Ca2+ entry through plasma membrane IP3 receptors

Inositol 1,4,5-trisphosphate receptors (IP3Rs) release calcium ions, Ca2+, from intracellular stores, but their roles in mediating Ca2+ entry are unclear. IP3 stimulated opening of very few (1.9 +/- 0.2 per cell) Ca2+-permeable channels in whole-cell patch-clamp recording of DT40 chicken or mouse B cells. Activation of the B cell receptor (BCR) in perforated-patch recordings evoked the same response. IP3 failed to stimulate intracellular or plasma membrane (PM) channels in cells lacking IP3R. Expression of IP3R restored both responses. Mutations within the pore affected the conductances of IP3-activated PM and intracellular channels similarly. An impermeant pore mutant abolished BCR-evoked Ca2+ signals, and PM IP3Rs were undetectable. After introduction of an alpha-bungarotoxin binding site near the pore, PM IP3Rs were modulated by extracellular alpha-bungarotoxin. IP(3)Rs are unusual among endoplasmic reticulum proteins in being also functionally expressed at the PM, where very few IP3Rs contribute substantially to the Ca2+ entry evoked by the BCR.     

Highly cooperative opening of calcium channels by inositol 1,4,5-trisphosphate

The kinetics of calcium release by inositol 1,4,5-trisphosphate (IP3) in permeabilized rat basophilic leukemia cells were studied to obtain insight into the molecular mechanism of action of this intracellular messenger of the phosphoinositide cascade. Calcium release from intracellular storage sites was monitored with fura-2, a fluorescent indicator. The dependence of the rate of calcium release on the concentration of added IP3 in the 4 to 40 nM range showed that channel opening requires the binding of at least three molecules of IP3. Channel opening occurred in the absence of added adenosine triphosphate, indicating that IP3 acts directly on the channel or on a protein that gates it. The channels were opened by IP3 in less than 4 seconds. The highly cooperative opening of calcium channels by nanomolar concentrations of IP3 enables cells to detect and amplify very small changes in the concentration of this messenger in response to hormonal, sensory, and growth control stimuli.     

Calcium as a coagonist of inositol 1,4,5-trisphosphate-induced calcium release

Inositol 1,4,5-trisphosphate (IP3)-induced calcium release from intracellular stores is a regulator of cytosolic-free calcium levels. The subsecond kinetics and regulation of IP3-induced calcium-45 release from synaptosome-derived microsomal vesicles were resolved by rapid superfusion. Extravesicular calcium acted as a coagonist, potentiating the transient IP3-induced release of calcium-45. Thus, rapid elevation of cytosolic calcium levels may trigger IP3-induced calcium release in vivo. Extravesicular calcium also produced a more slowly developing, reversible inhibition of IP3-induced calcium-45 release. Sequential positive and negative feedback regulation by calcium of IP3-induced calcium release may contribute to transients and oscillations of cytosolic-free calcium in vivo.     

IP_3敏感的钙离子通透性通道参与茉莉酸诱导的钙动员

[目的]研究茉莉酸诱导的钙动员通往IP3敏感的钙离子通透性通道的作用。[方法]以低温导入法将钙离子荧光探针Fluo-3/AM导入拟南芥叶片细胞中,利用LASAF（Leica Application Suite-Advanced Fluorescence）软件记录肝素对茉莉酸（JA）诱导的胞内钙离子荧光强度的变化。[结果]经不同浓度的肝素预处理后,拟南芥叶细胞中胞内钙离子的荧光强度降低,再用100μmol/LJA处理时,其荧光强度升高,但仅与未经肝素处理的荧光强度相当。[结论]肝素预处理可抑制JA诱导的胞内钙离子浓度的升高。     

PML regulates apoptosis at endoplasmic reticulum by modulating calcium release

The promyelocytic leukemia (PML) tumor suppressor is a pleiotropic modulator of apoptosis. However, the molecular basis for such a diverse proapoptotic role is currently unknown. We show that extranuclear Pml was specifically enriched at the endoplasmic reticulum (ER) and at the mitochondria-associated membranes, signaling domains involved in ER-to-mitochondria calcium ion (Ca(2+)) transport and in induction of apoptosis. We found Pml in complexes of large molecular size with the inositol 1,4,5-trisphosphate receptor (IP(3)R), protein kinase Akt, and protein phosphatase 2a (PP2a). Pml was essential for Akt- and PP2a-dependent modulation of IP(3)R phosphorylation and in turn for IP(3)R-mediated Ca(2+) release from ER. Our findings provide a mechanistic explanation for the pleiotropic role of Pml in apoptosis and identify a pharmacological target for the modulation of Ca(2+) signals.     

