled range method, activity of single large conductance Ca2+-activated K+ channels (KCa channels) was studied. For the sequences of alternating open and shut time intervals, the dependence R/S vs. N in the double logarithmic coordinates presented a curve with two slopes, H1 =0.60 ± 0.04, and H2 = 0.88 ± 0.21, where H1 and H2 characterized the Hurst exponents for shot and long time ranges, respectively. Similar results were obtained for reduced data sets consisting of only open or only shut intervals. Randomization of the experimental data resulted in a single slope, H, of 0.52 ± 0.02. Simulations were performed with eight-state Markovian model without memory. The calculated Hurst exponent presented in average 0.54 ± 0.02. The results suggest that the activity of single Ca2+-activated K+ channel exhibits two regimes, with slight positive correlation at short time ranges (H1 =0.6), and strong positive correlation at long time ranges (H2 = 0.88); therefore the channel gating as a whole is not a steady-state Markovian process. 相似文献
18.
The spatio-temporal properties of Ca2+ transients during excitation-contraction coupling and elementary Ca2+ release events (Ca2+ sparks) were studied in atrial and ventricular myocytes with ultra-fast confocal microscopy using a Zeiss LSM 5 LIVE system that allows sampling rates of up to 60 kHz. Ca2+ sparks which originated from subsarcolemmal junctional sarcoplasmic reticulum (j-SR) release sites in atrial myocytes were anisotropic and elongated in the longitudinal direction of the cell. Ca2+ sparks in atrial cells originating from non-junctional SR and in ventricular myocytes were symmetrical. Ca2+ spark recording in line scan mode at 40,000 lines/s uncovered step-like increases of [Ca2+]i. 2-D imaging of Ca2+ transients revealed an asynchronous activation of release sites and allowed the sequential recording of Ca2+ entry through surface membrane Ca2+ channels and subsequent activation of Ca2+-induced Ca2+ release. With a latency of 2.5 ms after application of an electrical stimulus, Ca2+ entry could be detected that was followed by SR Ca2+ release after an additional 3 ms delay. Maximum Ca2+ release was observed 4 ms after the beginning of release. The timing of Ca2+ entry and release was confirmed by simultaneous [Ca2+]i and membrane current measurements using the whole cell voltage-clamp technique. In atrial cells activation of discrete individual release sites of the j-SR led to spatially restricted Ca2+ release events that fused into a peripheral ring of elevated [Ca2+]i that subsequently propagated in a wave-like fashion towards the center of the cell. In ventricular myocytes asynchronous Ca2+ release signals from discrete sites with no preferential subcellular location preceded the whole-cell Ca2+ transient. In summary, ultra-fast confocal imaging allows investigation of Ca2+ signals with a time resolution similar to patch clamp technique, however in a less invasive fashion. 相似文献
19.
20.
Lukas Jaroslaw Motloch Robert Larbig Tina Gebing Sara Reda Astrid Schwaiger Johannes Leitner Martin Wolny Lars Eckardt Uta C. Hoppe 《PloS one》2016,11(2)