Feedback regulation between phosphatidylinositol-3,4,5-trisphosphate dependent Rac exchange factor 1 and transforming growth factor β1 and prognostic value in gastric cancer

BACKGROUND Phosphatidylinositol-3,4,5-trisphosphate dependent Rac exchange factor 1(PREX1)was reported to be overexpressed in some cancers and involved in cancer development,but its expression and significance in gastric cancer remain unclear.AIM To evaluate the expression of PREX1 in gastric cancer and its significance in the development of gastric cancer,especially to evaluate the potential mechanism of PREX1 in gastric cancer.METHODS Bioinformatic analysis was performed in order to examine the expression of PREX1 in gastric cancer.The relationship between the survival rate of gastric cancer patients and PREX1 expression was assessed by Kaplan Meier portal.The Gene Set Enrichment Analysis and the correlation between PREX1 and transforming growth factor(TGF)β1 pathway-related mediators were evaluated by cBioPortal for Cancer Genomics.Western blotting and reverse transcriptase polymerase chain reaction assay were used to test the role of TGFβ1 on the expression of PREX1.Western blotting and dual-luciferase reporter system was used to evaluate the effect of PREX1 on the activation of TGFβ1 pathway.Wound healing and Transwell assay were used to assess the effect of PREX1 on the metastasis activity of gastric cancer cells.RESULTS PREX1 was overexpressed in the gastric tumors,and the expression levels were positively associated with the development of gastric cancer.Also,the high expression of PREX1 revealed poor prognosis,especially for those advanced and specific intestinal gastric cancer patients.PREX1 was closely involved in the positive regulation of cell adhesion and positively correlated with TGFβ1-related mediators.Furthermore,TGFβ1 could induce the expression of PREX1 at both the protein and mRNA level.Also,PREX1 could activate the TGFβ1 pathway.The induced PREX1 could increase the migration and invasion activity of gastric cancer cells.CONCLUSION PREX1 is overexpressed in gastric cancer,and the high level of PREX1 predicts poor prognosis.PREX1 is closely associated with TGFβsignaling and promotes the metastasis of gastric cancer cells.

Altered expression of stromal interaction molecule(STIM)-calcium release-activated calcium channel protein(ORAI) and inositol1,4,5-trisphosphate receptors(IP_3Rs)in cancer:will they become a new battlefield for oncotherapy?

The stromal interaction molecule(STIM)-calcium release-activated calcium channel protein(ORAI) and inositol1,4,5-trisphosphate receptors(IP_3Rs) play pivotal roles in the modulation of Ca^(2+)-regulated pathways from gene transcription to cell apoptosis by driving calcium-dependent signaling processes.Increasing evidence has implicated the dysregulation of STIM-ORAI and IP_3Rs in tumorigenesis and tumor progression.By controlling the activities,structure,and/or expression levels of these Ca^(2+)-transporting proteins,malignant cancer cells can hijack them to drive essential biological functions for tumor development.However,the molecular mechanisms underlying the participation of STIM-ORAI and IP_3Rs in the biological behavior of cancer remain elusive.In this review,we summarize recent advances regarding STIM-ORAI and IP_3Rs and discuss how they promote cell proliferation,apoptosis evasion,and cell migration through temporal and spatial rearrangements in certain types of malignant cells.An understanding of the essential roles of STIM-ORAI and IP_3Rs may provide new pharmacologic targets that achieve a better therapeutic effect by inhibiting their actions in key intracellular signaling pathways.

Inositol 1,4,5-trisphosphate receptor in the liver:Expression and function

The liver is a complex organ that performs several functions to maintain homeostasis.These functions are modulated by calcium,a second messenger that regulates several intracellular events.In hepatocytes and cholangiocytes,which are the epithelial cell types in the liver,inositol 1,4,5-trisphosphate(InsP3)receptors(ITPR)are the only intracellular calcium release channels.Three isoforms of the ITPR have been described,named type 1,type 2 and type 3.These ITPR isoforms are differentially expressed in liver cells where they regulate distinct physiological functions.Changes in the expression level of these receptors correlate with several liver diseases and hepatic dysfunctions.In this review,we highlight how the expression level,modulation,and localization of ITPR isoforms in hepatocytes and cholangiocytes play a role in hepatic homeostasis and liver pathology.

