受体调节的钙内流不存在秘密通道

某些物质与受体结合后,激活磷脂酶C,促进4,5-二磷酸磷脂酰肌醇水解成1,4,5-三磷酸肌醇(IP_3),引起内质网钙库的排空,即钙的动员,钙库的排空进而又可刺激外钙的内流。受体调节的钙内流主要受钙库的排空控制。实验表明,激素敏感的钙库处于排空状态时较易与细胞外     

人白血病细胞系P2X7受体的表达及其介导的细胞内钙浓度变化

采用半定量RT-PCR和流式细胞术,在基因和蛋白水平研究了白血病细胞系U937、HL60和Ramos细胞P2X7受体的表达。荧光染料Fura-2／AM负载后,用荧光分光光度计测定P2X7受体激动剂三磷酸腺苷(adenosine 5′-triphosphate,ATP)和苯甲酰苯甲酸ATP(2′,3′-O-(4-benzoyl)benzoyl-ATP,BzATP)刺激前后细胞内钙离子浓度的变化,以确认其功能。结果表明：U937和HL60细胞系表达P2X7受体的mRNA和蛋白,Ramos不表达;在激动剂的刺激下,可引发U937和HL60细胞胞内钙浓度的显著升高,但对Ramos没有作用。当去除胞外钙离子时,ATP和BzATP刺激均不能引起U937和HL60细胞胞内钙离子浓度的升高。提示U937和HL60细胞表达P2X7受体的基因和功能蛋白,Ramos细胞则不表达该受体。     

1,4,5-三磷酸肌醇在细胞信号转换系统中的作用

肌醇磷酸脂代谢的中间产物1,4,5-三磷酸肌醇在细胞内外的信号转换系统中起着重要的媒介作用。各种不以cAMP 为第二信使的细胞外激动剂作用于靶细胞的相应受体时,首先激活细胞膜上特异的磷脂酶C,使4,5-二磷酸磷脂酰肌醇水解,释出1,4,5-三磷酸肌醇,后者进一步使细胞内Ca~(2 )贮释放,从而激活钙/钙调蛋白系统,引起细胞的各种生理效应。     

细胞外Ca2+对爪蟾脑片神经元微抑制性突触后电流的调制

应用盲法膜片钳全细胞记录技术,以爪蟾视顶盖神经元微抑制性突触后电流(miniature inhibitory postsyn-aptic currents,mIPSCs)为指标,观察了细胞外Ca^2 对爪蟾脑片神经元突触后mIPSC的调制。结果表明：用细胞外无钙或无钙含乙二醇双乙胺醚-N,N′-四乙酸(EGTA)(200nmol／L—2mmol／L)溶液灌流,均可使mIPSCs的发放频率降低;非特异性钙离子拮抗剂氯化铬(100μmol／L)也可使mIPSCs的频率降低;内质网钙泵抑制剂thapsigargin(TG)以及内质网ryanodine受体(RyR)激动剂ryanodine均可使mIPSCs频率升高,内质网RyR拮抗剂普鲁卡因则可降低mIPSCs的频率;磷脂酶C抑制剂U73122也可降低mIPSCs的频率,对三磷酸肌醇(inositol 1,4,5-triphosphate,IP3)水平有抑制作用的咖啡因亦可显著地降低mIPSCs,甚至完全抑制mIPSCs。从而表明：对突触前神经元及其末梢,细胞外钙离子可通过细胞膜上的钙通道进入细胞内,使细胞内钙浓度升高,突触前神经末梢释放出更多的神经递质。进而可能使突触后mIPSCs的频率增加;突触前细胞内钙储池上的Rya和IP3R均可介导钙从其中释放,并也可使突触前细胞内的钙离子浓度升高,进而可能使突触后mIPSCs的发放频率增加。     

FK-506结合蛋白对钙释放通道的调控

细胞内自由钙作为一种重要的细胞信使广泛地参与细胞生理功能调控.胞内钙库(内质网系和肌浆网系)对调节细胞内自由钙水平起着重要的作用.钙库膜上的钙释放通道(ryanodine受体和三磷酸肌醇受体)受许多因素调控,其中之一就是新近研究得相当多的FK506结合蛋白.免疫抑制剂FK506能特异地结合钙库上一种分子质量为12 ku左右的蛋白,这种FK506结合蛋白与钙释放通道形成一种紧密连接的复合体,在正常生理情况下对钙释放通道起着十分重要的调控作用.     

