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1.
Gierten J Hassel D Schweizer PA Becker R Katus HA Thomas D 《Biochimica et biophysica acta》2012,1818(1):33-41
Two-pore-domain potassium (K(2P)) channels mediate K(+) background currents that stabilize the resting membrane potential and contribute to repolarization of action potentials in excitable cells. The functional significance of K(2P) currents in cardiac electrophysiology remains poorly understood. Danio rerio (zebrafish) may be utilized to elucidate the role of cardiac K(2P) channels in vivo. The aim of this work was to identify and functionally characterize a zebrafish otholog of the human K(2P)10.1 channel. K(2P)10.1 orthologs in the D. rerio genome were identified by database analysis, and the full zK(2P)10.1 coding sequence was amplified from zebrafish cDNA. Human and zebrafish K(2P)10.1 proteins share 61% identity. High degrees of conservation were observed in protein domains relevant for structural integrity and regulation. K(2P)10.1 channels were heterologously expressed in Xenopus oocytes, and currents were recorded using two-electrode voltage clamp electrophysiology. Human and zebrafish channels mediated K(+) selective background currents leading to membrane hyperpolarization. Arachidonic acid, an activator of hK(2P)10.1, induced robust activation of zK(2P)10.1. Activity of both channels was reduced by protein kinase C. Similar to its human counterpart, zK(2P)10.1 was inhibited by the antiarrhythmic drug amiodarone. In summary, zebrafish harbor K(2P)10.1 two-pore-domain K(+) channels that exhibit structural and functional properties largely similar to human K(2P)10.1. We conclude that the zebrafish represents a valid model to study K(2P)10.1 function in vivo. 相似文献
2.
Constanze Schmidt Felix Wiedmann Frank Tristram Priya Anand Wolfgang Wenzel Patrick Lugenbiel Patrick A. Schweizer Hugo A. Katus Dierk Thomas 《Life sciences》2014
Aims
Effective management of atrial fibrillation (AF) often remains an unmet need. Cardiac two-pore-domain K+ (K2P) channels are implicated in action potential regulation, and their inhibition has been proposed as a novel antiarrhythmic strategy. K2P2.1 (TREK-1) channels are expressed in the human heart. This study was designed to identify and functionally express porcine K2P2.1 channels. In addition, we sought to analyze cardiac expression and AF-associated K2P2.1 remodeling in a clinically relevant porcine AF model.Main methods
Three pK2P2.1 isoforms were identified and amplified. Currents were recorded using voltage clamp electrophysiology in the Xenopus oocyte expression system. K2P2.1 remodeling was studied by quantitative real time PCR and Western blot in domestic pigs during AF induced by atrial burst pacing.Key findings
Human and porcine K2P2.1 proteins share 99% identity. Residues involved in phosphorylation or glycosylation are conserved. Porcine K2P2.1 channels carried outwardly rectifying K+ currents similar to their human counterparts. In pigs, K2P2.1 was expressed ubiquitously in the heart with predominance in the atrial tissue. AF was associated with time-dependent reduction of K2P2.1 protein in the RA by 70% (7 days of AF) and 80% (21 days of AF) compared to control animals in sinus rhythm. K2P2.1 expression in the left atrium, AV node, and ventricles was not affected by AF.Significance
Similarities between porcine and human K2P2.1 channels indicate that the pig may represent a valid model for mechanistic and preclinical studies. AF-related atrial K2P2.1 remodeling has potential implications for arrhythmia maintenance and antiarrhythmic therapy. 相似文献3.
Claudia Seyler Patrick A. SchweizerEdgar Zitron Hugo A. KatusDierk Thomas 《Biochemical and biophysical research communications》2014
Atrial fibrillation (AF) contributes significantly to cardiovascular morbidity and mortality. The growing epidemic is associated with cardiac repolarization abnormalities and requires the development of more effective antiarrhythmic strategies. Two-pore-domain K+ channels stabilize the resting membrane potential and repolarize action potentials. Recently discovered K2P17.1 channels are expressed in human atrium and represent potential targets for AF therapy. However, cardiac electropharmacology of K2P17.1 channels remains to be investigated. This study was designed to elucidate human K2P17.1 regulation by antiarrhythmic drugs. 相似文献
4.
