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排序方式: 共有741条查询结果,搜索用时 15 毫秒
41.
《Molecular membrane biology》2013,30(8):435-447
AbstractThe hERG potassium channel is a member of the voltage gated potassium (Kv) channel family, comprising a pore domain and four voltage sensing domains (VSDs). Like other Kv channels, the VSD senses changes in membrane voltage and transmits the signal to gates located in the pore domain; the gates open at positive potentials (activation) and close at negative potentials, thereby controlling the ion flux. hERG, however, differs from other Kv channels in that it is activated slowly but inactivated rapidly – a property that is crucial for the role it plays in the repolarization of the cardiac action potential. Voltage-gating requires movement of gating charges across the membrane electric field, which is accomplished by the transmembrane movement of the fourth transmembrane segment, S4, of the VSD containing the positively charged arginine or lysine residues. Here we ask if the functional differences between hERG and other Kv channels could arise from differences in the transmembrane movement of S4. To address this, we have introduced single cysteine residues into the S4 region of the VSD, expressed the mutant channels in Xenopus oocytes and examined the effect of membrane impermeable para-chloromercuribenzene sulphonate on function by the two-electrode voltage clamp technique. Our results show that depolarization results in the accessibility of seven consecutive S4 residues, including the first two charged residues, K525 and R528, to extracellularly applied reagent. These data indicate that the extent of S4 movement in hERG is similar to other Kv channels, including the archabacterial KvAP and the Shaker channel of Drosophila. 相似文献
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43.
Benchmarking sample preparation/digestion protocols reveals tube‐gel being a fast and repeatable method for quantitative proteomics
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Sample preparation, typically by in‐solution or in‐gel approaches, has a strong influence on the accuracy and robustness of quantitative proteomics workflows. The major benefit of in‐gel procedures is their compatibility with detergents (such as SDS) for protein solubilization. However, SDS‐PAGE is a time‐consuming approach. Tube‐gel (TG) preparation circumvents this drawback as it involves directly trapping the sample in a polyacrylamide gel matrix without electrophoresis. We report here the first global label‐free quantitative comparison between TG, stacking gel (SG), and basic liquid digestion (LD). A series of UPS1 standard mixtures (at 0.5, 1, 2.5, 5, 10, and 25 fmol) were spiked in a complex yeast lysate background. TG preparation allowed more yeast proteins to be identified than did the SG and LD approaches, with mean numbers of 1979, 1788, and 1323 proteins identified, respectively. Furthermore, the TG method proved equivalent to SG and superior to LD in terms of the repeatability of the subsequent experiments, with mean CV for yeast protein label‐free quantifications of 7, 9, and 10%. Finally, known variant UPS1 proteins were successfully detected in the TG‐prepared sample within a complex background with high sensitivity. All the data from this study are accessible on ProteomeXchange (PXD003841). 相似文献
44.
León D. Islas 《Channels (Austin, Tex.)》2016,10(3):202-213
Voltage-gated potassium channels or Kv's are membrane proteins with fundamental physiological roles. They are composed of 2 main functional protein domains, the pore domain, which regulates ion permeation, and the voltage-sensing domain, which is in charge of sensing voltage and undergoing a conformational change that is later transduced into pore opening. The voltage-sensing domain or VSD is a highly conserved structural motif found in all voltage-gated ion channels and can also exist as an independent feature, giving rise to voltage sensitive enzymes and also sustaining proton fluxes in proton-permeable channels. In spite of the structural conservation of VSDs in potassium channels, there are several differences in the details of VSD function found across variants of Kvs. These differences are mainly reflected in variations in the electrostatic energy needed to open different potassium channels. In turn, the differences in detailed VSD functioning among voltage-gated potassium channels might have physiological consequences that have not been explored and which might reflect evolutionary adaptations to the different roles played by Kv channels in cell physiology. 相似文献
45.
Sungmoo Lee Hong Hua Piao Masoud Sepheri-Rad Arong Jung Uhna Sung Yoon-Kyu Song Bradley J. Baker 《Journal of visualized experiments : JoVE》2016,(108)
Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of Förster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera. 相似文献
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47.