不同钙效应剂对长春花光合特性的影响

研究了细胞质膜钙通道阻断剂氯化镧(LaCl3)、胞内IP3通道阻断剂肝素(Heparin)、胞内CaM活性抑制剂三氟啦嗪(TFP)对长春花光合作用的影响,结果发现:Ca2+通道阻断剂处理后长春花叶片净光合速率(Pn)、PSII最大量子产量(Fv/Fm)、PSⅡ实际量子产量(Yield)、电子传递速率(ETR)、光化学淬灭系数(qP)均下降,非光化学淬灭系数(qN)及Chla、Chlb、Chla+Chlb含量上升,表明Ca2+通道阻断剂对长春花的光合作用有较大影响,且不是通过加速叶片光合色素降解或抑制其合成来实现抑制叶片的光合能力;其中Heparin处理的长春花叶片相关参数变化幅度最大,表明胞内钙库通过IP3通道释放的Ca2+在长春花叶片光合作用过程中发挥了更积极的作用.     

肌醇脂质和cAMP在血小板活化因子诱导血小板聚集中的作用

目的：探讨肌醇脂质和cAMP两个细胞内第二信使系统在血小板活化因子(PAF)诱导血小板聚集过程中所起到的作用和相互关系。方法：采用PKC激动剂PMA和Ca^2 通道A23187,以及PKA激动剂Sp-cAMPS和抑制剂Rp-cAMPS干预的方法,分析观察这两个信使系统在PAF诱导血小板聚集过程中的作用;采用^3H—Inositol和^14C-Adenine双标记液体闪烁技术测定PAF诱导血小板可逆聚集过程中肌醇-1,4,5-三磷酸酯(IP3)和cAMP的水平变化的方法,分析研究这两个信使系统在PAF诱导血小板聚集过程中的相互关系。结果：(1)PMA和A23187能分别增强PAF的血小板聚集效应,而且两者具有协同作用;(2)Rp-cAMP和Sp-cAMPS两者本身都不能引起血小板聚集,但能分别增强和抑制PAF的聚集效应;(3)IP3和cAMP的水平变化分别与血小板的聚集和解聚过程一致。结论：(1)肌醇脂质信使系统是细胞内转导PAF诱导血小板聚集的主要胞内信使系统。(2)降低血小板内cAMP浓度不能诱导聚集,但能增强肌醇脂质信使系统的聚集效应;升高cAMP水平能拮抗肌醇脂质信使系统的作用,这可能是使可逆相聚集的血小板解聚的一个重要机制。     

水稻OsV3IP2和OsV3IP3原核表达与鉴定

通过生物信息学分析确定OsV3IP2和OsV3IP3的可溶性肽段,获得可溶性的OsV3IP2和OsV3IP3外源表达蛋白质,将相应的编码区克隆于原核表达载体p ET32a中,进行IPTG诱导表达,利用SDS-PAGE和Western blot检测目的蛋白质。结果表明,利用生物信息学软件对蛋白质的二级结构进行预测,将OsV3IP2、OsV3IP3分别分为2个和4个部分。将其分别克隆到p ET32a后,采用0.2 mol/L IPTG在10、20、30℃诱导表达。经SDS-PAGE和Western Blot分析,证明OsV3IP2-1、OsV3IP 2-2、OsV3IP 3-1表达的融合蛋白存在于上清液中,而OsV3IP 3-2、OsV3IP 3-3表达的融合蛋白存在于沉淀中,获得了OsV3IP2-1、OsV3IP 2-2、OsV3IP 3-1可溶性目的蛋白,为进一步体外验证OsVDAC3与OsV3IP2、OsV3IP3的互作奠定了基础。     

