共查询到20条相似文献,搜索用时 31 毫秒
1.
Evans WH Martin PE 《BioEssays : news and reviews in molecular, cellular and developmental biology》2002,24(10):876-880
Gap junction intercellular communication channels permit the exchange of small regulatory molecules and ions between neighbouring cells and coordinate cellular activity in diverse tissue and organ systems. These channels have short half-lives and complex assembly and degradation pathways. Much of the recent work elucidating gap junction biogenesis has featured the use of connexins (Cx), the constituent proteins of gap junctions, tagged with reporter proteins such as Green Fluorescent Protein (GFP) and has illuminated the dynamics of channel assembly in live cells by high-resolution time-lapse microscopy. With some studies, however, there are potential short-comings associated with the GFP chimeric protein technologies. A recent report by Gaietta et al., has highlighted the use of recombinant proteins with tetracysteine tags attached to the carboxyl terminus of Cx43, which differentially labels 'old' and 'new' connexins thus opening up new avenues for studying temporal and spatial localisation of proteins and in situ trafficking events. 相似文献
2.
The combination of affinity purification and tandem mass spectrometry (MS) has emerged as a powerful approach to delineate biological processes. In particular, the use of epitope tags has allowed this approach to become scaleable and has bypassed difficulties associated with generation of antibodies. Single epitope tags and tandem affinity purification (TAP) tags have been used to systematically map protein complexes generating protein interaction data at a near proteome-wide scale. Recent developments in the design of tags, optimisation of purification conditions, experimental design and data analysis have greatly improved the sensitivity and specificity of this approach. Concomitant developments in MS, including high accuracy and high-throughput instrumentation together with quantitative MS methods, have facilitated large-scale and comprehensive analysis of multiprotein complexes. 相似文献
3.
Hughey RP Mueller GM Bruns JB Kinlough CL Poland PA Harkleroad KL Carattino MD Kleyman TR 《The Journal of biological chemistry》2003,278(39):37073-37082
The epithelial Na+ channel (ENaC) is a tetramer of two alpha-, one beta-, and one gamma-subunit, but little is known about its assembly and processing. Because co-expression of mouse ENaC subunits with three different carboxyl-terminal epitope tags produced an amiloride-sensitive sodium current in oocytes, these tagged subunits were expressed in both Chinese hamster ovary or Madin-Darby canine kidney type 1 epithelial cells for further study. When expressed alone alpha-(95 kDa), beta-(96 kDa), and gamma-subunits (93 kDa) each produced a single band on SDS gels by immunoblotting. However, co-expression of alphabetagammaENaC subunits revealed a second band for each subunit (65 kDa for alpha, 110 kDa for beta, and 75 kDa for gamma) that exhibited N-glycans that had been processed to complex type based on sensitivity to treatment with neuraminidase, resistance to cleavage by endoglycosidase H, and GalNAc-independent labeling with [3H]Gal in glycosylation-defective Chinese hamster ovary cells (ldlD). The smaller size of the processed alpha- and gamma-subunits is also consistent with proteolytic cleavage. By using alpha- and gamma-subunits with epitope tags at both the amino and carboxyl termini, proteolytic processing of the alpha- and gamma-subunits was confirmed by isolation of an additional epitope-tagged fragment from the amino terminus (30 kDa for alpha and 18 kDa for gamma) consistent with cleavage within the extracellular loop. The fragments remain stably associated with the channel as shown by immunoblotting of co-immunoprecipitates, suggesting that proteolytic cleavage represents maturation rather than degradation of the channel. 相似文献
4.
Anand R 《Biochemical and biophysical research communications》2000,276(1):157-161
At least two different models for the transmembrane topology of the glutamate receptor subunits have been proposed. We investigated some features of these two models for the GluR1 subunit by inserting epitope tags between residues Lys(502)-Pro(503), Ala(632)-Glu(633), Lys(712)-Pro(713), or after the C-terminal residue Leu(889). The accessibility of the tags then was detected using a tag-specific antibody before and after detergent-permeabilizing oocytes expressing the tagged subunits. The epitope tag inserted between residues Lys(712)-Pro(713) is extracellular and after Leu(889) intracellular. Epitope tags inserted between residues Lys(502)-Pro(503) and residues Ala(632)-Glu(633) were not detectable. Collectively, these results provide supporting evidence for a previously proposed topological model of GluR subunits containing an N-terminal extracellular domain, three transmembrane domains, the first two of which are bridged by a reentrant membrane pore-lining loop, and an intracellular C-terminal domain. 相似文献
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Development of an increasingly detailed understanding of the eucaryotic mitochondrial ATP synthase requires a detailed knowledge of the stoichiometry, structure and function of F(0) sector subunits in the contexts of the proton channel and the stator stalk. Still to be resolved are the precise locations and roles of other supernumerary subunits present in mitochondrial ATP synthase complexes, but not found in the bacterial or chloroplast enzymes. The highly developed system of molecular genetic manipulation available in the yeast Saccharomyces cerevisiae, a unicellular eucaryote, permits testing for gene function based on the effects of gene disruption or deletion. In addition, the genes encoding ATP synthase subunits can be manipulated to introduce specific amino acids at desired positions within a subunit, or to add epitope or affinity tags at the C-terminus, enabling questions of stoichiometry, structure and function to be addressed. Newly emerging technologies, such as fusions of subunits with GFP are being applied to probe the dynamic interactions within mitochondrial ATP synthase, between ATP synthase complexes, and between ATP synthase and other mitochondrial enzyme complexes. 相似文献
7.
