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1.
Acidosis is a common feature of brain in acute neurological injury, particularly in ischemia where low pH has been assumed
to play an important role in the pathological process. However, the cellular and molecular mechanisms underlying acidosis-induced
injury remain unclear. Recent studies have demonstrated that activation of Ca2+-permeable acid-sensing ion channels (ASIC1a) is largely responsible for acidosis-mediated, glutamate receptor-independent,
neuronal injury. In cultured mouse cortical neurons, lowering extracellular pH to the level commonly seen in ischemic brain
activates amiloride-sensitive ASIC currents. In the majority of these neurons, ASICs are permeable to Ca2+, and an activation of these channels induces increases in the concentration of intracellular Ca2+ ([Ca2+]i). Activation of ASICs with resultant [Ca2+]i loading induces time-dependent neuronal injury occurring in the presence of the blockers for voltage-gated Ca2+ channels and the glutamate receptors. This acid-induced injury is, however, inhibited by the blockers of ASICs, and by reducing
[Ca2+]o. In focal ischemia, intracerebroventricular administration of ASIC1a blockers, or knockout of the ASIC1a gene protects brain
from injury and does so more potently than glutamate antagonism. Furthermore, pharmacological blockade of ASICs has up to
a 5 h therapeutic time window, far beyond that of glutamate antagonists. Thus, targeting the Ca2+-permeable acid-sensing ion channels may prove to be a novel neuroprotective strategy for stroke patients. 相似文献
2.
《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
3.
Zhao-Wen Wang Masayuki Nara Yong-Xiao Wang Michael I. Kotlikoff 《The Journal of general physiology》1997,110(1):35-44
The effects of sulfhydryl reduction/oxidation on the gating of large-conductance, Ca2+-activated K+ (maxi-K) channels were examined in excised patches from tracheal myocytes. Channel activity was modified by sulfhydryl redox agents applied to the cytosolic surface, but not the extracellular surface, of membrane patches. Sulfhydryl reducing agents dithiothreitol, β-mercaptoethanol, and GSH augmented, whereas sulfhydryl oxidizing agents diamide, thimerosal, and 2,2′-dithiodipyridine inhibited, channel activity in a concentration-dependent manner. Channel stimulation by reduction and inhibition by oxidation persisted following washout of the compounds, but the effects of reduction were reversed by subsequent oxidation, and vice versa. The thiol-specific reagents N-ethylmaleimide and (2-aminoethyl)methanethiosulfonate inhibited channel activity and prevented the effect of subsequent sulfhydryl oxidation. Measurements of macroscopic currents in inside-out patches indicate that reduction only shifted the voltage/nPo relationship without an effect on the maximum conductance of the patch, suggesting that the increase in nPo following reduction did not result from recruitment of more functional channels but rather from changes of channel gating. We conclude that redox modulation of cysteine thiol groups, which probably involves thiol/disulfide exchange, alters maxi-K channel gating, and that this modulation likely affects channel activity under physiological conditions. 相似文献
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Activation of Plant Plasma Membrane Ca2+-Permeable Channels by Race-Specific Fungal Elicitors 总被引:4,自引:0,他引:4
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The response of plant cells to invading pathogens is regulated by fluctuations in cytosolic Ca2+ levels that are mediated by Ca2+-permeable channels located at the plasma membrane of the host cell. The mechanisms by which fungal elicitors can induce Ca2+ uptake by the host cell were examined by the application of conventional patch-clamp techniques. Whole-cell and single-channel experiments on tomato (Lycopersicon esculentum L.) protoplasts revealed a race-specific fungal elicitor-induced activation of a plasma membrane Ca2+-permeable channel. The presence of the fungal elicitor resulted in a greater probability of channel opening. Guanosine 5[prime]-[[beta]-thio]diphosphate, a GDP analog that locks heterotrimeric G-proteins into their inactivated state, abolished the channel activation induced by the fungal elicitor, whereas guanosine 5[prime][[gamma]-thio]triphosphate, a nonhydrolyzable GTP analog that locks heterotrimeric G-proteins into their activated state, produced an effect similar to that observed with the fungal elicitor. Mastoparan, which stimulates GTPase activity, mimicked the effect of GTP[[gamma]]S. The addition of HA1004 (a protein kinase inhibitor) in the presence of the elicitor totally abolished channel activity, whereas okadaic acid (a protein phosphatase inhibitor) moderately enhanced channel activity, suggesting that the activation of the channel by fungal elicitors is modulated by a heterotrimeric G-protein-dependent phosphorylation of the channel protein. 相似文献
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8.
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
9.
