共查询到20条相似文献,搜索用时 46 毫秒
1.
Gaurav Bajaj Andrew M. Hau Peter Hsu Philip R. Gafken Michael I. Schimerlik Jane E. Ishmael 《Biochemical and biophysical research communications》2014
N-methyl-d-aspartate (NMDA) receptors are calcium-permeable ion channels assembled from four subunits that each have a common membrane topology. The intracellular carboxyl terminal domain (CTD) of each subunit varies in length, is least conserved between subunits, and binds multiple intracellular proteins. We defined a region of interest in the GluN2A CTD, downstream of well-characterized membrane-proximal motifs, that shares only 29% sequence similarity with the equivalent region of GluN2B. GluN2A (amino acids 875–1029) was fused to GST and used as a bait to identify proteins from mouse brain with the potential to bind GluN2A as a function of calcium. Using mass spectrometry we identified calmodulin as a calcium-dependent GluN2A binding partner. Equilibrium fluorescence spectroscopy experiments indicate that Ca2+/calmodulin binds GluN2A with high affinity (5.2 ± 2.4 nM) in vitro. Direct interaction of Ca2+/calmodulin with GluN2A was not affected by disruption of classic sequence motifs associated with Ca2+/calmodulin target recognition, but was critically dependent upon Trp-1014. These findings provide new insight into the potential of Ca2+/calmodulin, previously considered a GluN1-binding partner, to influence NMDA receptors by direct association. 相似文献
2.
Commercial solvents such as toluene are commonly used as drugs of abuse by children and adolescents. The cellular and molecular sites and mechanisms of actions of these compounds are not well studied but their effects on behavior resemble those of central nervous system depressants such as alcohol, barbiturates and benzodiazepines. In this study, the effects of toluene on voltage-sensitive calcium channels (VSCCs) were measured in pheochromocytoma cells. The KCl-induced rise in intracellular calcium as measured by calcium imaging was almost completely blocked by the dihydropyridine calcium channel antagonist nifedipine verifying that undifferentiated pheochromocytoma cells express mainly the L-type of calcium channel. Toluene (0.3–3000 μM) by itself did not affect intracellular calcium levels in resting cells but dose-dependently inhibited the KCl-induced rise in calcium. This inhibition was substantially reversed upon washout of the toluene-containing solution. KCl-dependent increases in intracellular calcium in cells differentiated with nerve growth factor (NGF) were largely insensitive to nifedipine. Toluene produced a greater inhibition of the KCl response in NGF treated cells as compared with undifferentiated cells. A similar finding was obtained when whole-cell patch-clamp-electrophysiology was used to directly monitor the effects of toluene on voltage-activated calcium currents in undifferentiated and differentiated cells. These results show that dihydropyridine sensitive and insensitive calcium channels are inhibited by toluene and may represent important sites of action for this compound. 相似文献
3.
大电导钙激活钾通道(BKCa)及其开放剂研究进展 总被引:2,自引:0,他引:2
大电导钙激活钾通道(BKCa)广泛分布在哺乳动物各种组织(不含心肌细胞)中,并参与细胞内信号转导、细胞的兴奋及代谢调节等生理过程。BKCa功能异常牵涉到特发性癫痫、高血压等疾病的发生。BKCa通道是治疗高血压、尿失禁、哮喘、冠心病及缺血性脑中风等疾病的潜在药物靶点。探索高活性、高选择性、细胞通透性优良、类药性好的BKCa通道开放剂,不仅有助于阐明BKCa通道在生理病理条件下的作用机制,而且为治疗心脑血管疾病的药物研发奠定基础。对各类BKCa通道开放剂做一概述。 相似文献
4.