Insect nicotinic acetylcholine receptors (nAChRs) play a central role in mediating neuronal synaptic transmission and are the target sites for the increasingly important group of neonicotinoid insecticides. Six nicotinic acetylcholine receptor (nAChR) subunits (four alpha-type and two beta-type) have been cloned previously from the model insect species Drosophila melanogaster. Despite extensive efforts, it has not been possible to generate functional recombinant nAChRs by heterologous expression of any combination of these six subunits. It has, however, been possible to express functional hybrid receptors when Drosophila alpha subunits are co-expressed with vertebrate beta subunits. This has led to the assumption that successful heterologous expression might require an, as yet, uncloned beta-type insect subunit. Examination of the recently completed Drosophila genomic sequence data has identified a novel putative nAChR beta-type subunit. Here we report the molecular cloning, heterologous expression and characterization of this putative Drosophila nAChR subunit (Dbeta3). Phylogenetic comparisons with other ligand-gated ion channel subunit sequences support its classification as a nAChR subunit but show it to be a distantly related member of this neurotransmitter receptor subunit family. Evidence that the Dbeta3 subunit is able to coassemble with other Drosophila nAChR subunits and contribute to recombinant nAChRs has been obtained by both radioligand binding and coimmunoprecipitation studies in transfected Drosophila S2 cells. 相似文献
8.
W. R. Williams 《Journal of receptor and signal transduction research》2018,38(2):101-111
Purine nucleotides transduce cell membrane receptor responses and modulate ion channel activity. This is accomplished through conformational change in the structure of nucleotides and cell membrane associated proteins. The aim of this study is to enhance our understanding of nucleotide dependence in regard to signal transduction events, drug action and pharmacological promiscuity. Nucleotides and ligand structures regulating Gα protein subunits, voltage- and ligand-gated ion channels are investigated for molecular similarity using a computational program. Results differentiate agonist and antagonist structures, identify molecular similarity within nucleotide and ligand structures and demonstrate the potential of ligands to regulate nucleotide conformational change. Relative molecular similarity within nucleotides and the ligands of the major receptor classes provides insight into mechanisms of receptor and ion channel regulation. The nucleotide template model has some merit as an initial screening tool in the study and comparison of drug and hormone structures. 相似文献
9.
Tseng TT McMahon AM Johnson VT Mangubat EZ Zahm RJ Pacold ME Jakobsson E 《Journal of molecular microbiology and biotechnology》2007,12(3-4):249-262
Voltage-gated ion channels are well known for their functional roles in excitable tissues. Excitable tissues rely on voltage-gated ion channels and their auxiliary subunits to achieve concerted electrical activity in living cells. Auxiliary subunits are also known to provide functional diversity towards the transport and biogenesis properties of the principal subunits. Recent interests in pharmacological properties of these auxiliary subunits have prompted significant amounts of efforts in understanding their physiological roles. Some auxiliary subunits can potentially serve as drug targets for novel analgesics. Three families of sodium channel auxiliary subunits are described here: beta1 and beta3, beta2 and beta4, and temperature-induced paralytic E (TipE). While sodium channel beta-subunits are encoded in many animal genomes, TipE has only been found exclusively in insects. In this review, we present phylogenetic analyses, discuss potential evolutionary origins and functional data available for each of these subunits. For each family, we also correlate the functional specificity with the history of evolution for the individual auxiliary subunits. 相似文献
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Taanman JW 《Biochimica et biophysica acta》1999,1410(2):103-123
12.