Yi‐zhou Tan Dong‐dong Fei Xiao‐ning He Ji‐min Dai Rong‐chen Xu Xin‐yue Xu Jun‐jie Wu Bei Li 《Cell proliferation》2019,52(4)
L‐type voltage‐gated calcium ion channels (L‐VGCCs) have been demonstrated to be the mediator of several significant intracellular activities in excitable cells, such as neurons, chromaffin cells and myocytes. Recently, an increasing number of studies have investigated the function of L‐VGCCs in non‐excitable cells, particularly stem cells. However, there appear to be no systematic reviews of the relationship between L‐VGCCs and stem cells, and filling this gap is prescient considering the contribution of L‐VGCCs to the proliferation and differentiation of several types of stem cells. This review will discuss the possible involvement of L‐VGCCs in stem cells, mainly focusing on osteogenesis mediated by mesenchymal stem cells (MSCs) from different tissues and neurogenesis mediated by neural stem/progenitor cells (NSCs). Additionally, advanced applications that use these channels as the target for tissue engineering, which may offer the hope of tissue regeneration in the future, will also be explored. 相似文献
48.
Usherwood PN Davies TG Mellor IR O'Reilly AO Peng F Vais H Khambay BP Field LM Williamson MS 《FEBS letters》2007,581(28):5485-5492
Mutations in the DIIS4-S5 linker and DIIS5 have identified hotspots of pyrethroid and DDT interaction with the Drosophila para sodium channel. Wild-type and mutant channels were expressed in Xenopus oocytes and subjected to voltage-clamp analysis. Substitutions L914I, M918T, L925I, T929I and C933A decreased deltamethrin potency, M918T, L925I and T929I decreased permethrin potency and T929I, L925I and I936V decreased fenfluthrin potency. DDT potency was unaffected by M918T, but abolished by T929I and reduced by L925I, L932F and I936V, suggesting that DIIS5 contains at least part of the DDT binding domain. The data support a computer model of pyrethroid and DDT binding. 相似文献
49.
Alvinella pompejana, the so-called Pompeii worm (Desbruyères and Laubier, 1980), is found exclusively in association to high temperature venting,
at the surface of hydrothermal chimneys of the East Pacific Rise. The main characteristics of this emblematic species is its
tolerance to high temperature but its ability to colonize extremely hot substrates has been the subject of much controversy.
In the last decade, new tools allowing in situ and in vivo investigation have been determinant in the understanding of the
strategies and adaptations required to colonize such an extremely hot environment. New data relative to the characterization
of the animal habitat conditions, on one hand, to the molecular adaptations of this organism and the colonization processes
by this species, on the other hand, are now available. Advanced methods and tools, that have fostered the physico-chemical
characterization of vent habitats in recent years, are first reviewed. Factors controlling the physico-chemical variability
of vent habitats and the threats A. pompejana might effectively face are discussed. The exceptional thermotolerance of this species and the maximum temperature it could
sustain are then considered in the light of molecular data relative to its collagen stability. Life history traits as well
as biological controls on tube micro-habitat conditions are discussed on the basis of new in situ and in vivo experiments
and characterization. Finally, the current knowledge and opened questions related to the molecular adaptations to chemical
stresses are briefly stated. The ability of Alvinella pompejana to colonize these substrates is far from being fully understood, but the exceptional properties of its extracellular biopolymers
and the behavior of the worm can be now considered as major clues in the colonization process. Alvinella pompejana could thus stand at the limits authorized for its biological machinery in a highly dynamic environment where temperature
can readily reach lethal values, but where temperature regulation by the animal itself would prevent exposure to deleterious
thermal spikes. The dynamic system associating this pioneer species and its associated microflora might be viewed as a key
to the subsequent colonization of these environments by less tolerant species, highlighting A. pompejana as a new type of ecosystem bioengineer. 相似文献
50.
Mitochondrial uncoupling protein 2 (UCP2) is highly abundant in rapidly proliferating cells that utilize aerobic glycolysis, such as stem cells, cancer cells, and cells of the immune system. However, the function of UCP2 has been a longstanding conundrum. Considering the strict regulation and unusually short life time of the protein, we propose that UCP2 acts as a “signaling protein” under nutrient shortage in cancer cells. We reveal that glutamine shortage induces the rapid and reversible downregulation of UCP2, decrease of the metabolic activity and proliferation of neuroblastoma cells, that are regulated by glutamine per se but not by glutamine metabolism. Our findings indicate a very rapid (within 1?h) metabolic adaptation that allows the cell to survive by either shifting its metabolism to the use of the alternative fuel glutamine or going into a reversible, more quiescent state. The results imply that UCP2 facilitates glutamine utilization as an energetic fuel source, thereby providing metabolic flexibility during glucose shortage. The targeting UCP2 by drugs to intervene with cancer cell metabolism may represent a new strategy for treatment of cancers resistant to other therapies. 相似文献