《Cell calcium》2019
Cancer stem cells (CSC’s) have emerged as a key area of investigation due to associations with cancer development and treatment resistance, related to their ability to remain quiescent, self-renew and terminally differentiate. Targeting CSC’s in addition to the tumour bulk could ensure complete removal of the cancer, lessening the risk of relapse and improving patient survival. Understanding the mechanisms supporting the functions of CSC’s is essential to highlight targets for the development of therapeutic strategies. Changes in intracellular calcium through calcium channel activity is fundamental for integral cellular processes such as proliferation, migration, differentiation and survival in a range of cell types, under both normal and pathological conditions. Here in we highlight how calcium channels represent a key mechanism involved in CSC function. It is clear that expression and or function of a number of channels involved in calcium entry and intracellular store release are altered in CSC’s. Correlating with aberrant proliferation, self-renewal and differentiation, which in turn promoted cancer progression and treatment resistance. Research outlined has demonstrated that targeting altered calcium channels in CSC populations can reduce their stem properties and induce terminal differentiation, sensitising them to existing cancer treatments. Overall this highlights calcium channels as emerging novel targets for CSC therapies. 相似文献
5.
Andrew P. Stewart 《Biochemical and biophysical research communications》2010,394(2):383-2025
Several members of the transient receptor potential (TRP) channel superfamily have been shown to assemble as tetramers. Here we have determined the subunit stoichiometry of the transient receptor potential M8 (TRPM8) channel using atomic force microscopy (AFM). TRPM8 channels were isolated from transfected cells, and complexes were formed between the channels and antibodies against a V5 epitope tag present on each subunit. The complexes were then subjected to AFM imaging. A frequency distribution of the molecular volumes of antibody decorated channels had a peak at 1305 nm3, close to the expected size of a TRPM8 tetramer. The frequency distribution of angles between pairs of bound antibodies had two peaks, at 93° and 172°, confirming that the channel assembles as a tetramer. We suggest that this assembly pattern is common to all members of the TRP channel superfamily. 相似文献
6.
7.
Rita E. Sirrieh David M. MacLean Vasanthi Jayaraman 《The Journal of biological chemistry》2015,290(20):12812-12820
The N-methyl-d-aspartate (NMDA) subtype of the ionotropic glutamate receptors is the primary mediator of calcium-permeable excitatory neurotransmission in the central nervous system. Subunit composition and binding of allosteric modulators to the amino-terminal domain determine the open probability of the channel. By using luminescence resonance energy transfer with functional receptors expressed in CHO cells, we show that the cleft of the amino-terminal domain of the GluN2B subunit, which has a lower channel open probability, is on average more closed than the GluN2A subunit, which has a higher open probability. Furthermore, the GluN1 amino-terminal domain adopts a more open conformation when coassembled with GluN2A than with GluN2B. Binding of spermine, an allosteric potentiator, opens the amino-terminal domain cleft of both the GluN2B subunit and the adjacent GluN1 subunit. These studies provide direct structural evidence that the inherent conformations of the amino-terminal domains vary based on the subunit and match the reported open probabilities for the receptor. 相似文献
8.
Imaging single-channel calcium microdomains 总被引:3,自引:0,他引:3
The Ca(2+) microdomains generated around the mouth of open ion channels represent the basic building blocks from which cytosolic Ca(2+) signals are constructed. Recent improvements in optical imaging techniques now allow these microdomains to be visualized as single channel calcium fluorescence transients (SCCaFTs), providing information about channel properties that was previously accessible only by electrophysiological patch-clamp recordings. We review recent advances in single channel Ca(2+) imaging methodologies, with emphasis on total internal reflection fluorescence microscopy (TIRFM) as the technique of choice for recording SCCaFTs from voltage- and ligand-gated plasmalemmal ion channels. This technique of 'optical patch-clamp recording' is massively parallel, permitting simultaneous imaging of hundreds of channels; provides millisecond resolution of gating kinetics together with sub-micron spatial resolution of channel locations; and is applicable to diverse families of membrane channels that display partial permeability to Ca(2+) ions. 相似文献
9.