Dagmar Knoflach Vasily Kerov Simone B Sartori Gerald J Obermair Claudia Schmuckermair Xiaoni Liu Vithiyanjali Sothilingam Marina Garcia Garrido Sheila A Baker Martin Gl?smann Klaus Schicker Mathias Seeliger Amy Lee Alexandra Koschak 《Channels (Austin, Tex.)》2013,7(6):503-508
It is generally expected that 2-pore domain K+ (K2P) channels are open or outward rectifiers in asymmetric physiological K+ gradients, following the Goldman-Hodgkin-Katz (GHK) current equation. Although cloned K2P channels have been extensively studied, their current-voltage (I-V) relationships are not precisely characterized and previous definitions are contradictory. Here we study all the functional channels from 6 mammalian K2P subfamilies in transfected Chinese hamster ovary cells with patch-clamp technique, and examine whether their I-V relationships are described by the GHK current equation. K2P channels display 2 distinct types of I-V curves in asymmetric physiological K+ gradients. Two K2P isoforms in the TWIK subfamily conduct large inward K+ currents and have a nearly linear I-V curve. Ten isoforms from 5 other K2P subfamilies conduct small inward K+ currents and exhibit open rectification, but fits with the GHK current equation cannot precisely reveal the differences in rectification among K2P channels. The Rectification Index, a ratio of limiting I-V slopes for outward and inward currents, is used to quantitatively describe open rectification of each K2P isoform, which is previously qualitatively defined as strong or weak open rectification. These results systematically and precisely classify K2P channels and suggest that TWIK K+ channels have a unique feature in regulating cellular function. 相似文献
5.
Haijun Chen Dongchuan Zuo Jianing Zhang Min Zhou Liqun Ma 《Channels (Austin, Tex.)》2014,8(6):503-508
It is generally expected that 2-pore domain K+ (K2P) channels are open or outward rectifiers in asymmetric physiological K+ gradients, following the Goldman-Hodgkin-Katz (GHK) current equation. Although cloned K2P channels have been extensively studied, their current-voltage (I-V) relationships are not precisely characterized and previous definitions are contradictory. Here we study all the functional channels from 6 mammalian K2P subfamilies in transfected Chinese hamster ovary cells with patch-clamp technique, and examine whether their I-V relationships are described by the GHK current equation. K2P channels display 2 distinct types of I-V curves in asymmetric physiological K+ gradients. Two K2P isoforms in the TWIK subfamily conduct large inward K+ currents and have a nearly linear I-V curve. Ten isoforms from 5 other K2P subfamilies conduct small inward K+ currents and exhibit open rectification, but fits with the GHK current equation cannot precisely reveal the differences in rectification among K2P channels. The Rectification Index, a ratio of limiting I-V slopes for outward and inward currents, is used to quantitatively describe open rectification of each K2P isoform, which is previously qualitatively defined as strong or weak open rectification. These results systematically and precisely classify K2P channels and suggest that TWIK K+ channels have a unique feature in regulating cellular function. 相似文献
6.
Zhuren Wang Nathan C. Wong Yvonne Cheng Steven J. Kehl David Fedida 《The Journal of general physiology》2009,133(4):361-374
Crystal structures of potassium (K+) channels reveal that the selectivity filter, the narrow portion of the pore, is only ∼3-Å wide and buttressed from behind, so that its ability to expand is highly constrained, and the permeation of molecules larger than Rb+ (2.96 Å in diameter) is prevented. N-methyl-d-glucamine (NMDG+), an organic monovalent cation, is thought to be a blocker of Kv channels, as it is much larger (∼7.3 Å in mean diameter) than K+ (2.66 Å in diameter). However, in the absence of K+, significant NMDG+ currents could be recorded from human embryonic kidney cells expressing Kv3.1 or Kv3.2b channels and Kv1.5 R487Y/V, but not wild-type channels. Inward currents were much larger than outward currents due to the presence of intracellular Mg2+ (1 mM), which blocked the outward NMDG+ current, resulting in a strong inward rectification. The NMDG+ current was inhibited by extracellular 4-aminopyridine (5 mM) or tetraethylammonium (10 mM), and largely eliminated in Kv3.2b by an S6 mutation that prevents the channel from opening (P468W) and by a pore helix mutation in Kv1.5 R487Y (W472F) that inactivates the channel at rest. These data indicate that NMDG+ passes through the open ion-conducting pore and suggest a very flexible nature of the selectivity filter itself. 0.3 or 1 mM K+ added to the external NMDG+ solution positively shifted the reversal potential by ∼16 or 31 mV, respectively, giving a permeability ratio for K+ over NMDG+ (PK+/PNMDG+) of ∼240. Reversal potential shifts in mixtures of K+ and NMDG+ are in accordance with PK+/PNMDG+, indicating that the ions compete for permeation and suggesting that NMDG+ passes through the open state. Comparison of the outer pore regions of Kv3 and Kv1.5 channels identified an Arg residue in Kv1.5 that is replaced by a Tyr in Kv3 channels. Substituting R with Y or V allowed Kv1.5 channels to conduct NMDG+, suggesting a regulation by this outer pore residue of Kv channel flexibility and, as a result, permeability. 相似文献
7.
