异型缝隙连接通道和磷酸化对心脏细胞通讯的调节作用

为了检测缝隙连接蛋白 (Cx) 4 3和Cx4 5组成的多种异型缝隙连接通道和磷酸化对缝隙连接 (GJ)里细胞通讯的调节作用 ,将转染了编码为Cx4 3或Cx4 5的DNA后的Hela细胞放置在一起共同培养组成双侧和单侧异型GJ通道。显微注射荧光素黄 (LY)后 ,利用紫外光检测经 2 0 0nmol/L十四 (烷 )酰佛波醇乙酸酯 (TPA)处理前后由Cx4 3和Cx4 5所组成的多种异型GJ通道对荧光染料的偶联率。结果 :在不同的GJ中 ,同型GJ通道Cx4 3(HoCx4 3)偶联率最高。单侧异型GJ通道Cx4 3(MH4 3)和单侧异型GJ通道Cx4 5 (MH4 5 )的偶联率较之相关的HoCx4 3和同型GJ通道Cx4 5 (HoCx4 5 )低。从Cx4 5侧注入荧光染料的MH4 5偶联率较之从Cx4 3/ 4 5侧注射的MH4 5、双侧异型GJ通道Cx4 3/4 5 (BH4 3/ 4 5 )和HoCx4 5等的偶联率都低。TPA处理后HOCx4 3的偶联率降低 ,而当Cx4 3和Cx4 5组合成异型BH4 3/4 5和MH4 3通道其偶联率下降更显著。结论 :Cx4 3和Cx4 5共同表达可构成BH4 3/ 4 5、MH4 3和MH4 5等通道 ,而这些异型GJ通道可降低细胞间通信和对磷酸化的敏感性。有关MH4 5通道 ,其偶联率大小决定于荧光染料的注射方向 ,此可能与心律失常再折返的解剖学的机制有关。     

异型缝隙连接通道和磷酸化对心脏缝隙连接通透性的影响

缝隙连接 (GJ)是连接相邻细胞的通道 ,它由细胞间的缝隙连接蛋白 (Cx)构成。这些通道在体内参与了生物信息传播等重要的生理过程。由不同种Cx组成的GJ通道明显不同于由单种Cx组成的同型GJ通道的通透特性。Cx密度和分布的改变与心律失常相关。磷酸化作用可使GJ通道尤其是异型GJ通道的通透性下降。     

异型缝隙连接通道对心脏电生理功能的调控作用

由多种不同的缝隙连接蛋白(Cx)组成的异型缝隙连接(GJ)通道的电生理``特性不同于由单一种Cx组成的同型GJ通道,前者对心脏细胞间通信的调节作用也有别于后者。在病理情况下,由于心脏内Cx的再分布所致的异型GJ通道的电生理特性改变可诱发心律失常的发生。     

缝隙连接蛋白43与心肌缺血再灌注心律失常的研究进展

近年来,心肌缺血再灌注损伤(myocardial ischemic-reperfusion injury,MIRI)的治疗和预防是心血管领域新兴的研究课题之一。缝隙连接(gap junction,GJ)是心脏电生理的基本结构。缝隙连接蛋白(connexin,Cx)是缝隙连接的基本单位。缝隙连接蛋白43(connexin 43,Cx43)是心脏Cx家族中最丰富的成员,Cx43的正常表达对于心脏发育、电耦联的心肌细胞活性和心肌功能的协调至关重要。Cx43与MIRI之间的关系已成为当前研究的重点。该文就Cx43与心肌缺血再灌注心律失常的关系作一综述。     

心肌缝隙连接蛋白43与心血管疾病的关系

心脏缝隙连接（gap junction,GJ）是细胞间进行电化学偶联及信息交换的重要途径,是介导心脏维持正常的协调性和同步性功能的重要桥梁及特殊通道。GJ主要由连接蛋白组成,而在心肌细胞水平上主要包括连接蛋白（connexin,Cx）43、Cx40和Cx45,但各自在心脏部位的表达分布和功能上却有所不同。现已有研究报道分析在许多心血管疾病中,GJ在结构和功能上的改变或异常与主要心血管事件的发生有直接关联。Cx含量及分布的减少、GJ调控因素的影响及GJ的侧一侧连接导致传导异向性变化等,均可能使心脏整体功能活动不同程度地下降或受到抑制,从而引起心脏疾病的发生和发展。     