Inositol 1,4,5-trisphosphate 3-kinases:functions and regulations

Inositol 1,4,5-trisphosphate 3-kinase (IP3 3-kinase/IP3K) plays an important role in signal transduction in animal cellsby phosphorylating inositol 1,4,5-trisphosphate (IP3) to inositol 1,3,4,5-tetrakisphosphate (IP4). Both IP3 and IP4 arecritical second messengers which regulate calcium (Ca2+) homeostasis. Mammalian IP3Ks are involved in many biologicalprocesses, including brain development, memory, learning and so on. It is widely reported that Ca2+ is a canonicalsecond messenger in higher plants. Therefore, plant IP3K should also play a crucial role in plant development. Recently,we reported the identification of plant IP3K gene (AtIpk2β/AtIP3K) from Arabidopsis thaliana and its characterization.Here, we summarize the molecular cloning, biochemical properties and biological functions of IP3Ks from animal, yeastand plant. This review also discusses potential functions of IP3Ks in signaling crosstalk, inositol phosphate metabolism,gene transcriptional control and so on.

High expression of type I inositol 1,4,5-trisphosphate receptor in the kidney of rats with hepatorenal syndrome

AIM To detect the expression of typeⅠ inositol 1,4,5-trisphosphate receptor(IP3 RI) in the kidney of rats with hepatorenal syndrome(HRS).METHODS One hundred and twenty-five Sprague-Dawley rats were randomly divided into four groups to receive an intravenous injection of D-galactosamine(D-Gal N) plus lipopolysaccharide(LPS; group G/L, n = 50), D-Gal N alone(group G, n = 25), LPS alone(group L, n = 25), and normal saline(group NS, n = 25), respectively.At 3, 6, 9, 12, and 24 h after injection, blood, liver, and kidney samples were collected. Hematoxylineosin staining of liver tissue was performed to assess hepatocyte necrosis. Electron microscopy was used to observe ultrastructural changes in the kidney. Western blot analysis and real-time PCR were performed to detect the expression of IP3 RI protein and m RNA in the kidney, respectively.RESULTS Hepatocyte necrosis was aggravated gradually, which was most significant at 12 h after treatment with D-galactosamine/lipopolysaccharide, and was characterized by massive hepatocyte necrosis. At the same time, serum levels of biochemical indicators including liver and kidney function indexes were all significantly changed. The structure of the renal glomerulus and tubules was normal at all time points. Western blot analysis indicated that IP3 RI protein expression began to rise at 3 h(P < 0.05) and peaked at 12 h(P < 0.01). Real-time PCR demonstrated that IP3 RI m RNA expression began to rise at 3 h(P < 0.05) and peaked at 9 h(P < 0.01).CONCLUSION IP3 RI protein expression is increased in the kidney of HRS rats, and may be regulated at the transcriptional level.

Phosphatidylinositol-3,4,5-trisphosphate dependent Rac exchange factor 1 is a diagnostic and prognostic biomarker for hepatocellular carcinoma