NG2 cells, a fourth type of glial cell in the mammalian CNS, undergo reactive changes in response to a wide variety of brain insults. Recent studies have demonstrated that neuronally expressed acid-sensing ion channels (ASICs) are implicated in various neurological disorders including brain ischemia and seizures. Acidosis is a common feature of acute neurological conditions. It is postulated that a drop in pH may be the link between the pathological process and activation of NG2 cells. Such postulate immediately prompts the following questions: Do NG2 cells express ASICs? If so, what are their functional properties and subunit composition? Here, using a combination of electrophysiology, Ca2+ imaging and immunocytochemistry, we present evidence to demonstrate that NG2 cells of the rat hippocampus express high density of Ca2+-permeable ASIC1a channels compared with several types of hippocampal neurons. First, nucleated patch recordings from NG2 cells revealed high density of proton-activated currents. The magnitude of proton-activated current was pH dependent, with a pH for half-maximal activation of 6.3. Second, the current-voltage relationship showed a reversal close to the equilibrium potential for Na+. Third, psalmotoxin 1, a blocker specific for the ASIC1a channel, largely inhibited proton-activated currents. Fourth, Ca2+ imaging showed that activation of proton-activated channels led to an increase of [Ca2+]i. Finally, immunocytochemistry showed co-localization of ASIC1a and NG2 proteins in the hippocampus. Thus the acid chemosensor, the ASIC1a channel, may serve for inducing membrane depolarization and Ca2+ influx, thereby playing a crucial role in the NG2 cell response to injury following ischemia. 相似文献
10.
Anna Amcheslavsky Mona?L. Wood Andriy?V. Yeromin Ian Parker J.?Alfredo Freites Douglas?J. Tobias Michael?D. Cahalan 《Biophysical journal》2015,108(2):237-246
Upon endoplasmic reticulum Ca2+ store depletion, Orai channels in the plasma membrane are activated directly by endoplasmic reticulum-resident STIM proteins to generate the Ca2+-selective, Ca2+ release-activated Ca2+ (CRAC) current. After the molecular identification of Orai, a plethora of functional and biochemical studies sought to compare Orai homologs, determine their stoichiometry, identify structural domains responsible for the biophysical fingerprint of the CRAC current, identify the physiological functions, and investigate Orai homologs as potential therapeutic targets. Subsequently, the solved crystal structure of Drosophila Orai (dOrai) substantiated many findings from structure-function studies, but also revealed an unexpected hexameric structure. In this review, we explore Orai channels as elucidated by functional and biochemical studies, analyze the dOrai crystal structure and its implications for Orai channel function, and present newly available information from molecular dynamics simulations that shed light on Orai channel gating and permeation. 相似文献
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Klaus Schuhmann Christoph Romanin Werner Baumgartner Klaus Groschner 《The Journal of general physiology》1997,110(5):503-513
Modulation of L-type Ca2+ channels by tonic elevation of cytoplasmic Ca2+ was investigated in intact cells and inside-out patches from human umbilical vein smooth muscle. Ba2+ was used as charge carrier, and run down of Ca2+ channel activity in inside-out patches was prevented with calpastatin plus ATP. Increasing cytoplasmic Ca2+ in intact cells by elevation of extracellular Ca2+ in the presence of the ionophore A23187 inhibited the activity of L-type Ca2+ channels in cell-attached patches. Measurement of the actual level of intracellular free Ca2+ with fura-2 revealed a 50% inhibitory concentration (IC50) of 260 nM and a Hill coefficient close to 4 for Ca2+- dependent inhibition. Ca2+-induced inhibition of Ca2+ channel activity in intact cells was due to a reduction of channel open probability and availability. Ca2+-induced inhibition was not affected by the protein kinase inhibitor H-7 (10 μM) or the cytoskeleton disruptive agent cytochalasin B (20 μM), but prevented by cyclosporin A (1 μg/ ml), an inhibitor of protein phosphatase 2B (calcineurin). Elevation of Ca2+ at the cytoplasmic side of inside-out patches inhibited Ca2+ channels with an IC50 of 2 μM and a Hill coefficient close to unity. Direct Ca2+-dependent inhibition in cell-free patches was due to a reduction of open probability, whereas availability was barely affected. Application of purified protein phosphatase 2B (12 U/ml) to the cytoplasmic side of inside-out patches at a free Ca2+ concentration of 1 μM inhibited Ca2+ channel open probability and availability. Elevation of cytoplasmic Ca2+ in the presence of PP2B, suppressed channel activity in inside-out patches with an IC50 of ∼380 nM and a Hill coefficient of ∼3; i.e., characteristics reminiscent of the Ca2+ sensitivity of Ca2+ channels in intact cells. Our results suggest that L-type Ca2+ channels of smooth muscle are controlled by two Ca2+-dependent negative feedback mechanisms. These mechanisms are based on (a) a protein phosphatase 2B-mediated dephosphorylation process, and (b) the interaction of intracellular Ca2+ with a single membrane-associated site that may reside on the channel protein itself. 相似文献
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14.