Odor stimuli trigger influx of calcium into olfactory neurons of the channel catfish

Olfactory transduction is thought to be mediated by a G protein-coupled increase in intracellular adenosine 3',5'-monophosphate (cAMP) that triggers the opening of cAMP-gated cation channels and results in depolarization of the plasma membrane of olfactory neurons. In olfactory neurons isolated from the channel catfish, Ictalurus punctatus, stimulation with olfactory stimuli (amino acids) elicits an influx of calcium that leads to a rapid increase in intracellular calcium. In addition, in a reconstitution assay a plasma membrane calcium channel has been identified that is gated by inositol-1,4,5-trisphosphate (IP3), which could mediate this calcium influx. Together with previous studies indicating that stimulation with olfactory stimuli leads to stimulation of phosphoinositide turnover in olfactory cilia, these data suggest that an influx of calcium triggered by odor stimulation of phosphoinositide turnover may be an alternate or additional mechanism of olfactory transduction.     

Structure of the human dopamine D3 receptor in complex with a D2/D3 selective antagonist

Dopamine modulates movement, cognition, and emotion through activation of dopamine G protein-coupled receptors in the brain. The crystal structure of the human dopamine D3 receptor (D3R) in complex with the small molecule D2R/D3R-specific antagonist eticlopride reveals important features of the ligand binding pocket and extracellular loops. On the intracellular side of the receptor, a locked conformation of the ionic lock and two distinctly different conformations of intracellular loop 2 are observed. Docking of R-22, a D3R-selective antagonist, reveals an extracellular extension of the eticlopride binding site that comprises a second binding pocket for the aryl amide of R-22, which differs between the highly homologous D2R and D3R. This difference provides direction to the design of D3R-selective agents for treating drug abuse and other neuropsychiatric indications.     

Generation of calcium oscillations in fibroblasts by positive feedback between calcium and IP3

A wide variety of nonexcitable cells generate repetitive transient increases in cytosolic calcium ion concentration ([Ca2+]i) when stimulated with agonists that engage the phosphoinositide signalling pathway. Current theories regarding the mechanisms of oscillation disagree on whether Ca2+ inhibits or stimulates its own release from internal stores and whether inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DG) also undergo oscillations linked to the Ca2+ spikes. In this study, Ca2+ was found to stimulate its own release in REF52 fibroblasts primed by mitogens plus depolarization. However, unlike Ca2+ release in muscle and nerve cells, this amplification was insensitive to caffeine or ryanodine and required hormone receptor occupancy and functional IP3 receptors. Oscillations in [Ca2+]i were accompanied by oscillations in IP3 concentration but did not require functional protein kinase C. Therefore, the dominant feedback mechanism in this cell type appears to be Ca2+ stimulation of phospholipase C once this enzyme has been activated by hormone receptors.     

Effect of phospholipase C-gamma overexpression on PDGF-induced second messengers and mitogenesis

Platelet-derived growth factor (PDGF) stimulates phospholipase C (PLC) activity and the phosphorylation of the gamma isozyme of PLC (PLC-gamma) in vitro and in living cells. The role of PLC-gamma in the phosphoinositide signaling pathway was addressed by examining the effect of overexpression of PLC-gamma on cellular responses to PDGF. Overexpression of PLC-gamma correlated with PDGF-induced tyrosine phosphorylation of PLC-gamma and with PDGF-induced breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2). However, neither bradykinin- nor lysophosphatidic acid-induced phosphoinositide metabolism was enhanced in the transfected cells, suggesting that the G protein-coupled phosphoinositide responses to these ligands are mediated by other PLC isozymes. The enhanced PDGF-induced generation of inositol trisphosphate (IP3) did not enhance intracellular calcium signaling or influence PDGF-induced DNA synthesis. Thus, enzymes other than PLC-gamma may limit PDGF-induced calcium signaling and DNA synthesis. Alternatively, PDGF-induced calcium signaling and DNA synthesis may use biochemical pathways other than phosphoinositide metabolism for signal transduction.     