BACKGROUND Phosphatidylinositol-3,4,5-trisphosphate dependent Rac exchange factor 1(PRex1)was reported to be a risk factor in several cancers,including breast cancer,lung cancer,and melanoma,but its expression and role in hepatocellular carcinoma(HCC)have not yet been fully studied.AIM To explore the expression of P-Rex1 in HCC,and further evaluate its potential application in the diagnosis and prognosis of HCC,especially in hepatitis B virus(HBV)-related patients.METHODS P-Rex1 expression in HCC was evaluated by real-time-quantitative polymerase chain reaction.The expression of P-Rex1 was subjected to correlation analysis with clinical features,such as lymph node invasion,distant metastasis,HBV infection,patient’s age and gender.Receiver operating characteristic analysis was used to examine the potential role of P-Rex1 as a diagnostic biomarker in HCC.Kaplan-Meier analysis was used to determine the association between P-Rex1 expression and overall survival,progression-free survival and relapse-free survival.Bioinformatic analysis was used to validate the clinical findings.RESULTS P-Rex1 expression was significantly increased in HCC tumors than adjacent tissues.The expression of P-Rex1 was higher in HCC patients with lymph node invasion,distant metastasis,HBV infection and positive alpha-fetoprotein,respectively.The receiver operating characteristic analysis showed that P-Rex1 was a diagnostic biomarker with a higher area under the curve value,especially in patients with HBV infection.Survival analysis showed that patients with higher P-Rex1 expression had a favorable survival rate,even in early-stage patients.CONCLUSION P-Rex1 expression was highly increased in HCC,and the expression level of PRex1 was positively correlated with tumor progression.P-Rex1 has a higher efficiency in the diagnosis of HBV-related HCC,and could also be used as a favorable prognostic factor for HCC patients.

Expression of Inositol 1,4,5-trisphosphate Receptor mRNA in Myocardium of Spontaneous Hypertension Rats and Cultured Vascular Smooth Muscle Cells of Rats

Objective\ To investigate expression of inositol 1,4,5 trisphosphate receptor (IP\-3R) mRNA on sacroplasmic reticular in myocardium of spontaneous hypertension rats (SHRs) and cultured vascular smooth muscle cells (VSMC) of rats and effects of perindopril and urapidil on them. Methods\ SHRs were orally given perindopril (1.0 mg·kg\+\{ 1\}·d\+\{ 1\}) or urapidil (15 mg·kg\+\{ 1\}·d\+\{ 1\}) for 24 weeks, respectively. Expression of IP\-3R mRNA was examined by semi quantitative reverse transcription polymers chain reaction (RT PCR) using three oligonuclotide primers for each subtype of IP\-3R with β actin as internal label. Results\ All subtypes of IP\-3R were expressed in myocardium of SHR, WKY and cultured VSMC. Expression of IP\-3R mRNA in left ventricle of SHR was markedly enhanced. Urapidil could down regulate expression of IP\-3R Ⅰand IP\-3R Ⅲ, perindopril slightly increased expression of IP\-3R Ⅱ and decreased expression of IP\-3R Ⅰand IP\-3R Ⅲ in myocardium of SHR. Conclusion\ Our results suggest that expression of IP\-3R mRNA in cardiovascular system could be regulated by urapidil and perindopril.

1,4,5-三磷酸肌醇受体及其在休克中的作用

1,4,5-三磷酸肌醇(IP3)受体(IP3R)是内质网释放钙离子(Ca2+)的主要通道之一,在维持细胞内钙稳态方面发挥着重要作用。休克作为一种严重的、发生器官功能障碍与结构损伤的病理过程,钙稳态失调是其重要的中间环节。鉴于IP3R功能或表达异常引起钙稳态失调参与了多种病理过程的发生、进展,本文对IP3R结构与调节的研究进展进行综述,总结IP3R在失血性休克、感染性休克中的作用,期望以IP3R为切入点,为防治重症休克提供理论基础,并探索靶向IP3R防治休克的新策略。