Imaging calcium entering the cytosol through a single opening of plasma membrane ion channels: SCCaFTs—fundamental calcium events 总被引:2,自引:2,他引:0
Hui Zou Lawrence M. Lifshitz Richard A. Tuft Kevin E. Fogarty Joshua J. Singer 《Cell calcium》2004,35(6):523
Recently, it has become possible to record the localized fluorescence transient associated with the opening of a single plasma membrane Ca2+ permeable ion channel using Ca2+ indicators like fluo-3. These Single Channel Ca2+ Fluorescence Transients (SCCaFTs) share some of the characteristics of such elementary events as Ca2+ sparks and Ca2+ puffs caused by Ca2+ release from intracellular stores (due to the opening of ryanodine receptors and IP3 receptors, respectively). In contrast to intracellular Ca2+ release events, SCCaFTs can be observed while simultaneously recording the unitary channel currents using patch-clamp techniques to verify the channel openings. Imaging SCCaFTs provides a way to examine localized Ca2+ handling in the vicinity of a channel with a known Ca2+ influx, to obtain the Ca2+ current passing through plasma membrane cation channels in near physiological solutions, to localize Ca2+ permeable ion channels on the plasma membrane, and to estimate the Ca2+ currents underlying those elementary events where the Ca2+ currents cannot be recorded. Here we review studies of these fluorescence transients associated with caffeine-activated channels, L-type Ca2+ channels, and stretch-activated channels. For the L-type Ca2+ channel, SCCaFTs have been termed sparklets. In addition, we discuss how SCCaFTs have been used to estimate Ca2+ currents using the rate of rise of the fluorescence transient as well as the signal mass associated with the total fluorescence increase. 相似文献
10.
Brevetoxin-3 (PbTx-3), described to increase the open probability of voltage-dependent sodium channels, caused trains of action potentials and fast oscillatory changes in fluorescence intensity of fluo-3-loaded rat skeletal muscle cells in primary culture, indicating that the toxin increased intracellular Ca(2+) levels. PbTx-3 did not elicit calcium transients in dysgenic myotubes (GLT cell line), lacking the alpha1 subunit of the dihydropyridine receptor (DHPR), but after transfection of the alpha1DHPR cDNA to GLT cells, PbTx-3 induced slow calcium transients that were similar to those of normal cells. Ca(2+) signals evoked by PbTx-3 were inhibited by blocking either IP(3) receptors, with 2-aminoethoxydiphenyl borate, or phospholipase C with U73122. PbTx-3 caused a tetrodotoxin-sensitive increase in intracellular IP(3) mass levels, dependent on extra-cellular Na(+). A similar increase in IP(3) mass was induced by high K(+) depolarization but no action potential trains (nor calcium signals) were elicited by prolonged depolarization under current clamp conditions. The increase in IP(3) mass induced by either PbTx-3 or K(+) was also detected in Ca(2+)-free medium. These results establish that the effect of the toxin on both intracellular Ca(2+) and IP(3) levels occurs via a membrane potential sensor instead of directly by Na(+) flux and supports the notion of a train of action potentials being more efficient as a stimulus than sustained depolarization, suggesting that tetanus is the physiological stimulus for the IP(3)-dependent calcium signal involved in regulation of gene expression. 相似文献
11.
NMDA receptors are ligand-gated ion channels with a regulatory intracellular C-terminal domain (CTD). In GluN2B, the CTD is the largest domain in the protein but is intrinsically disordered. The GluN2B subunit is the major tyrosine-phosphorylated protein in synapses. Src kinase phosphorylates the GluN2B CTD, but it is unknown how this affects channel activity. In disordered proteins, phosphorylation can tip the balance between order and disorder. Transitions can occur in both directions, so it is not currently possible to predict the effects of phosphorylation. We used single molecule fluorescence to characterize the effects of Src phosphorylation on GluN2B. Scanning fluorescent labeling sites throughout the domain showed no positional dependence of the energy transfer. Instead, efficiency only scaled with the separation between labeling sites suggestive of a relatively featureless conformational energy landscape. Src phosphorylation led to a general expansion of the polypeptide, which would result in greater exposure of known protein-binding sites and increase the physical separation between contiguous sites. Phosphorylation makes the CTD more like a random coil leaving open the question of how Src exerts its effects on the NMDA receptor. 相似文献
12.