Milan Brumen Aleš Fajmut Andrej Dobovišek Etienne Roux 《Journal of biological physics》2005,31(3-4):515-524
The action of different agonists such as acetylcholine on the membrane of airway smooth muscle cells may induce cytosolic
Ca2+ oscillations which can be a part of the Ca2+ signalling pathway, eventually leading to cell contraction. The aim of the present study is to present a mathematical model
of the possible effect of the initial Ca2+ distribution within the cell on the form and frequency of induced Ca2+ oscillations. It takes into account intracellular Ca2+ stores such as sarcoplasmic reticulum and cytosolic proteins as well as Ca2+ exchange across the plasma membrane. We are able to demonstrate a closer agreement of model predictions with observed Ca2+ traces for a significantly wider range of parameter values, as was previously reported. We show also that the total cellular
Ca2+ content is an important system parameter especially because of the content in sarcoplasmic reticulum. At a total Ca2+ increase of about 20%, the oscillation frequency increases by 25%; also, damped oscillations become sustained. Cases are
indicated in which such a situation could occur. 相似文献
15.
TRP channels and Ca2+ signaling 总被引:3,自引:0,他引:3
Minke B 《Cell calcium》2006,40(3):261-275
There is a rapidly growing interest in the family of transient receptor potential (TRP) channels because TRP channels are not only important for many sensory systems, but they are crucial components of the function of neurons, epithelial, blood and smooth muscle cells. These facts make TRP channels important targets for treatment of diseases arising from the malfunction of these channels in the above cells and for treatment of inflammatory pain. TRP channels are also important for a growing number of genetic diseases arising from mutations in various types of TRP channels. The Minerva-Gentner Symposium on TRP channels and Ca(2+) signaling, which took place in Eilat, Israel (February 24-28, 2006) has clearly demonstrated that the study of TRP channels is a newly emerging field of biomedicine with prime importance. In the Eilat symposium, investigators who have contributed seminal publications and insight into the TRP field presented their most recent, and in many cases still unpublished, studies. The excellent presentations and excitement generated by them demonstrated that much progress has been achieved. Nevertheless, it was also evident that the field of TRP channels is still in its infancy in comparison to other fields of ion channels, and even the fundamental knowledge of the gating mechanism of TRP channels is still unsolved. The beautiful location of the symposium, together with informal intensive discussions among the participants, contributed to the success of this meeting. 相似文献
16.
Jörg Mitterdorfer Manfred Grabner Richard L. Kraus Steffen Hering Heino Prinz Hartmut Glossmann Jörg Striessnig 《Journal of bioenergetics and biomembranes》1998,30(4):319-334
Different types of voltage-gated Ca2+ channels exist in the plasma membrane of electrically excitable cells. By controlling depolarization-induced Ca2+ entry into cells they serve important physiological functions, such as excitation-contraction coupling, neurotransmitter and hormone secretion, and neuronal plasticity. Their function is fine-tuned by a variety of modulators, such as enzymes and G-proteins. Block of so-called L-type Ca2+ channels by drugs is exploited as a therapeutic principle to treat cardiovascular disorders, such as hypertension. More recently, block of so-called non-L-type Ca2+ channels was found to exert therapeutic effects in the treatment of severe pain and ischemic stroke. As the subunits of different Ca2+ channel types have been cloned, the modulatory sites for enzymes, G-proteins, and drugs can now be determined using molecular engineering and heterologous expression. Here we summarize recent work that has allowed us to determine the sites of action of L-type Ca2+ channel modulators. Together with previous biochemical, electrophysiological, and drug binding data these results provide exciting insight into the molecular pharmacology of this voltage-gated Ca2+ channel family. 相似文献
17.
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
18.
Diabetes mellitus, a debilitating chronic disease, affects ~100?million people. Peripheral neuropathy is one of the most common early complications of diabetes in ~66?% of these patients. Altered Ca(2+) handling and Ca(2+) signaling were detected in a huge variety of preparations isolated from animals with experimentally induced type 1 and 2 diabetes as well as patients suffering from the disease. We reviewed the role of Ca(2+) signaling through cation channels and oxidative stress on diabetic neuropathic pain in sensory neurons. The pathogenesis of diabetic neuropathy involves the polyol pathway, advanced glycation end products, oxidative stress, protein kinase C activation, neurotrophism, and hypoxia. Experimental studies with respect to oxidative stress and Ca(2+) signaling, inhibitor roles of antioxidants in diabetic neuropathic pain are also summarized in the review. We hypothesize that deficits in insulin, triggers alterations of sensory neurone phenotype that are critical for the development of abnormal Ca(2+) homeostasis and oxidative stress and associated mitochondrial dysfunction. The transient receptor potential channels are a large family of proteins with six main subfamilies. The sheer number of different TRPs with distinct functions supports the statement that these channels are involved in a wide range of processes ranging in diabetic neuropathic pain and it seems that the TRPC, TRPM and TRPV groups are mostly responsible from diabetic neuropathic pain. In conclusion, the accumulating evidence implicating Ca(2+) dysregulation and over production of oxidative stress products in diabetic neuropathic pains, along with recent advances in understanding of genetic variations in cation channels such as TRP channels, makes modulation of neuronal Ca(2+) handling an increasingly viable approach for therapeutic interventions against the painful and degenerative aspects of many diabetic neuropathies. 相似文献
19.