缝隙连接、连接蛋白43及其与心律失常的关系

缝隙连接(GJ)通道是介导心肌细胞间电化学信息交流,保证心脏整体活动的协调性和同步性的特殊通道。在致心律失常发生上,GJ通道介导的细胞间电耦联障碍甚至比膜离子通道功能紊乱起了更重要的作用。连接蛋白43(Cx43)是心室GJ通道的主要构成成份,其表达和分布的异常将导致心室肌细胞的整体性异常,从而传导速度和传导各向异性发生改变,产生折返和传导阻滞。以GJ通道为作用靶点的新一代抗心律失常药的出现将为心律失常的治疗带来新的希望。     

缝隙连接与高血压

<正>缝隙连接(gap junction,GJ),又称间隙连接、通讯连接,是由连接相邻两个细胞之间的连接通道排列而成的一种特殊膜结构。细胞间的通讯方式可分为间接与直接方式。相邻细胞间通过GJ所介导的细胞间的通讯为直接通讯,又称缝隙连接细胞间通讯(gap junction intercellular communication,GJIC)。相邻细胞通过直接通讯进行信息、能量和物质交换,     

缝隙连接蛋白与心律失常的相关性研究进展

以往认为心律失常的发生主要与心肌细胞兴奋性异常有关但近年来的研究表明细胞间电耦联障碍是心律失常的另一个重要原因之一,其所起的致心律失常作用甚至大于兴奋性异常。由心肌缝隙连接蛋白(connexin,Cx)构成的缝隙连接(gapjunction,GJ)通道是心肌细胞之间的一种特殊连接通道,     

脑利钠肽对新生大鼠心室肌细胞缝隙连接蛋白Cx43含量的影响

观察脑利钠肽(BNP)对缝隙连接蛋白Cx43含量的影响,探讨BNP对心室肌细胞电生理特性的直接效应。以培养新生大鼠心室肌细胞为实验模型,分为实验组和对照组,实验组以不同浓度(1×10-8,1×10-7,1×10-6mol/L)的BNP处理心肌细胞24h后,通过免疫印迹法(Westernblot)反映心肌细胞Cx43总蛋白含量变化;通过免疫组化法在激光共聚焦显微镜下观察GJ通道的密度变化。结果:Westernblot显示BNP处理组和对照组比较Cx43条带显色密度减弱并有统计学意义(P<0.01),且随着BNP的剂量越大,Cx43条带密度减弱越明显。在共聚焦显微镜下观察到的Cx43的特异性荧光经过强度定量分析后得出实验组GJ通道Cx43密度比对照组减弱(P<0.01)。结论:一定浓度的BNP可使GJ蛋白Cx43的含量下调。BNP有可能参予心室肌细胞GJ的重构,从而在“致心律失常基质”的形成中起作用。     

连接蛋白、缝隙连接的结构功能及其与心血管疾病

1　引言细胞间的通讯方式可分为间接与直接方式。以体循环远程分泌、旁分泌或自分泌方式经第二信使途径完成一系列生理、生化功能的调节方式称为间接通讯 ;而以细胞间的缝隙连接 (gap junction)为途径进行的细胞间直接的信息交流 ,称为直接通讯 ,又称缝隙连接细胞间通讯 (gap junction inter- cellularcommunication/GJIC)。2　缝隙连接的组成、分布与结构[1]缝隙连接由相邻细胞膜上的两个连接子 (con-nexon)相互锚定组成 ,而连接子是一个六聚体 ,由六个亚单位 -连接蛋白 (connexin/Cx)组成。连接蛋白是由十余个成员组成的一个保守大家族 …     

Cardiac gap junction channels show quantitative differences in selectivity

Several proteins including connexin40 (Cx40) and connexin43 (Cx43) form gap junctions between cells of the heart; they may be found separately or may be coexpressed. These connexins form channels with differing conductance and permeability properties. Cx40 and Cx43 are each required for normal electrical conduction between cells in different regions of the heart. We hypothesized that the major difference between these connexins might be in their selective intercellular passage of small molecules such as second messengers, which can be assessed using biologically inert fluorescent probes. Therefore, we designed experimental paradigms to quantitate the permeability properties of these cardiac connexins using simultaneous measurement of junctional conductance (g(j)) by the double whole-cell patch-clamp technique and intercellular transfer of Lucifer Yellow (LY) by fluorescence microscopy. These studies were performed in HeLa cells stably transfected with Cx40 or Cx43 or cotransfected with both connexins. We found that homotypic Cx43 channels were about 5 times more permeable to LY than homotypic Cx40 channels (flux of approximately 1560 versus approximately 300 molecules/channel per second). Channels between heterotypic (Cx40-Cx43) cell pairs and between pairs of coexpressing cells exhibited intermediate LY permeability. The permeability ratio for LY relative to monovalent cation (K+) ranged from 0.0025 for Cx40 to 0.028 for Cx43. These permeability ratios suggest that the connexins are highly selective for solutes in the size and charge range of many second messengers. Moreover, the data indicate that coexpression of connexins does not generate unique permeability characteristics, but rather results in an intermediate permeability for solutes involved in metabolic/biochemical coupling.     