Donald D. F. Loo Peter D. Brown Ernest M. Wright 《The Journal of membrane biology》1988,105(3):221-231
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
8.
Potassium channels allow the selective flux of K+ excluding the smaller, and more abundant in the extracellular solution, Na+ ions. Here we show that Shab is a typical K+ channel that excludes Na+ under bi-ionic, Nao/Ki or Nao/Rbi, conditions. However, when internal K+ is replaced by Cs+ (Nao/Csi), stable inward Na+ and outward Cs+ currents are observed. These currents show that Shab selectivity is not accounted for by protein structural elements alone, as implicit in the snug-fit model of selectivity. Additionally, here we report the block of Shab channels by external Ca2+ ions, and compare the effect that internal K+ replacement exerts on both Ca2+ and TEA block. Our observations indicate that Ca2+ blocks the channels at a site located near the external TEA binding site, and that this pore region changes conformation under conditions that allow Na+ permeation. In contrast, the latter ion conditions do not significantly affect the binding of quinidine to the pore central cavity. Based on our observations and the structural information derived from the NaK bacterial channel, we hypothesize that Ca2+ is probably coordinated by main chain carbonyls of the pore´s first K+-binding site. 相似文献
9.
《Free radical biology & medicine》1999,26(3-4):253-259
Reactive oxygen species (ROS) play a crucial role in pathophysiology of the cardiovascular system. The present study was designed to analyze the redox sensitivity of G-protein-activated inward rectifier K+ (GIRK) channels, which control cardiac contractility and excitability. GIRK1 subunits were heterologously expressed in Xenopus laevis oocytes and the resulting K+ currents were measured with the two-electrode voltage clamp technique. Oxygen free radicals generated by the hypoxanthine/xanthine oxidase system led to a marked increase in the current through GIRK channels, termed superoxide-induced current (ISO). Furthermore, ISO did not depend on G-protein-dependent activation of GIRK currents by coexpressed muscarinic m2-receptors, but could also be observed when no agonist was present in the bathing solution. Niflumic acid at a concentration of 0.5 mmol/l did not abolish ISO, whereas 100 μmol/l Ba2+ attenuated ISO completely. Catalase (106 i.u./l) failed to suppress ISO, whereas H2O2 concentration was kept close to zero, as measured by chemiluminescence. Hence, we conclude that O2•− or a closely related species is responsible for ISO induction. Our results demonstrate a significant redox sensitivity of GIRK1 channels and suggest redox-activation of G-protein-activated inward rectifier K+ channels as a key mechanism in oxidative stress-associated cardiac dysfunction. 相似文献
10.
Katy Morgan Roach Stephen Mark Duffy William Coward Carol Feghali-Bostwick Heike Wulff Peter Bradding 《PloS one》2013,8(12)
Background
Idiopathic pulmonary fibrosis (IPF) is a common, progressive and invariably lethal interstitial lung disease with no effective therapy. We hypothesised that KCa3.1 K+ channel-dependent cell processes contribute to IPF pathophysiology.Methods
KCa3.1 expression in primary human lung myofibroblasts was examined using RT-PCR, western blot, immunofluorescence and patch-clamp electrophysiology. The role of KCa3.1 channels in myofibroblast proliferation, wound healing, collagen secretion and contraction was examined using two specific and distinct KCa3.1 blockers (TRAM-34 and ICA-17043 [Senicapoc]).Results
Both healthy non fibrotic control and IPF-derived human lung myofibroblasts expressed KCa3.1 channel mRNA and protein. KCa3.1 ion currents were elicited more frequently and were larger in IPF-derived myofibroblasts compared to controls. KCa3.1 currents were increased in myofibroblasts by TGFβ1 and basic FGF. KCa3.1 was expressed strongly in IPF tissue. KCa3.1 pharmacological blockade attenuated human myofibroblast proliferation, wound healing, collagen secretion and contractility in vitro, and this was associated with inhibition of TGFβ1-dependent increases in intracellular free Ca2+.Conclusions
KCa3.1 activity promotes pro-fibrotic human lung myofibroblast function. Blocking KCa3.1 may offer a novel approach to treating IPF with the potential for rapid translation to the clinic. 相似文献11.