Since the first Hodgkin and Huxley ion channel model was described in the 1950s, there has been an explosion in mathematical models to describe ion channel function. As experimental data has become richer, models have concomitantly been improved to better represent ion channel kinetic processes, although these improvements have generally resulted in more model complexity and an increase in the number of parameters necessary to populate the models. Models have also been developed to explicitly model drug interactions with ion channels. Recent models of drug-channel interactions account for the discrete kinetics of drug interaction with distinct ion channel state conformations, as it has become clear that such interactions underlie complex emergent kinetics such as use-dependent block. Here, we describe an approach for developing a model for ion channel drug interactions. The method describes the process of extracting rate constants from experimental electrophysiological function data to use as initial conditions for the model parameters. We then describe implementation of a parameter optimization method to refine the model rate constants describing ion channel drug kinetics. The algorithm takes advantage of readily available parallel computing tools to speed up the optimization. Finally, we describe some potential applications of the platform including the potential for gaining fundamental mechanistic insights into ion channel function and applications to in silico drug screening and development. 相似文献
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Channelopathies of inwardly rectifying potassium channels. 总被引:6,自引:0,他引:6
Mutations in genes encoding ion channels have increasingly been identified to cause disease conditions collectively termed channelopathies. Recognizing the molecular basis of an ion channel disease has provided new opportunities for screening, early diagnosis, and therapy of such conditions. This synopsis provides an overview of progress in the identification of molecular defects in inwardly rectifying potassium (Kir) channels. Structurally and functionally distinct from other channel families, Kir channels are ubiquitously expressed and serve functions as diverse as regulation of resting membrane potential, maintenance of K(+) homeostasis, control of heart rate, and hormone secretion. In humans, persistent hyperinsulinemic hypoglycemia of infancy, a disorder affecting the function of pancreatic beta cells, and Bartter's syndrome, characterized by hypokalemic alkalosis, hypercalciuria, increased serum aldosterone, and plasma renin activity, are the two major diseases linked so far to mutations in a Kir channel or associated protein. In addition, the weaver phenotype, a neurological disorder in mice, has also been associated with mutations in a Kir channel subtype. Further genetic linkage analysis and full understanding of the consequence that a defect in a Kir channel would have on disease pathogenesis are among the priorities in this emerging field of molecular medicine. 相似文献
15.
Stewart AP Haerteis S Diakov A Korbmacher C Edwardson JM 《The Journal of biological chemistry》2011,286(37):31944-31952
The epithelial sodium channel (ENaC) is a member of the ENaC/degenerin superfamily. ENaC is a heteromultimer containing three homologous subunits (α, β, and γ); however, the subunit stoichiometry is still controversial. Here, we addressed this issue using atomic force microscopy imaging of complexes between isolated ENaC and antibodies/Fab fragments directed against specific epitope tags on the α-, β- and γ-subunits. We show that for α-, β- and γ-ENaC alone, pairs of antibodies decorate the channel at an angle of 120°, indicating that the individual subunits assemble as homotrimers. A similar approach demonstrates that αβγ-ENaC assembles as a heterotrimer containing one copy of each subunit. Intriguingly, all four subunit combinations also produce higher-order structures containing two or three individual trimers. The trimer-of-trimers organization would account for earlier reports that ENaC contains eight to nine subunits. 相似文献
16.
Hool LC 《European biophysics journal : EBJ》2005,34(5):369-376
The first ion channels demonstrated to be sensitive to changes in oxygen tension were K+ channels in glomus cells of the carotid body. Since then a number of hypoxia-sensitive ion channels have been identified. However, not all K+ channels respond to hypoxia alike. This has raised some debate about how cells detect changes in oxygen tension. Because ion channels respond rapidly to hypoxia it has been proposed that the channel is itself an oxygen sensor. However, channel function can also be modified by thiol reducing and oxidizing agents, implicating reactive oxygen species as signals in hypoxic events. Cardiac ion channels can also be modified by hypoxia and redox agents. The rapid and slow components of the delayed rectifier K+ channel are differentially regulated by hypoxia and -adrenergic receptor stimulation. Mutations in the genes that encode the subunits for the channel are associated with Long QT syndrome and sudden cardiac death. The implications with respect to effects of hypoxia on the channel and triggering of cardiac arrhythmia will be discussed. 相似文献
17.