钾离子通道是分布最为广泛、种类繁多的一类离子通道,因其生理功能的多样性,已成为许多疾病的药物作用靶点。近年来,许 多化学结构不同的药物均因钾离子通道阻滞引起的严重心肌毒性而被撤出市场,使得小分子药物的钾通道抑制活性筛选面临重大挑战。 介绍检测钾离子通道的小分子荧光探针的研究进展,并总结小分子荧光探针的作用机制,为今后小分子荧光探针的设计提供思路,使得 小分子荧光探针可以广泛应用于候选药物的高通量筛选、钾离子通道的活体成像与检测。 相似文献
13.
Toshio Ohta Takashi Suzuki Hironobu Sasano 《Biochemical and biophysical research communications》2010,394(3):464-469
The β subunits of voltage-dependent calcium channels are known to modify calcium channel currents through pore-forming α1 subunits. The β3 subunit is expressed in the adrenal gland and participates in forming various calcium channel types. We performed a series of experiments in β3-null mice to determine the role of the β3 subunit in catecholamine release from the adrenal chromaffin system.Protein levels of N-type channel forming CaV2.2 and L-type forming CaV1.2 decreased. The β3-null mice showed a decreased baroreflex, suggesting decreased sympathetic tonus, whereas plasma catecholamine levels did not change. Pulse-voltage stimulation revealed significantly increased amperometrical currents in β3-null mice, while patch-clamp recordings showed a significant reduction in Ca2+-currents due to reduced L- and N-type currents, indicating facilitated exocytosis. A biochemical analysis revealed increased InsP3 production.In conclusion, our results indicate the importance of the β3 subunit in determining calcium channel characteristics and catecholamine release in adrenal chromaffin cells. 相似文献
14.
Voltage-gated calcium channel subunits from platyhelminths: potential role in praziquantel action 总被引:3,自引:0,他引:3
Voltage-gated calcium (Ca2+) channels provide the pathway for Ca2+ influxes that underlie Ca2+ -dependent responses in muscles, nerves and other excitable cells. They are also targets of a wide variety of drugs and toxins. Ca2+ channels are multisubunit protein complexes consisting of a pore-forming alpha(1) subunit and other modulatory subunits, including the beta subunit. Here, we review the structure and function of schistosome Ca2+ channel subunits, with particular emphasis on variant Ca2+ channel beta subunits (Ca(v)betavar) found in these parasites. In particular, we examine the role these beta subunits may play in the action of praziquantel, the current drug of choice against schistosomiasis. We also present evidence that Ca(v)betavar homologs are found in other praziquantel-sensitive platyhelminths such as the pork tapeworm, Taenia solium, and that these variant beta subunits may thus represent a platyhelminth-specific gene family. 相似文献
15.
S Wijetunge A C Dolphin A D Hughes 《Biochemical and biophysical research communications》2002,290(4):1246-1249
Voltage-operated calcium channels are modulated by tyrosine kinases in different cell types. In this study, I(Ba) was measured by the whole cell voltage-clamp technique in single COS-7 cells overexpressing the Ca(v)2.2 calcium channels encoding N-type currents. Bath application of genistein, a nonselective PTK inhibitor (50-300 microM), concentration-dependently inhibited calcium channel currents, whereas the inactive structural analogue, daidzein, was without effect over the same concentration range. Similarly, PP1, a src family-selective tyrosine kinase inhibitor, inhibited I(Ba) in a concentration-dependent manner (500 nM-5 microM) over a range of test potentials. Expression of the Ca(v)2.2alpha1 (alpha(1B)) subunit alone gave rise to functional channels, and genistein (100 microM) also inhibited currents elicited by the alpha(1B) subunit alone. These results indicate that tyrosine kinase inhibitors are likely to inhibit Ca(v)2.2 calcium channels via an action on the pore-forming alpha(1) subunit and suggest that an endogenous member of the Src family may play a physiological role in modulating these channels. 相似文献
16.