Sun-Hye Choi Byung-Hwan Lee Hyeon-Joong Kim Seok-Won Jung Hyun-Sook Kim Ho-Chul Shin Jun-Hee Lee Hyoung-Chun Kim Hyewhon Rhim Sung-Hee Hwang Tal soo Ha Hyun-Ji Kim Hana Cho Seung-Yeol Nah 《Molecules and cells》2014,37(9):656-663
Gintonin, a novel, ginseng-derived G protein-coupled lysophosphatidic acid (LPA) receptor ligand, elicits [Ca2+]i transients in neuronal and non-neuronal cells via pertussis toxin-sensitive and pertussis toxin-insensitive G proteins. The slowly activating delayed rectifier K+ (IKs) channel is a cardiac K+ channel composed of KCNQ1 and KCNE1 subunits. The C terminus of the KCNQ1 channel protein has two calmodulin-binding sites that are involved in regulating IKs channels. In this study, we investigated the molecular mechanisms of gintonin-mediated activation of human IKs channel activity by expressing human IKs channels in Xenopus oocytes. We found that gintonin enhances IKs channel currents in concentration- and voltage-dependent manners. The EC50 for the IKs channel was 0.05 ± 0.01 μg/ml. Gintonin-mediated activation of the IKs channels was blocked by an LPA1/3 receptor antagonist, an active phospholipase C inhibitor, an IP3 receptor antagonist, and the calcium chelator BAPTA. Gintonin-mediated activation of both the IKs channel was also blocked by the calmodulin (CaM) blocker calmidazolium. Mutations in the KCNQ1 [Ca2+]i/CaM-binding IQ motif sites (S373P, W392R, or R539W)blocked the action of gintonin on IKs channel. However, gintonin had no effect on hERG K+ channel activity. These results show that gintonin-mediated enhancement of IKs channel currents is achieved through binding of the [Ca2+]i/CaM complex to the C terminus of KCNQ1 subunit. 相似文献
12.
In whole cell patch clamp recordings, we found that normal human adrenal zona fasciculata (AZF) cells express voltage-gated, rapidly inactivating Ca2+ and K+ currents and a noninactivating, leak-type K+ current. Characterization of these currents with respect to voltage-dependent gating and kinetic properties, pharmacology, and modulation by the peptide hormones adrenocorticotropic hormone (ACTH) and AngII, in conjunction with Northern blot analysis, identified these channels as Cav3.2 (encoded by CACNA1H), Kv1.4 (KCNA4), and TREK-1 (KCNK2). In particular, the low voltage–activated, rapidly inactivating and slowly deactivating Ca2+ current (Cav3.2) was potently blocked by Ni2+ with an IC50 of 3 µM. The voltage-gated, rapidly inactivating K+ current (Kv1.4) was robustly expressed in nearly every cell, with a current density of 95.0 ± 7.2 pA/pF (n = 64). The noninactivating, outwardly rectifying K+ current (TREK-1) grew to a stable maximum over a period of minutes when recording at a holding potential of −80 mV. This noninactivating K+ current was markedly activated by cinnamyl 1-3,4-dihydroxy-α-cyanocinnamate (CDC) and arachidonic acid (AA) and inhibited almost completely by forskolin, properties which are specific to TREK-1 among the K2P family of K+ channels. The activation of TREK-1 by AA and inhibition by forskolin were closely linked to membrane hyperpolarization and depolarization, respectively. ACTH and AngII selectively inhibited the noninactivating K+ current in human AZF cells at concentrations that stimulated cortisol secretion. Accordingly, mibefradil and CDC at concentrations that, respectively, blocked Cav3.2 and activated TREK-1, each inhibited both ACTH- and AngII-stimulated cortisol secretion. These results characterize the major Ca2+ and K+ channels expressed by normal human AZF cells and identify TREK-1 as the primary leak-type channel involved in establishing the membrane potential. These findings also suggest a model for cortisol secretion in human AZF cells wherein ACTH and AngII receptor activation is coupled to membrane depolarization and the activation of Cav3.2 channels through inhibition of hTREK-1. 相似文献
13.