Formation of heterotypic gap junction channels by connexins 40 and 43

Gap junctions formed between transfected cells expressing connexin (Cx) 40 and Cx43 (Cx43-RIN, Cx40-HeLa, and Cx43-HeLa) revealed a relationship, g(j)=f(V(j)), at steady state, that is typified by a nonsymmetrical behavior similar to that previously reported for other heterotypic channels (gap junction conductance [g(j)]; transjunctional voltage [V(j)]). The unitary conductance of the channels was sensitive to the polarity of V(j). A main state conductance of 61 pS was found when the Cx43 cell was stepped positively or the Cx40 cell negatively (V(j)=70 mV); the reverse polarities yielded a conductance of 100 pS. These heterotypic channels were permeable to carboxyfluorescein. In addition, two other heterotypic forms are illustrated to demonstrate that endogenous Cx45 expression cannot explain the results. The demonstration of heterotypic Cx40-Cx43 channels may have implications for the propagation of the electrical impulse in heart. For example, they may contribute to the slowing of the impulse propagation through the junctions between Purkinje fibers and ventricular muscle.     

Selectivity of connexin 43 channels is regulated through protein kinase C-dependent phosphorylation

Coordinated contractile activation of the heart and resistance to ischemic injury depend, in part, on the intercellular communication mediated by Cx43-composed gap junctions. The function of these junctions is regulated at multiple levels (assembly to degradation) through phosphorylation at specific sites in the carboxyl terminus (CT) of the Cx43 protein. We show here that the selective permeability of Cx43 junctions is regulated through protein kinase C (PKC)-dependent phosphorylation at serine 368 (S368). Selective permeability was measured in several Cx43-expressing cell lines as the rate constant for intercellular dye diffusion relative to junctional conductance. The selective permeability of Cx43 junctions under control conditions was quite variable, as was the open-state behavior of the comprising channels. Coexpression of the CT of Cx43 as a distinct protein, treatment with a PKC inhibitor, or mutation of S368 to alanine, all reduced (or eliminated) phosphorylation at S368, reduced the incidence of 55- to 70-pS channels, and reduced by 10-fold the selective permeability of the junctions for a small cationic dye. Because PKC activation during preischemic conditioning is cardioprotective during subsequent ischemic episodes, we examined no-flow, ischemic hearts for Cx43 phosphorylated at S368 (pS368). Consistent with early activation of PKC, pS368-Cx43 was increased in ischemic hearts; despite extensive lateralization of total Cx43, pS368-Cx43 remained predominantly at intercalated disks. Our data suggest that the selectivity of gap junction channels at intercalated disks is increased early in ischemia.     

Connexin43 and connexin45 form heteromeric gap junction channels in which individual components determine permeability and regulation

Two gap junction proteins, connexin43 (Cx43) and connexin45 (Cx45), are coexpressed in many cardiac and other cells. Homomeric channels formed by these proteins differ in unitary conductance, permeability, and regulation. We sought to determine the ability of Cx43 and Cx45 to oligomerize with each other to form heteromeric gap junction channels and to determine the functional and regulatory properties of these heteromeric channels. HeLa cells were transfected with Cx45 or (His)(6)-tagged Cx43 or sequentially transfected with both connexins. Immunoblots verified production of the transfected connexins, and immunofluorescence demonstrated that they were colocalized in the HeLa-Cx43(His)(6)/Cx45 cells. Connexons were solubilized from HeLa-Cx43(His)(6)/Cx45 cells by using Triton X-100 and were applied to a Ni(2+)-NTA column, which binds the His(6) sequence. Cx45 was coeluted from the column with Cx43(His)(6), demonstrating that some hemichannels contain both connexins. Single-channel recordings showed that the HeLa-Cx43(His)(6)/Cx45 cells exhibited single-channel conductances that were not observed in cells expressing either connexin alone. Dye-coupling experiments showed that HeLa-Cx43(His)(6) cells readily passed Lucifer yellow and N-(2-aminoethyl)biotinamide hydrochloride (neurobiotin); in contrast, HeLa-Cx45 and HeLa-Cx43(His)(6)/Cx45 cells showed extensive intercellular passage of neurobiotin but little coupling with Lucifer yellow. Treatment with the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate reduced junctional conductance in cells expressing Cx43, Cx45, or both connexins, but it reduced the extent of neurobiotin transfer only in HeLa-Cx43(His)(6) and HeLa-Cx43(His)(6)/Cx45 cells but not in the HeLa-Cx45 cells. Thus, biochemical and electrophysiological evidence suggests that Cx43 and Cx45 extensively mix to form heteromeric channels; however, individual connexin components dominate aspects of the physiological behavior of these channels.     