In this study, several methodological aspects of the pepscan strategy have been investigated with the objective to delineate the amino acid sequences of peptide segments that form the epitopes of thyrotropin beta-subunit (TSHbeta) recognised by monoclonal antibodies. Hitherto, the pepscan strategy has found application as an effective method to identify linear sequence regions that constitute contiguous epitopes within the primary structure of some proteins. However, with heterodimeric glycoprotein hormones and their subunits such as TSHbeta, as well as for many other globular proteins, the majority of the epitopes recognised by anti-protein antibodies will be derived from discontinuous segments that collectively form the epitope. In these cases the pepscan technique will only be able to identify individual segments of the overall discontinuous epitope site as linear peptides, some of which may interact with relatively low binding affinity. Consequently, additional attention must thus be given to the optimisation of the specific binding and detection conditions. Knowledge of the structures of these peptide segments can, however, provide a valuable basis to develop peptide structures that more closely mimic the topographical features of the epitope in the mature, folded protein. In an attempt to identify functional segments involved in the epitopes recognised by the anti-hTSH monoclonal antibodies, mAb279 and mAb299, the impact of various experimental conditions on the efficacy of the pepscan strategy has been investigated. The strategy involved the synthesis of a series of overlapping pin-bound octapeptides with amino acid sequences derived from the TSH beta-subunit. The ability of these pin-bound octapeptides to bind to either mAb279 or mAb299 in ELISA-based assay was then determined under conditions involving different concentrations of the primary and/or secondary antibodies, and changes in buffer composition, incubation times and washing procedures. Theresults of this study illustrate some of the constraints and limitations of the pepscan technique when used to delineate discontinuous epitopes of globular proteins, as well as providing insight into potential avenues to optimise and refine this method. 相似文献
18.
S-peptide epitope tagging for protein purification, expression monitoring, and localization in mammalian cells 总被引:1,自引:0,他引:1
Epitope tags are widely used in cell biology and biochemistry research. The S-peptide/S-protein interaction has previously been utilized to purify polypeptides expressed in bacteria. We have now re-engineered the S-peptide/S-protein system to allow isolation of S-peptide-tagged polypeptides and their binding partners from eukaryotic cells with S-protein-agarose. In addition, two anti-S-peptide monoclonal antibodies have been generated for analysis of expression and subcellular localization of S-peptide-tagged polypeptides. These reagents make the S-peptide/S-protein system an attractive alternative to currently available epitope tagging methods. 相似文献
19.
The epithelial Na(+) channel (ENaC) and acid-sensitive ion channel (ASIC) branches of the ENaC/degenerin superfamily of cation channels have drawn increasing attention as potential therapeutic targets in a variety of diseases and conditions. Originally thought to be solely expressed in fluid absorptive epithelia and in neurons, it has become apparent that members of this family exhibit nearly ubiquitous expression. Therapeutic opportunities range from hypertension, due to the role of ENaC in maintaining whole body salt and water homeostasis, to anxiety disorders and pain associated with ASIC activity. As a physiologist intrigued by the fundamental mechanics of salt and water transport, it was natural that Dale Benos, to whom this series of reviews is dedicated, should have been at the forefront of research into the amiloride-sensitive sodium channel. The cloning of ENaC and subsequently the ASIC channels has revealed a far wider role for this channel family than was previously imagined. In this review, we will discuss the known and potential roles of ENaC and ASIC subunits in the wide variety of pathologies in which these channels have been implicated. Some of these, such as the role of ENaC in Liddle's syndrome are well established, others less so; however, all are related in that the fundamental defect is due to inappropriate channel activity. 相似文献
20.
Wilson Edwards Wai-Ping Fung-Leung Chichi Huang Ellen Chi Nancy Wu Yi Liu Michael P. Maher Rachelle Bonesteel Judith Connor Ross Fellows Elena Garcia Jerry Lee Lu Lu Karen Ngo Brian Scott Hong Zhou Ronald V. Swanson Alan D. Wickenden 《The Journal of biological chemistry》2014,289(33):22704-22714
Ion channels are an attractive class of drug targets, but progress in developing inhibitors for therapeutic use has been limited largely due to challenges in identifying subtype selective small molecules. Animal venoms provide an alternative source of ion channel modulators, and the venoms of several species, such as scorpions, spiders and snails, are known to be rich sources of ion channel modulating peptides. Importantly, these peptides often bind to hyper-variable extracellular loops, creating the potential for subtype selectivity rarely achieved with small molecules. We have engineered scorpion venom peptides and incorporated them in fusion proteins to generate highly potent and selective Kv1.3 inhibitors with long in vivo half-lives. Kv1.3 has been reported to play a role in human T cell activation, and therefore, these Kv1.3 inhibitor fusion proteins may have potential for the treatment of autoimmune diseases. Our results support an emerging approach to generating subtype selective therapeutic ion channel inhibitors. 相似文献