1,4,5-三磷酸肌醇受体与神经变性疾病

1,4,5-三磷酸肌醇受体(inositol 1,4,5-trisphosphate receptors,IP3Rs)是细胞内质网上的钙离子(cal⁃cium ion,Ca^(2+))通道,通过调控Ca^(2+)参与细胞生物学功能,是维持中枢神经系统正常功能的关键分子。近年来,越来越多的研究发现,IP3Rs结构和功能异常与神经变性疾病如阿尔茨海默病、帕金森病、亨廷顿病、脊髓小脑共济失调等的发病机制密切相关,这些结构和功能异常如何影响IP3Rs功能,及相关钙信号,并且如何在这些疾病的发病和严重程度中发挥作用,仍尚不清楚。IP3Rs如何在神经变性疾病中发挥作用将于本文中进行综述。

大鼠心肌缺血再灌注时心肌细胞核1,4,5三磷酸肌醇受体结合特性改变的研究

目的观察大鼠心肌缺血再灌注时心肌细胞核1,4,5三磷酸肌醇受体(IP3R)的结合特性改变,进一步探索心肌缺血再灌注时细胞凋亡的病理机制。方法TUNEL技术检测心肌细胞核凋亡率。蔗糖密度梯度离心法提纯心肌细胞核,放射配体分析法检测心肌细胞核膜上IP3R的结合特性变化。结果①缺血再灌注损伤(IRI)组大鼠心肌细胞凋亡率高于对照组(P<0.01)。②大鼠心肌缺血再灌注损伤时,心肌细胞核IP3R的最大结合容量Bmax较假手术组增加2.6倍;而其亲和力Kd值无明显变化。③IRI组心肌细胞核IP3R经激活的PKC磷酸化后结合特性增强1.54倍,对照组核IP3R经PKC磷酸化后结合特性无明显改变。④[Ca2+]对IRI组及假手术组心肌细胞核IP3R的调节均呈双相性,胞浆钙超载状态下([Ca2+]为5μmol·L-1时)较正常胞浆钙浓度下([Ca2+]为100nmol·L-1时),两组核IP3R结合特性均增强,[Ca2+]为100μmol·L-1时,两组核IP3R结合特性降低。结论大鼠心肌心肌缺血再灌注损伤病理情况下心肌细胞核IP3R结合特性增强,致核内钙浓度([Ca2+]n)增高,这可能是心肌缺血再灌注损伤中心肌细胞凋亡的主要病理机制之一。

骨形成蛋白(BMP)-2/4,IA型BMP受体及Smad1、4、5在Tca8113舌癌细胞中的表达及意义

目的 认识和分析BMPs及其信号传导因子与口腔鳞状细胞癌发生、发展的可能关系。方法 用免疫组织化学方法检测BMP - 2 / 4 ,IA型BMP受体及Smad1,4 ,5在Tca8113细胞中表达并分析其意义。结果 BMP - 2 / 4 ,IA型BMP受体及Smad1,4 ,5在Tca8113细胞中有表达。其中 ,在胞浆中发现有BMP - 2 / 4的阳性信号 ,在胞膜中有IA型BMP受体的阳性信号 ,在胞质、胞核中有Smad1,4 ,5的强阳性表达。结论 BMPs及其信号传导因子与口腔鳞状细胞癌发生。

内质网蛋白44(ERp44)通过1,4,5-三磷酸肌醇受体介导基因转录(英文)

细胞核内钙离子浓度的增加可以引起包括钙离子激活的基因转录在内的很多生理功能.运用Western blot、免疫荧光、实时定量聚合酶链反应、钙成像以及外源三磷酸腺苷刺激细胞释放钙离子等试验方法,发现1,4,5-三磷酸肌醇受体和内质网蛋白44(ERp44)在内质网和核膜上都有很好的共定位.外源三磷酸腺苷可以通过1,4,5-三磷酸肌醇受体刺激核内钙瞬变并磷酸化环磷酸腺苷反应原件结合蛋白(CREB)、刺激原癌基因c-Myc的表达.但是,这些功能都能被1,4,5-三磷酸肌醇受体抑制剂2-氨乙氧基二苯酯硼酸(2-APB)和过表达内质网蛋白44(ERp44)所抑制.这些结果均提示在子宫颈癌HeLa细胞中内质网蛋白44(ERp44)通过1,4,5-三磷酸肌醇受体而介导基因转录.