CD44基因的分子生物学特性及其与肿瘤关系的研究进展

CD44是一种细胞表面跨膜糖蛋白分子 ,在许多细胞上均有分布 ,如淋巴细胞、单核细胞、红细胞、成纤维细胞、上皮细胞、平滑肌细胞、神经胶质细胞及肿瘤细胞等[1-3 ] 。CD44蛋白属于未分类的粘附分子 ,其正常功能是作为受体识别透明质酸(HA)和胶原蛋白 I、II等 ,主要参与淋巴细胞的激活以及细胞 -细胞 ,细胞 -基质之间的特异性粘连过程 ,CD44基因的变异性、多样性表达与肿瘤的生长及转移有密切的相关性 [4～ 7] 。现将 CD44基因的分子生物学特性、主要功能、在常见肿瘤中的表达及其与肿瘤发生、浸润、转移的关系和可能的转移机制作简要…     

构建钙荧光探针细胞用于探究胞浆和线粒体钙对ATP刺激的响应

目的:构建表达基因编辑钙探针(GECIs)的细胞系HeLa-GECIs,探究细胞应答外界ATP刺激中钙离子在细胞内的响应和变化。方法:分别用能够直接通过荧光强度反映细胞胞浆内和线粒体内钙离子相对浓度的2种钙探针cyto-GCaMP6和4mt-GCaMP6感染HeLa细胞,获得2种表达钙离子探针的HeLa细胞系;在感染了2种腺病毒探针24 h后,用共聚焦荧光显微镜检测荧光探针在HeLa细胞内的表达情况;在表达2种钙探针的细胞的培养基中加入外源ATP,用Time-lapse成像动态观测技术观察HeLa细胞内钙离子对外环境中ATP的响应。结果:共聚焦荧光显微镜观察,确定95%以上的细胞表达了对应的钙离子指示荧光探针;Time-lapse成像动态观测技术观察发现,在细胞培养基中加入ATP后,细胞胞浆钙探针荧光强度瞬时(3～6 s)升至10倍,200 s后逐渐降低到基础水平;线粒体钙到达峰值(4倍)的时间稍滞后(5～8 s),并且回落更慢,300 s时至1.5倍。在ATP受体P2X7抑制剂A438079预处理的实验组,上述胞浆钙和线粒体钙浓度上升不明显。结论:构建了能在活体细胞内通过荧光探针实时监测钙离子响应胞外ATP刺激的细胞实验体系,为进一步深入探究ATP等危险信号导致细胞的炎性损伤机制奠定了基础。     

气相色谱法检测大鼠脑及颌下腺磷脂酰肌醇代谢环路中肌醇的含量

磷脂酰肌醇(PI)约占细胞总磷脂的5～10％,其代谢十分活跃。近几年来,人们发现PI代谢是许多膜受体信号跨膜转导的重要途径。乙酰胆碱等多种神经递质激动其受体后,经三磷酸鸟苷结合蛋白转导激活细胞膜磷脂酶C,催化PI水解生成两种信使物质:1,4,5一三磷酸肌醇(IP_3)及二酰基甘油,其中IP_3经特异性     

受体调节钙通道有三种机制

几乎所有的组织都有受体调节的钙通道,它们参与多种细胞功能,第二信使诱导的电压门控性钙通道开放是近年来提出的一种较完善的机制,以解释受体调节的钙电导变化。1,4,5-三磷酸肌醇(InsP_3)作为第二信使,使细胞内贮存的钙释放到胞浆内,使细胞内钙浓度([Ca](?))升高,从而引起钙内流,也就是钙通道开放。这种机制已经在几个系统内得到证实,但是在乳腺细胞中却没有发现这种电压门控性钙通道,而乳腺细胞上肯定存在着受体调节的钙通道。最近 Kuno,Reuter 和 Irvine 分别提出了另外三种机制,解释受体介导的钙内流。     

亚硝酸钠诱导的草鱼肝细胞凋亡中内质网应激IRE1通路的作用研究

为探究亚硝酸钠诱导草鱼肝细凋亡中内质网应激IRE1通路的作用, 草鱼(Ctenopharyngodon idellus)肝细胞L8824分别置于亚硝酸钠浓度为0、5、20和50 mg/L暴露12h和24h。同时采用三磷酸肌醇受体拮抗剂2-APB和IRE1α抑制剂STF-083010分别和20 mg/L亚硝酸钠共同孵育L8824细胞24h。检测细胞凋亡以及c-Jun氨基末端激酶(c-Jun N-terminal kinase, jnk)、B淋巴细胞瘤-2(B-cell lymphoma-2, bcl-2)、bcl-2相关X蛋白(bcl-2 associated X protein, bax)、caspase9、caspase3、肌醇酶1α (inositol requiring enzyme 1α, ire1α)、X盒结合蛋白 1s (X-box binding protein 1s, xbp1s)和葡萄糖调节蛋白78 (glucose-regulated protein78, grp78)基因的表达和细胞质内钙离子浓度。结果表明: 与对照组相比, 亚硝酸钠处理组细胞凋亡率显著上升, jnk、bax、caspase9、caspase3、ire1α、xbp1s和grp78 mRNA的表达显著升高, bcl-2 mRNA的表达量显著下降, 细胞质内钙离子浓度显著上升。与亚硝酸钠单处理组相比, STF-083010处理组细胞凋亡率显著下降, ire1α、xbp1s、grp78、jnk、bax、caspase3 mRNA表达量显著下降, bcl-2 mRNA表达量显著上升, 2-APB和STF-083010两个处理组细胞质内钙离子浓度均显著下降。结果显示, 高浓度的亚硝酸钠可诱导草鱼肝细胞凋亡和钙离子紊乱, 内质网应激IRE1通路发挥了作用。     