NMDA receptors (NMDARs) form glutamate-gated ion channels that have central roles in neuronal communication and plasticity throughout the brain. Dysfunctions of NMDARs are involved in several central nervous system disorders, including stroke, chronic pain and schizophrenia. One hallmark of NMDARs is that their activity can be allosterically regulated by a variety of extracellular small ligands. While much has been learned recently regarding allosteric inhibition of NMDARs, the structural determinants underlying positive allosteric modulation of these receptors remain poorly defined. Here, we show that polyamines, naturally occurring polycations that selectively enhance NMDARs containing the GluN2B subunit, bind at a dimer interface between GluN1 and GluN2B subunit N-terminal domains (NTDs). Polyamines act by shielding negative charges present on GluN1 and GluN2B NTD lower lobes, allowing their close apposition, an effect that in turn prevents NTD clamshell closure. Our work reveals the mechanistic basis for positive allosteric modulation of NMDARs. It provides the first example of an intersubunit binding site in this class of receptors, a discovery that holds promise for future drug interventions. 相似文献
17.
Fukumori R Takarada T Kambe Y Nakazato R Fujikawa K Yoneda Y 《Neurochemistry international》2012,61(4):498-505
We have previously shown the possible involvement of mitochondrial membrane potential disruption in the mechanisms underlying the neurotoxicity seen after activation of N-methyl-d-aspartate (NMDA) receptors (NMDAR) in primary cultured rat hippocampal neurons. In this study, we attempted to demonstrate a pivotal role of mitochondrial uncoupling protein-2 (UCP2) as a determinant of the NMDA neurotoxicity by using acquired NMDAR channels artificially orchestrated in HEK293 cells. In cells with overexpression of UCP2, immunoreactive UCP2 was exclusively detected at intracellular locations stained with the mitochondrial marker MitoTracker. In cells with acquired NMDAR channels, exposure to either NMDA or the calcium ionophore A23187 similarly led to a significant increase in cytosolic Ca(2+) levels determined by Fluo-3 imaging irrespective of the overexpression of UCP2. By contrast, NMDA, but not A23187, was significantly more effective in increasing mitochondrial Ca(2+) levels determined by Rhod-2 fluorescence imaging in cells transfected with NMDAR subunit and UCP2 expression vectors than in those without UCP2 overexpression. Overexpression of UCP2 significantly increased the number of cells stained with propidium iodide in cultures with acquired NMDAR channels, but failed to significantly affect that in cells exposed to A23187. Immunocytochemical and immunoprecipitation analyses similarly revealed the possible interaction between GluN1 subunit and UCP2 in HEK293 cells with acquired NMDAR channels and UCP2 overexpression. These results suggest that UCP2 could play a role as a determinant of the neurotoxicity mediated by NMDAR through a mechanism related to the unidentified interaction with the essential GluN1 subunit toward modulation of mitochondrial Ca(2+) levels in neurons. 相似文献
18.