糖尿病肾病大鼠肾脏组织中Ⅰ型1,4,5-三磷酸肌醇受体的表达

目的:检测糖尿病肾病(DN)大鼠肾脏组织中Ⅰ型1,4,5-三磷酸肌醇受体(IP3R)的表达。方法:35只健康雄性清洁级SD大鼠随机分成正常对照组(NC组,n=10)和DN组(以新鲜配制的枸橼酸缓冲液稀释链脲佐菌素成10g/L的溶液,按60mg/kg腹腔注射进行造模,当血糖值超过16.7mmol/L,表明糖尿病模型成功。普通饮食继续喂养3周、6周、12周,用放射免疫法测定24h尿蛋白排泄量,≥30mg时确认DN大鼠模型成功),后将DN大鼠随机分为DN3周组(n=9)、DN6周组(n=8)和DN12周组(n=8)。应用免疫组织化学法、RT-PCR测定4组大鼠肾脏组织中Ⅰ型IP3R蛋白及mRNA的表达。结果:Ⅰ型IP3R蛋白主要分布于肾小球系膜区、毛细血管袢和血管平滑肌细胞内,而肾小管未见阳性染色。与NC组相比,各DN组Ⅰ型IP3R蛋白阳性细胞率和mRNA的表达明显增高,差异有统计学意义(F=41.536、53.635,P<0.001)。结论:肾脏组织中IP3R表达增强可能是导致DN产生的重要因素之一。

肺动脉高压大鼠肺动脉平滑肌细胞1,4,5-三磷酸肌醇受体表达的变化

目的 :检测肺动脉高压 ( PAH)大鼠肺动脉平滑肌细胞 ( PASMCs)上 1,4,5三磷酸肌醇受体 ( IP3 R)的亚型及 IP3 R各亚型在 PAH时表达水平的变化。方法 :采用大鼠一次性腹腔注射野百合碱 ( MCT) 6 0 mg/kg复制 PAH动物模型 ,用 Western blot法检测 IP3 R亚型的表达及在 PAH状态下其蛋白水平表达的变化。结果 :大鼠 PASMCs共同表达 3个 IP3 R亚型 ,在 PAH时 IP3 R1表达水平明显增高 ,约为对照组的 2倍 ( P<0 .0 1) ,而 IP3 R2和 IP3 R3的表达水平无明显变化。结论 :PASMCs的 IP3 R1表达增强可能参与了

肿瘤坏死因子-α增强肝肾综合征时肾脏I型1,4,5-三磷酸肌醇受体表达

目的:通过观察TNF-α对肾组织中Ⅰ型IP3R表达的影响来探讨肝肾综合征的发病机制.方法:制备大鼠离体肾灌注模型,随机分成对照组(A组)、肝素处理组(B组)、TNF-α处理组(C组),留取的标本应用免疫组织化学技术、Westernblot及实时定量PCR检测肾组织Ⅰ型IP3R蛋白的定位、表达及mRNA的变化.结果:Ⅰ型IP3R主要分布于肾小球系膜细胞和血管平滑肌细胞的胞质内.免疫组化结果显示,C组与A组相比棕褐色颗粒着色的阳性细胞明显增多,有显著性差异(U=2.26,P<0.05);B组与A组相比阳性染色细胞无明显减少(P>0.05);Western blot结果与免疫组织化学结果相一致:C组与A组相比Ⅰ型IP3R蛋白表达水平明显增高,有显著性差异(1.89±0.11vs0.55±0.03,P<0.05);B组与A组相比无显著性差异(P>0.05);实时定量PCR结果显示:C组与A组相比Ⅰ型IP3R mRNA的表达水平明显增高,有显著性差异(7.99±0.12vs1.00±0.05,P<0.05);B组与A组相比无显著性差异(P>0.05).结论:TNF-α可增强肾小球系膜细胞和血管平滑肌细胞Ⅰ型IP3R蛋白的表达,且Ⅰ型IP3R mRNA也呈增加趋势.