Subtype-specific and ER lumenal environment-dependent regulation of inositol 1,4,5-trisphosphate receptor type 1 by ERp44

Inositol 1,4,5-trisphosphate receptors (IP(3)Rs) are intracellular channel proteins that mediate Ca(2+) release from the endoplasmic reticulum (ER) and are involved in many biological processes and diseases. IP(3)Rs are differentially regulated by a variety of cytosolic proteins, but their regulation by ER lumenal protein(s) remains largely unexplored. In this study, we found that ERp44, an ER lumenal protein of the thioredoxin family, directly interacts with the third lumenal loop of IP(3)R type 1 (IP(3)R1) and that the interaction is dependent on pH, Ca(2+) concentration, and redox state: the presence of free cysteine residues in the loop is required. Ca(2+)-imaging experiments and single-channel recording of IP(3)R1 activity with a planar lipid bilayer system demonstrated that IP(3)R1 is directly inhibited by ERp44. Thus, ERp44 senses the environment in the ER lumen and modulates IP(3)R1 activity accordingly, which should in turn contribute to regulating both intralumenal conditions and the complex patterns of cytosolic Ca(2+) concentrations.     

Inositol 1,4,5-trisphosphate IP(3) receptors and their role in neuronal cell function

Inositol 1,4,5-trisphosphate (IP(3)) receptor is a Ca(2+) release channel localized on the endoplasmic reticulum (ER) and plays an important role in neuronal function. IP(3) receptor was discovered as a developmentally regulated protein missing in the cerebellar mutant mice. Recent studies indicate that IP(3)Rs are involved in early development and neuronal plasticity. IP(3) works to release IRBIT from the IP(3) binding core in addition to release Ca(2+). IRBIT binds to and activates Na, Bicarbonate cotransporter. Electron microscopic study show the IP(3) receptor has allosteric property to change its form from square to windmill in the presence of Ca(2+). IP(3)R associates with ERp44, a redox sensor, Homer, other proteins and is transported as vesicular ER on microtubules. All these data suggests IP(3) receptor/CA(2+) channel works as a signaling center inside cells.     

Uranyl acetate modulates gene expression and protein levels of the type 2, but not type 1 inositol 1,4,5-trisphosphate receptors in mouse kidney

Nephrotoxic effect of uranium is already well documented. Nevertheless, little is known about the effect of uranium on calcium homeostasis and calcium transport systems. Calcium released from endoplasmic reticulum through special calcium release channels--inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs)--serves as a main source of cytosolic calcium signaling in the majority of cell types. To contribute to understanding mechanism of toxicity of the uranyl acetate (UA), we focused on modulation of the gene expression, protein levels and activity of IP3 receptor's intracellular calcium channels by UA in mouse kidney. We have found that UA did not affect mRNA and protein levels of the type 1 IP3Rs, but increased mRNA and also protein levels of the type 2 IP3 receptors in kidney. Nevertheless, IP3-induced calcium release was decreased by addition of UA. We assume that decreased activity of IP3 receptors due to the acute exposure to UA results in feedback, which triggers activation of IP3R2 expression. Thus, inhibition of calcium release and increased levels of the type 2 IP3 receptors might participate, at least partially, in UA-induced nephrotoxicity.     