The K+ channel KZM2 is involved in stomatal movement by modulating inward K+ currents in maize guard cells 下载免费PDF全文
Yong‐Qiang Gao Wei‐Hua Wu Yi Wang 《The Plant journal : for cell and molecular biology》2017,92(4):662-675
Stomata are the major gates in plant leaf that allow water and gas exchange, which is essential for plant transpiration and photosynthesis. Stomatal movement is mainly controlled by the ion channels and transporters in guard cells. In Arabidopsis, the inward Shaker K+ channels, such as KAT1 and KAT2, are responsible for stomatal opening. However, the characterization of inward K+ channels in maize guard cells is limited. In the present study, we identified two KAT1‐like Shaker K+ channels, KZM2 and KZM3, which were highly expressed in maize guard cells. Subcellular analysis indicated that KZM2 and KZM3 can localize at the plasma membrane. Electrophysiological characterization in HEK293 cells revealed that both KZM2 and KZM3 were inward K+ (Kin) channels, but showing distinct channel kinetics. When expressed in Xenopus oocytes, only KZM3, but not KZM2, can mediate inward K+ currents. However, KZM2 can interact with KZM3 forming heteromeric Kin channel. In oocytes, KZM2 inhibited KZM3 channel conductance and negatively shifted the voltage dependence of KZM3. The activation of KZM2–KZM3 heteromeric channel became slower than the KZM3 channel. Patch‐clamping results showed that the inward K+ currents of maize guard cells were significantly increased in the KZM2 RNAi lines. In addition, the RNAi lines exhibited faster stomatal opening after light exposure. In conclusion, the presented results demonstrate that KZM2 functions as a negative regulator to modulate the Kin channels in maize guard cells. KZM2 and KZM3 may form heteromeric Kin channel and control stomatal opening in maize. 相似文献
14.
Pablo Martínez-López Claudia L. Treviño Juan J. Acevedo Lawrence B. Salkoff 《Biochemical and biophysical research communications》2009,381(2):204-153
Slo3 channels belong to the high conductance Slo K+ channel family. They are activated by voltage and intracellular alkalinization, and have a K+/Na+ permeability ratio (PK/PNa) of only approximately 5. Slo3 channels have only been found in mammalian sperm. Here we show that Slo3 channels expressed in Xenopus oocytes are also stimulated by elevated cAMP levels through PKA dependent phosphorylation. Capacitation, a maturational process required by mammalian sperm to enable them to fertilize eggs, involves intracellular alkalinization and an increase in cAMP. Our mouse sperm patch clamp recordings have revealed a K+ current that is time and voltage dependent, is activated by intracellular alkalinization, has a PK/PNa ? 5, is weakly blocked by TEA and is very sensitive to Ba2+. This current is also stimulated by cAMP. All of these properties match those displayed by heterologously expressed Slo3 channels, suggesting that the native current we observe in sperm is indeed carried by Slo3 channels. 相似文献
15.
G. Bkaily M. Peyrow T. Yamamoto A. Sculptoreanu D. Jacques N. Sperelakis 《Molecular and cellular biochemistry》1989,80(1-2):59-72
Summary The whole-cell voltage clamp technique was used to study the slow inward currents and K+ outward currents in single heart cells of embryonic chick and in rabbit aortic cells. In single heart cells of 3-day-old chick embryo three types of slow inward Na+ currents were found. The kinetics and the pharmacology of the slow INa, were different from those of the slow Ica in older embryos. Two types of slow inward currents were found in aortic single cells of rabbit; angiotensin 11 increased the sustained type and d-cAMP and d-cGMP decreased the slow transient component. Two types of outward K+ currents were found in both aortic and heart cells. Single channel analysis demonstrated the presence of a high single K+ channel conductance in aortic cells. In cardiac and vascular smooth muscles, slow inward currents do share some pharmacological properties, although the regulation of these channels by cyclic nucleotides and several drugs seems to be different. 相似文献
16.
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
17.