TNF-α增强主动脉平滑肌细胞内1型1,4,5-三磷酸肌醇受体表达参与感染性休克的发生机制

目的研究TNF-α对主动脉平滑肌细胞(VSMC)内1型1,4,5-三磷酸肌醇受体(IP3RⅠ)表达的影响,揭示TNF-α影响VSMC收缩功能参与感染性休克的发生机制。方法原代分离培养大鼠VSMC。按TNF-α处理的不同时间点(0、4、8、24h)分4组。分别应用Western blot、免疫荧光、RT-PCR、双荧光素酶检测方法,观察TNF-α对IP3RⅠmRNA、蛋白表达及其启动子活性的影响。结果 TNF-α处理组细胞内IP3RⅠ分布未见变化,8、24h组荧光强度增强提示IP3RⅠ蛋白含量增加,IP3RⅠ蛋白表达升高(4h:1.059±0.005 vs 1.000±0.002,P=0.010;8h:2.416±0.042 vs 1.000±0.002,P<0.01;24h:2.138±0.010vs 1.000±0.002,P<0.01,n=9),IP3RⅠmRNA表达明显增加(4h:2.260±0.889 vs 1.00±0.02,P=0.193;8h:5.449±2.279 vs1.00±0.02,P=0.000;24h:3.049±1.684 vs 1.00±0.02,P=0.042,n=9)。转染PGL3-IP3RⅠpromoter质粒后TNF-α组IP3RⅠ启动子活性明显增强(3.56±0.65 vs 1.00±0.05,P=0.020,n=9)。结论 TNF-α可上调IP3RⅠ基因启动子活性,从而引起IP3RⅠ蛋白表达升高,增强VSMC内IP3Rs系统介导的Ca2+释放作用,这可能是TNF-α影响VSMC收缩功能参与感染性休克血管调控的机制之一。

替米沙坦对糖尿病肾病大鼠肾组织Ⅰ型1,4,5-三磷酸肌醇受体表达的影响

目的:探讨替米沙坦对糖尿病肾病(DN)大鼠肾组织Ⅰ型1,4,5-三磷酸肌醇受体(IP3R)蛋白及mRNA表达的影响。方法:50只SD大鼠分为5组,即正常对照组(NC组)、DN6周组(D6组)、DN12周组(D12组)、替米沙坦治疗6周组(T6组)和替米沙坦治疗12周组(T12组),各组10只。D6、T6、D12和T12组大鼠制作DN动物模型,造模成功后T6和T12组分别给予替米沙坦治疗6和12周。采用免疫组织化学SP法检测各组大鼠肾组织Ⅰ型ⅠP3R蛋白的表达,RT-PCR测定其mRNA的表达。结果:5组大鼠肾组织Ⅰ型IP3R蛋白和mRNA表达的比较,差异有统计学意义(F=42.321和59.482,P均<0.001);T6、D6、T12和D12组较NC升高,T6组较D6组下降,T12组较D12组下降(P均<0.05)。结论:替米沙坦可能通过降低Ⅰ型IP3R的表达治疗DN。

Participation of the inositol 1,4,5-trisphosphategated calcium channel in the zona pellucida- and progesterone-induced acrosome reaction and calcium influx in human spermatozoa