The Role of Extracellular Ca2+Influx, Intracellular Ca2+ Release and Calmodulin in Mouse Egg Fertilization

The effects of various Ca^2 -modifying drugs on moue egg fertilization were studied.Ca^2 chelator,ethylen glycol-bis-(2-aminoethyl)-tetracetic acid(EGTA),and calmodulin(CaM) antagonist,trifluoperzaine (TFP),inhibited fertilization in a dose-dependent manner,whild Ca^2 channel bolcker,verspamil,did not have any effect.When intracellular Ca^2 release was blocked by 8-(N,N-diethylamino) octy 1-3,4,5-trimethoxy-benzonate(TME-8) or the Ca^2 oscillations were inhibited by an inhibitor of endoplasmic reticulum Ca^2 -At-Pase,thapsigargin,the second polar body emission and pronuclear formation were significantly decreased.In contrast,inhibition of intracellular Ca^2 release via bolckage of inositol 1,4,5-triphosphate (IP3) production by neomycin or lithium did not affect fertilization.The results sugest that both extracellular influx,intracellular Ca^2 release and CaM activation are required for mormal fertilization.However,extracellular influx through voltage-gated Ca^2 channel and intracellular release induced by IP3 and not the only pathways for producing Ca^2 transients in moue eggs.     

Coupling diurnal cytosolic Ca2+ oscillations to the CAS-IP3 pathway in Arabidopsis

Various signaling pathways rely on changes in cytosolic calcium ion concentration ([Ca2+]i). In plants, resting [Ca2+]i oscillates diurnally. We show that in Arabidopsis thaliana, [Ca2+]i oscillations are synchronized to extracellular Ca2+ concentration ([Ca2+]o) oscillations largely through the Ca2+-sensing receptor CAS. CAS regulates concentrations of inositol 1,4,5-trisphosphate (IP3), which in turn directs release of Ca2+ from internal stores. The oscillating amplitudes of [Ca2+]o and [Ca2+]i are controlled by soil Ca2+ concentrations and transpiration rates. The phase and period of oscillations are likely determined by stomatal conductance. Thus, the internal concentration of Ca2+ in plant cells is constantly being actively revised.     

HIV-1-infected T cells show a selective signaling defect after perturbation of CD3/antigen receptor

The binding of antigen or monoclonal antibody to the T cell receptor for antigen or the closely associated CD3 complex causes increases in the concentration of intracellular ionized calcium and subsequent cell proliferation. By measuring second messenger production in primary cultures of human immunodeficiency virus (HIV-1)--infected T cells stimulated with monoclonal antibodies specific for either CD3 or CD2, a specific impairment of membrane signaling was revealed. The HIV-1--infected T cells were unable to mobilize Ca2+ after stimulation with anti-CD3, whereas CD2-induced calcium mobilization remained intact. Furthermore, the HIV-1--infected cells proliferated poorly after CD3 stimulation, although the cells retained normal DNA synthesis in response to interleukin-2 stimulation. These results show that the signals initiated by CD2 and CD3 can be regulated independently within the same T cell; uncoupling of signal transduction after antigen-specific stimulation provides a biochemical mechanism to explain, in part, the profound immunodeficiency of patients with HIV-1 infection.     

Signaling of rat Frizzled-2 through phosphodiesterase and cyclic GMP

The Frizzled-2 receptor (Rfz2) from rat binds Wnt proteins and can signal by activating calcium release from intracellular stores. We show that wild-type Rfz2 and a chimeric receptor consisting of the extracellular and transmembrane portions of the beta2-adrenergic receptor with cytoplasmic domains of Rfz2 also signaled through modulation of cyclic guanosine 3',5'-monophosphate (cGMP). Activation of either receptor led to a decline in the intracellular concentration of cGMP, a process that was inhibited in cells treated with pertussis toxin, reduced by suppression of the expression of the heterotrimeric GTP-binding protein (G protein) transducin, and suppressed through inhibition of cGMP-specific phosphodiesterase (PDE) activity. Moreover, PDE inhibitors blocked Rfz2-induced calcium transients in zebrafish embryos. Thus, Frizzled-2 appears to couple to PDEs and calcium transients through G proteins.     