Relating specific connexin co-expression ratio to connexon composition and gap junction function

Cardiac connexin 43 (Cx43), Cx40 and Cx45 are co-expressed at distinct ratios in myocytes. This pattern is considered a key factor in regulating the gap junction channels composition, properties and function and remains poorly understood.This work aims to correlate gap junction function with the connexin composition of the channels at accurate ratios Cx43:Cx40 and Cx43:Cx45.Rat liver epithelial cells that endogenously express Cx43 were stably transfected to induce expression of accurate levels of Cx40 or Cx45 that may be present in various areas of the heart (e.g. atria and ventricular conduction system). Induction of Cx40 does not increase the amounts of junctional connexins (Cx43 and Cx40), whereas induction of Cx45 increases the amounts of junctional connexins (Cx43 and Cx45). Interestingly, the non-junctional fraction of Cx43 remains unaffected upon induction of Cx40 and Cx45. Co-immunoprecipitation studies show low level of Cx40/Cx43 heteromerisation and undetectable Cx45/Cx43 heteromerisation. Functional characterisation shows that induction of Cx40 and Cx45 decreases Lucifer Yellow transfer. Electrical coupling is decreased by Cx45 induction, whereas it is decreased at low induction of Cx40 and increased at high induction.These data indicate a fine regulation of the gap junction channel make-up in function of the type and the ratio of co-expressed Cxs that specifically regulates chemical and electrical coupling. This reflects specific gap junction function in regulating impulse propagation in the healthy heart, and a pro-arrhythmic potential of connexin remodelling in the diseased heart.     

Connexin 43 and ischemic preconditioning

Connexin 43 (Cx43) is the essential protein to form hemichannels and gap junctions in the myocardium. The phosphorylation status of Cx43 which is regulated by a variety of protein kinases and phosphatases determines hemichannel and/or gap junction conductance and permeability. Gap junctions are involved in cell-cell coupling while hemichannels contribute to cardiomyocyte volume regulation. Cx43-formed channels are involved in ischemia/reperfusion injury, since blockade of a large portion of Cx43-formed channels attenuates ischemic hypercontracture, infarct development and post myocardial infarction remodeling. Ischemic preconditioning's protection also depends on functional Cx43-formed channels, since uncoupling of channels or genetic Cx43 deficiency abolishes infarct size reduction by ischemic preconditioning. The exact underlying mechanism(s) how Cx43 mediates protection remain to be established.     

17beta-estradiol reduces the effect of metabolic inhibition on gap junction intercellular communication in rat cardiomyocytes via the estrogen receptor

The effects of 17beta-estradiol (E2) on gap junction intercellular communication (GJIC) were assessed by Lucifer yellow dye coupling in cultured neonatal rat cardiomyocytes after metabolic inhibition (MI) using potassium cyanide and sodium iodoacetate. MI significantly reduced dye coupling of cardiomyocytes to 8.5% +/- 0.6% of control levels, and pretreatment with E2, but not its inactive isomer 17alpha-estradiol, dose-dependently (EC(50) = 0.41 microM) increased the dye coupling up to 76% +/- 15% of control levels. The effect of E2 on MI-induced dye uncoupling was abolished by tamoxifen, a potent estrogen receptor (ER) antagonist. The ligand, E2-BSA-FITC, labeled the cardiomyocyte surface, whereas BSA-FITC did not, suggesting the presence of membrane-associated E2 receptors. Double immunofluorescence microscopy showed that MI-induced the accumulation of non-phosphorylated Cx43 at the gap junction and that this was prevented by E2 pretreatment. Labeling of Lucifer yellow-microinjected cardiomyocytes with antibodies specific for Ser368-phosphorylated Cx43 (Ser368Cx43) or non-phosphorylated Cx43 confirmed that E2 reduced the MI-induced inhibition of dye coupling and accumulation of non-phosphorylated Cx43 concomitant with the reappearance of Ser368Cx43 at the gap junction. MI caused a decrease in Ser368Cx43 protein levels, and pretreatment with E2 significantly increased the levels of Ser368Cx43. Inhibition of protein kinase C (PKC) with chelerythrine blocked the E2-induced increase of Ser368Cx43 levels in MI-treated cardiomyocytes. These results suggest that E2 attenuates the inhibitory effect of MI on GJIC in cardiomyocytes by affecting the phosphorylation of Cx43, possibly mediated by activation of PKC via a membrane-associated signaling mechanism.     