IP3 receptor/Ca2+ channel: from discovery to new signaling concepts

Inositol 1,4,5-trisphosphate (IP(3)) is a second messenger that induces the release of Ca(2+) from the endoplasmic reticulum (ER). The IP(3) receptor (IP(3)R) was discovered as a developmentally regulated glyco-phosphoprotein, P400, that was missing in strains of mutant mice. IP(3)R can allosterically and dynamically change its form in a reversible manner. The crystal structures of the IP(3)-binding core and N-terminal suppressor sequence of IP(3)R have been identified. An IP(3) indicator (known as IP(3)R-based IP(3) sensor) was developed from the IP(3)-binding core. The IP(3)-binding core's affinity to IP(3) is very similar among the three isoforms of IP(3)R; instead, the N-terminal IP(3) binding suppressor region is responsible for isoform-specific IP(3)-binding affinity tuning. Various pathways for the trafficking of IP(3)R have been identified; for example, the ER forms a meshwork upon which IP(3)R moves by lateral diffusion, and vesicular ER subcompartments containing IP(3)R move rapidly along microtubles using a kinesin motor. Furthermore, IP(3)R mRNA within mRNA granules also moves along microtubules. IP(3)Rs are involved in exocrine secretion. ERp44 works as a redox sensor in the ER and regulates IP(3)R1 activity. IP(3) has been found to release Ca(2+), but it also releases IRBIT (IP(3)R-binding protein released with IP(3)). IRBIT is a pseudo-ligand for IP(3) that regulates the frequency and amplitude of Ca(2+) oscillations through IP(3)R. IRBIT binds to pancreas-type Na, bicarbonate co-transporter 1, which is important for acid-base balance. The presence of many kinds of binding partners, like homer, protein 4.1N, huntingtin-associated protein-1A, protein phosphatases (PPI and PP2A), RACK1, ankyrin, chromogranin, carbonic anhydrase-related protein, IRBIT, Na,K-ATPase, and ERp44, suggest that IP(3)Rs form a macro signal complex and function as a center for signaling cascades. The structure of IP(3)R1, as revealed by cryoelectron microscopy, fits closely with these molecules.     

Protein kinase A and two phosphatases are components of the inositol 1,4,5-trisphosphate receptor macromolecular signaling complex

The inositol 1,4,5-trisphosphate receptor (IP3R) is a ubiquitously expressed intracellular calcium (Ca(2+)) release channel on the endoplasmic reticulum. IP3Rs play key roles in controlling Ca(2+) signals that activate numerous cellular functions including T cell activation, neurotransmitter release, oocyte fertilization and apoptosis. There are three forms of IP3R, all of which are ligand-gated channels activated by the second messenger inositol 1,4,5-trisphosphate. Channel function is modulated via cross-talk with other signaling pathways including those mediated by kinases and phosphatases. In particular IP3Rs are known to be regulated by cAMP-dependent protein kinase (PKA) phosphorylation. In the present study we show that PKA and the protein phosphatases PP1 and PP2A are components of the IP3R1 macromolecular signaling complex. PKA phosphorylation of IP3R1 increases channel activity in planar lipid bilayers. These studies indicate that regulation of IP3R1 function via PKA phosphorylation involves components of a macromolecular signaling complex.     

ERp44, a novel endoplasmic reticulum folding assistant of the thioredoxin family

In human cells, Ero1-Lalpha and -Lbeta (hEROs) regulate oxidative protein folding by selectively oxidizing protein disulfide isomerase. Specific protein--protein interactions are probably crucial for regulating the formation, isomerization and reduction of disulfide bonds in the endoplasmic reticulum (ER). To identify molecules involved in ER redox control, we searched for proteins interacting with Ero1-Lalpha. Here, we characterize a novel ER resident protein (ERp44), which contains a thioredoxin domain with a CRFS motif and is induced during ER stress. ERp44 forms mixed disulfides with both hEROs and cargo folding intermediates. Whilst the interaction with transport-competent Ig-K chains is transient, ERp44 binds more stably with J chains, which are retained in the ER and eventually degraded by proteasomes. ERp44 does not bind a short-lived ribophorin mutant lacking cysteines. Its overexpression alters the equilibrium of the different Ero1-Lalpha redox isoforms, suggesting that ERp44 may be involved in the control of oxidative protein folding.     

ERp44 C160S/C212S mutants regulate IP3R1 channel activity

Previous studies have indicated that ERp44 inhibits inositol 1,4,5-trisphosphate (IP₃)-induced Ca²⁺ release (IICR) via IP₃R₁, but the mechanism remains largely unexplored. Using extracellular ATP to induce intracellular calcium transient as an IICR model, Ca²⁺ image, pull down assay, and Western blotting experiments were carried out in the present study. We found that extracellular ATP induced calcium transient via IP₃Rs (IICR) and the IICR were markedly decreased in ERp44 over-expressed Hela cells. The inhibitory effect of C160S/C212S but not C29S/T396A/ΔT(331--377) mutants of ERp44 on IICR were significantly decreased compared with ERp44. However, the binding capacity of ERp44 to L3V domain of IP₃R₁ (1L3V) was enhanced by ERp44 C160S/C212S mutation. Taken together, these results suggest that the mutants of ERp44, C160/C212, can more tightly bind to IP₃R₁ but exhibit a weak inhibition of IP₃R₁ channel activity in Hela cells.