盐胁迫下植物的分子信号传导

盐胁迫下,植物体内的离子浓度和渗透压发生变化,诱导其产生第二信使(如IP3和活性氧分子等).第二信使调控细胞内Ca2 的水平,引发蛋白磷酸化级联反应,直接诱导或间接由转录因子诱导盐胁迫相关基因的表达,最终植物体表现耐受胁迫、生长受阻或死亡.     

小鼠TLR3基因双敲除打靶载体构建及巨噬细胞RAW264.7TLR3-/-细胞系的建立

[目的]建立Toll样受体3(TLR3)基因缺失的小鼠巨噬细胞RAW264.7细胞系,为探索狂犬病毒感染机体过程中TLR3在固有免疫反应中的作用机制提供理论依据.[方法]采用Golden Gate Kit试剂盒组装转录激活样效应因子核酸酶(TALEN)打靶载体pTALEN-TLR3,经酶切和测序验证其连接正确后,通过脂质体瞬时转染RAW264.7细胞,转染后提取细胞DNA,用T7核酸内切酶酶切验证TALEN质粒剪切活性.[结果]TALENs左右臂分两部分连接,首先完成A、B部分的各自连接,然后分别将T1LA与T1LB、T1RA与T1RB、T2LA与T2LB、T2RA与T2RB连接,TALEN模块经过两次连接后的PCR鉴定结果显示,T1L、T1R和T2L的4个克隆均呈阳性,T2R有3个克隆呈阳性.T2L和T2R质粒共转染RAW264.7细胞后提取DNA为模板,经PCR扩增后用T7核酸内切酶进行酶切,酶切后的DNA电泳结果显示TALEN2剪切活性较强,共获得3条条带(931、555和376 bp).TALEN打靶载体pTALEN-TLR3转染RAW264.7细胞24 h后用胰酶进行消化,并加入800μg/mL G418进行筛选,7 d后获得细胞单克隆;挑选阳性细胞克隆进行T7核酸内切酶酶切鉴定及测序,结果发现4-1和4-40号细胞克隆为双敲细胞系,均缺失7 bp的核苷酸碱基,为非3整数倍碱基缺失,可造成后续基因移码突变,使细胞基因功能失活.[结论]通过TALEN技术可成功构建TLR3基因双敲除的小鼠巨噬细胞RAW264.7TLR3-/-细胞系,且可用于狂犬病毒感染细胞后细胞因子和TLR3间的关系研究.     

In vivo Ca2+ dynamics in a cricket auditory neuron: an example of chemical computation

Fura-2 calcium imaging in the cricket omega neuron revealed increased intracellular free calcium ion concentration in response to simulated cricket calling songs and other sound stimuli. The time course of the increase and decrease in intracellular calcium coincided with the time course of forward masking, a time-dependent modulation of auditory sensitivity. The buffering of calcium transients with high concentrations of a kinetically fast calcium buffer eliminated the post-stimulus hyperpolarization associated with forward masking, whereas the uncaging of calcium inside the neuron produced a hyperpolarization. The results suggest that sound-stimulated intracellular calcium accumulation acts by means of a calcium-activated hyperpolarizing current to produce forward masking. These findings underscore the importance of chemical dynamics in neural computation by demonstrating a behaviorally relevant role of calcium dynamics in vivo.     

Phosphorylation of proteins by inositol pyrophosphates

The inositol pyrophosphates IP7 and IP8 contain highly energetic pyrophosphate bonds. Although implicated in various biologic functions, their molecular sites of action have not been clarified. Using radiolabeled IP7, we detected phosphorylation of multiple eukaryotic proteins. We also observed phosphorylation of endogenous proteins by endogenous IP7 in yeast. Phosphorylation by IP7 is nonenzymatic and may represent a novel intracellular signaling mechanism.