《Bioorganic & medicinal chemistry letters》2019,29(13):1601-1604
This letter describes a focused, multi-dimensional optimization campaign around BL-1249, a fenamate class non-steroidal anti-inflammatory and a known activator of the K2P potassium channels TREK-1 (K2P2.1) and TREK-2 (K2P10.1). While BL-1249 has been widely profiled in vitro as a dual TREK-1/2 activator, poor physicochemical and DMPK properties have precluded a deeper understanding of the therapeutic potential of these key K2P channels across a broad spectrum of peripheral and central human disease. Here, we report multi-dimensional SAR that led to a novel TREK-1/2 dual activator chemotype, exemplified by ONO-2960632/VU6011992, with improved DMPK properties, representing a new lead for further optimization towards robust in vivo tool compounds. 相似文献
18.
Summary. Patch-clamp whole-cell and single-channel recording techniques were used to investigate the regulation of outward K+ channels by external and internal protons in Brassica chinensis pollen protoplasts. Outward K+ currents and conductance were insensitive to external pH (pHo) except at pH 4.5. Maximal conductance (G
max) for the outward K+ currents was inhibited at acidic external pH. Half-activation voltage (E
1/2) for the outward K+ currents shifted to more positive voltages along with the decrease in pHo. E
1/2 can be described by a modified Henderson–Hasselbalch equation expected from a single titratable binding site. The activation
kinetics of the outward K+ channels was largely insensitive to pHo. An internal pH (pHi) of 4.5 significantly increased outward K+ currents and conductance. G
max for the outward K+ currents decreased with elevations in pHi. In contrast to the effect of pHo, E
1/2 was shifted to more positive voltages with elevations in pHi. The outward K+ currents, G
max and E
1/2 can be described by the modified Henderson–Hasselbalch equation. Furthermore, acidifying pHi accelerated the activation of the outward K+ currents significantly. The differences in electro-physiological properties among previously reported and currently described
plant outward K+ channels may reflect differences in the structure of these channels.
Received May 7, 2002; accepted July 9, 2002; published online November 29, 2002 相似文献
19.
Previous studies have shown that 17β-estradiol has a pivotal function by blocking voltage-gated K+ (Kv) channels in several different types of cells such as cardiac myocytes and neurons. Outward Kv currents can also be measured in osteoblasts, although little is known about the effects of 17β-estradiol on these currents. In human osteoblast-like MG63 cells, we found that 17β-estradiol inhibits peak and end Kv currents, with IC50 values of 480 and 325 nM, respectively. To elucidate the mechanism of inhibition, the kinetics of Kv currents were investigated. The half-maximum activation potential (V 1/2) was 1.3 mV and was shifted left to ?4.4 mV after application of 500 nM 17β-estradiol. For steady-state inactivation, the V 1/2 was –55.0 mV and weakly shifted left to –58.2 mV. To identify the molecular basis of outward Kv currents in MG63 cells, we performed RT-PCR analyses. The expression of Kv2.1 channels appeared to dominate over that of other Kv channels in MG63 cells. In COS-7 cells with heterologously expressed Kv2.1 channels, 17β-estradiol also inhibits macroscopic currents of Kv2.1. Our data indicate that 17β-estradiol inhibits Kv currents in human osteoblast-like MG63 cells and that Kv2.1 is a potential molecular correlate of outward Kv currents in these cells. 相似文献
20.
Extracellular acidification and reduction of extracellular K+ are known to decrease the currents of some voltage-gated potassium channels. Although the macroscopic conductance of WT hKv1.5 channels is not very sensitive to [K+]o at pH 7.4, it is very sensitive to [K+]o at pH 6.4, and in the mutant, H463G, the removal of K+
o virtually eliminates the current at pH 7.4. We investigated the mechanism of current regulation by K+
o in the Kv1.5 H463G mutant channel at pH 7.4 and the wild-type channel at pH 6.4 by taking advantage of Na+ permeation through inactivated channels. Although the H463G currents were abolished in zero [K+]o, robust Na+ tail currents through inactivated channels were observed. The appearnnce of H463G Na+ currents with a slow rising phase on repolarization after a very brief depolarization (2 ms) suggests that channels could
activate directly from closed-inactivated states. In wild-type channels, when intracellular K+ was replaced by NMG+ and the inward Na+ current was recorded, addition of 1 mM K+ prevented inactivation, but changing pH from 7.4 to 6.4 reversed this action. The data support the idea that C-type inactivation
mediated at R487 in Kv1.5 channels is influenced by H463 in the outer pore. We conclude that both acidification and reduction
of [K+]o inhibit Kv1.5 channels through a common mechananism (i.e., by increasing channel inactivation, which occurs in the resting
state or develops very rapidly after activation). 相似文献