Alterations of intercellular communication in neonatal cardiac myocytes from connexin43 null mice

Objective: To compare gap junction expression and intercellular coupling in wildtype neonatal cardiac myocytes to those from mice lacking the most abundant cardiac gap junction protein (connexin43, Cx43). Methods: Northern and Western blots compared connexin mRNA and protein levels, immunocytochemistry evaluated connexin distribution in neonatal Cx43 null(-/-), heterozygous(+/-) and wildtype(+/+) mouse hearts. Ca(2+) imaging, dye coupling and electrophysiological methods evaluated intercellular communication. Results: Similar levels of Cx40 and Cx45 were detected in all genotypes, although in adult cardiac tissue from wildtype mice, Cx43 expression was higher than in heterozygotes. After culturing dissociated cells for 3-4 days, cardiocytes beat spontaneously; in Cx43(+/+) and (+/-) cultures, the beating was generally quite synchronous. In Cx43(-/-) mice, interbeat intervals were on average twice as long and more variable than in Cx43(+/+) or Cx43(+/-) cultures. Junctional conductance was lower by about 60% in Cx43(-/-) as compared to Cx43(+/-) and (+/+) littermates; Lucifer Yellow dye coupling was virtually absent in Cx43(-/-) cardiomyocytes but was comparably strong in wildtype and heterozygous siblings. Macroscopic junctional conductance measurements on Cx43(-/-) cardiocytes showed slightly stronger voltage sensitivity in these cells than in Cx43(+/+) cardiocytes. Unitary junctional conductance measurements revealed distinct populations of channels contributing to macroscopic conductance for Cx43(+/+) and Cx43(-/-) genotypes. Conclusions: In Cx43-deficient cardiac myocytes, the expression of other connexins only partially compensates for the functional loss, with dye coupling and spontaneous beating being strongly impaired.     

Coupling asymmetry of heterotypic connexin 45/ connexin 43-EGFP gap junctions: properties of fast and slow gating mechanisms

Although fast and slow gating mechanisms have been described in gap junctions (GJs), their relative contributions to dependence on transjunctional voltage, V(j), is still unclear. We used cell lines expressing wild-type connexin 45 (Cx45) and connexin 43 fused with enhanced green fluorescent protein (Cx43-EGFP) to examine mechanisms of gating in homo- and heterotypic GJs formed of these connexins. Macroscopically Cx45/Cx45 channels show high sensitivity to V(j). Cx45 channels demonstrate two types of gating: fast transitions between open and residual states and slow transitions between open and completely closed states. Single-channel conductance of the Cx45 channel is approximately 32 pS for the open state and approximately 4 pS for the residual state. Cx45/Cx43-EGFP heterotypic junctions exhibit very asymmetrical V(j) gating with the maximum junctional conductance shifted to V(j) positive on the Cx45 side. Conductance of single Cx45/Cx43-EGFP channels is approximately 55 pS for the open state and approximately 4 pS for the residual state, values consistent with the simple-series connection of Cx45 and Cx43-EGFP hemichannels. At V(j) = 0, the slow gate of many Cx45 hemichannels is closed in both homotypic Cx45/Cx45 and heterotypic Cx45/Cx43-EGFP junctions. Fast and slow V(j) gates of both Cx45 and Cx43 hemichannels close for relative negativity at their cytoplasmic end. Coupling mediated by Cx45/Cx43-EGFP junctions can exhibit asymmetry that can be strongly modulated by small changes in difference of holding potentials. Cx45/Cx43 junctions are likely to be found in brain and heart and may mediate rectifying electrical transmission or modulatable chemical communication.