Imaging Ca(2+) entering the cytoplasm through a single opening of a plasma membrane cation channel. 总被引:1,自引:0,他引:1
H Zou L M Lifshitz R A Tuft K E Fogarty J J Singer 《The Journal of general physiology》1999,114(4):575-588
Discrete localized fluorescence transients due to openings of a single plasma membrane Ca(2+) permeable cation channel were recorded using wide-field digital imaging microscopy with fluo-3 as the Ca(2+) indicator. These transients were obtained while simultaneously recording the unitary channel currents using the whole-cell current-recording configuration of the patch-clamp technique. This cation channel in smooth muscle cells is opened by caffeine (Guerrero, A., F.S. Fay, and J.J. Singer. 1994. J. Gen. Physiol. 104:375-394). The localized fluorescence transients appeared to occur at random locations on the cell membrane, with the duration of the rising phase matching the duration of the channel opening. Moreover, these transients were only observed in the presence of sufficient extracellular Ca(2+), suggesting that they are due to Ca(2+) influx from the bathing solution. The fluorescence transient is characterized by an initial fast rising phase when the channel opens, followed by a slower rising phase during prolonged openings. When the channel closes there is an immediate fast falling phase followed by a slower falling phase. Computer simulations of the underlying events were used to interpret the time course of the transients. The rapid phases are mainly due to the establishment or removal of Ca(2+) and Ca(2+)-bound fluo-3 gradients near the channel when the channel opens or closes, while the slow phases are due to the diffusion of Ca(2+) and Ca(2+)-bound fluo-3 into the cytoplasm. Transients due to short channel openings have a "Ca(2+) spark-like" appearance, suggesting that the rising and early falling components of sparks (due to openings of ryanodine receptors) reflect the fast phases of the fluorescence change. The results presented here suggest methods to determine the relationship between the fluorescence transient and the underlying Ca(2+) current, to study intracellular localized Ca(2+) handling as might occur from single Ca(2+) channel openings, and to localize Ca(2+) permeable ion channels on the plasma membrane. 相似文献
19.
N-Methyl-D-aspartate (NMDA) receptors (NRs) are glutamate-gated channels critical for the functioning of the nervous system. They are assembled from two types of subunits, the essential GluN1 and at least one type of GluN2 (A, B, C, D) subunit. Nickel (Ni) modulates the NR current in a way dependent on the GluN2 subunit present. Besides voltage-dependent and voltage-independent inhibition, in GluN2B-containing channels Ni enhances channel activity. We have recently identified several domains of the channel involved in Ni interaction, but many aspects of this modulation remain elusive. The purpose of the present work is to investigate the role of calcium (Ca) in the effect of Ni on the NR current measured by voltage- and patch-clamp in RNA-injected Xenopus laevis oocytes or in transiently transfected mammalian HEK293 cells expressing GluN1/GluN2B recombinant receptors. In both expression systems, in the presence of a physiological concentration of Ca (1.8 mM), Ni increased the NR current with EC(50) in the μM range, but this potentiation was reduced by decreasing Ca concentration or when Ca was substituted with Ba. In injected oocytes, the effect of Ni in 0.3 mM external Ba was only inhibitory (IC(50) = 65 μM). Increasing the internal calcium buffering by EGTA and BAPTA application, as well as incubation with cytoskeleton perturbing agents, colchicine and cytochalasin-D, did not produce major modifications in the Ni effect. These observations indicate that Ni-mediated potentiation is not dependent on Ca influx and internal Ca concentration, but it is dependent on external Ca, which possibly interacts with the extracellular portion of the protein through a modulatory binding site. 相似文献
20.
Kobayashi T Yamada Y Fukao M Shiratori K Tsutsuura M Tanimoto K Tohse N 《Biochemical and biophysical research communications》2007,360(3):679-683
The β subunits of voltage-dependent calcium channels bind the pore-forming α1 subunit and play an important role in the regulation of calcium channel function. Recently, we have identified a new splice variant of the β4 subunit, which we have termed the β4d subunit. The β4d subunit is a truncated splice variant of the β4b subunit and lacks parts of the guanylate kinase (GK) domain and the C-terminus. The calcium current in BHK cells expressing α1C and α2δ with the β4d subunit was as small as that without the β4d subunit. Western blot analysis revealed that β4d protein was expressed to a lesser extent that the β4b protein. In addition, a GST pull down assay showed that the β4d subunit could not interact with the α1 subunit of the calcium channel. Collectively, our results suggest that the GK domain of the β subunit is essential for the expression of the functional calcium channel. 相似文献