Using a patch-clamp technique in the whole-cell configuration, we studied the effect of a nitric oxide (NO) donor, nitroglycerin (NG), on outward transmembrane ion current in isolated smooth muscle cells (SMC) of the main pulmonary artery of the rabbit. We also studied the characteristics of unitary high-conductance Ca2+-dependent K+ channels (KCa channels) in the SMC membrane in the cell-attached and outside-out configurations. Nitroglycerin in a 10 M concentration increased the amplitude and intensified oscillations of outward transmembrane current induced by step depolarization. In this case, the threshold of activation of the current (–40 mV) did not change. If the potential was +70 mV, the transmembrane current in the presence of NG increased, as compared with the control, by 32.6 ± 19.4% (n = 6), on average. Simultaneous addition of 10 M NG and 1 mM tetraethylammonium chloride (TEA), a blocker of KCa channels, to the external solution at the potential of +70 mV decreased the amplitude of outward transmembrane current with respect to the control by 25.2 ± 11% (n = 6) and suppressed oscillations of this current. In the series of experiments carried out in the outside-out configuration (concentration of K+ ions in the external solution was 5.9 mM), we calculated the conductance of a single KCa channel, which was approximately 150 pS. In the case where the potential was equal to +40 mV, 1 mM TEA suppressed completely the current through unitary KCa channels. In the series of experiments performed in the cell-attached configuration, 100 M NG to a considerable extent intensified the activity of unitary high-conductance KCa channels by increasing the probability of the channel open state (P
0), on average, by 80 ± 1%, as compared with the control. In this case, NG did not influence the conductance of single KCa channels. We concluded that the NO donor NG increases the amplitude of outward transmembrane current in SMC of the rabbit main pulmonary artery by stimulation of the activity of TEA-sensitive high-conductance KCa channels. Our experiments carried out on single KCa channels demonstrated that the activating effect of NG on KCa channels is realized at the expense of an increase in the P
0 of these channels, but not of a change in the conductance of single channels. 相似文献
20.
Marnie L. Olson Susan Chalmers John G. McCarron 《The Journal of biological chemistry》2010,285(3):2040-2050
Smooth muscle activities are regulated by inositol 1,4,5-trisphosphate (InsP3)-mediated increases in cytosolic Ca2+ concentration ([Ca2+]c). Local Ca2+ release from an InsP3 receptor (InsP3R) cluster present on the sarcoplasmic reticulum is termed a Ca2+ puff. Ca2+ released via InsP3R may diffuse to adjacent clusters to trigger further release and generate a cell-wide (global) Ca2+ rise. In smooth muscle, mitochondrial Ca2+ uptake maintains global InsP3-mediated Ca2+ release by preventing a negative feedback effect of high [Ca2+] on InsP3R. Mitochondria may regulate InsP3-mediated Ca2+ signals by operating between or within InsP3R clusters. In the former mitochondria could regulate only global Ca2+ signals, whereas in the latter both local and global signals would be affected. Here whether mitochondria maintain InsP3-mediated Ca2+ release by operating within (local) or between (global) InsP3R clusters has been addressed. Ca2+ puffs evoked by localized photolysis of InsP3 in single voltage-clamped colonic smooth muscle cells had amplitudes of 0.5–4.0 F/F0, durations of ∼112 ms at half-maximum amplitude, and were abolished by the InsP3R inhibitor 2-aminoethoxydiphenyl borate. The protonophore carbonyl cyanide 3-chloropheylhydrazone and complex I inhibitor rotenone each depolarized ΔΨM to prevent mitochondrial Ca2+ uptake and attenuated Ca2+ puffs by ∼66 or ∼60%, respectively. The mitochondrial uniporter inhibitor, RU360, attenuated Ca2+ puffs by ∼62%. The “fast” Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acted like mitochondria to prolong InsP3-mediated Ca2+ release suggesting that mitochondrial influence is via their Ca2+ uptake facility. These results indicate Ca2+ uptake occurs quickly enough to influence InsP3R communication at the intra-cluster level and that mitochondria regulate both local and global InsP3-mediated Ca2+ signals. 相似文献