The acrosome reaction is a prerequisite for fertilization,and its signaling pathway has been investigated for decades.Regardless of the type of inducers present,the acrosome reaction is ultimately mediated by the elevation of cytosolic calcium.Inositol 1,4,5-trisphosphate-gated calcium channels are important components of the acrosome reaction signaling pathway and have been confirmed by several researchers.In this study,we used a novel permeabilization tool BioPORTER?and first demonstrated its effectiveness in spermatozoa.The inositol 1,4,5-trisphosphate type-1 receptor antibody was introduced into spermatozoa by BioPORTER?and significantly reduced the calcium influx and acrosome reaction induced by progesterone,solubilized zona pellucida,and the calcium ionophore A23187.This finding indicates that the inositol 1,4,5-trisphosphate type-1 receptor antibody is a valid inositol 1,4,5-trisphosphate receptor inhibitor and provides evidence of inositol 1,4,5-trisphosphate-gated calcium channel involvement in the acrosome reaction in human spermatozoa.Moreover,we demonstrated that the transfer of 1,4,5-trisphosphate into spermatozoa induced acrosome reactions,which provides more reliable evidence for this process.In addition,by treating the spermatozoa with inositol 1,4,5-trisphosphate/BioPORTER?in the presence or absence of calcium in the culture medium,we showed that the opening of inositol 1,4,5-trisphosphate-gated calcium channels led to extracellular calcium influx.This particular extracellular calcium influx may be the major process of the final step of the acrosome reaction signaling pathway.

Roles of protein kinase C and inositol1,4,5-trisphosphate in the pathogenesis of hypoxic-ischemic brain injury in neonatal rats

目的探讨第二信使蛋白激酶C和1,4,5-三磷酸肌醇在新生大鼠缺氧缺血性脑损伤(HIBI)中的作用.方法蛋白激酶C蛋白定量采用Lowry法,蛋白激酶C活性测定采用γ-32P分别掺入细胞浆、细胞膜特殊底物肽的催化活性测定法.1,4,5-三磷酸肌醇测定采用放射受体竞争结合法.结果与对照组相比,缺血缺氧20分钟后0、4、12、24、48、72小时、7、14天组新生鼠脑皮质,海马神经细胞膜蛋白激酶C活性降低,脑皮质神经细胞胞浆蛋白激酶C活性增高,海马神经细胞胞浆活性变化不大,上述改变在缺血缺氧后21天组基本恢复正常.与此同时,脑皮质、海马神经细胞1,4,5-三磷酸肌醇含量降低,丘脑含量升高.经统计学分析,细胞浆蛋白激酶C活性变化与1,4,5-三磷酸肌醇变化不相关.结论缺血缺氧可导致蛋白激酶C及第二信使三磷酸肌醇发生变化,二者在缺氧缺血性脑损伤的病理生理机制中发挥一定作用.

Temperature-dependence and conformational basis of inositol 1,4,5-trisphosphate receptor regulated by Ca^(2+)

The inositol 1,4,5-trisphosphate (lnsP3) receptor was purified from bovine cerebellum and reconstituted in liposomes composed of phosphatidylcholine (PC) and phosphatidylethanola-mine (PE) (1:1) successfully. No effect of Ca2+ concentration on [3H]-lnsP3 binding to unreconsti-tuted lnsP3 receptor could be observed either at 4℃ or at 25℃, whereas the effect of [Ca2+] on reconstituted lnsP3 receptor depended on the temperature. The Ca2+ concentration outside the proteolipsome ([Ca2+]o) had no detectable effect on lnsP3 binding to lnsP3 receptor at 4℃. In contrast, with increase of [Ca2+]o from 0 to 100 nmol/L at 25℃, the lnsP3 binding activity increased gradually. Then the lnsP3 binding activity was decreased drastically at higher [Ca2+]0 and inhibited entirely at 50 nmol/L [Ca2+]. Conformational studies on intrinsic fluorescence of the reconstituted lnsP3 receptor and its quenching by Kl and HB indicated that the global conformation of reconstituted lnsP3 receptor could not be affected by [Ca2+]o at 4℃. While at 25℃, the effects of 10μmol/L [Ca2+]0 on global, membrane and cytoplasmic conformation of the reconstituted lnsP3 receptor were different significantly from that of 100 nmol/L [Ca2+]0.