间隙连接分子Cx43相关蛋白及其功能研究进展

间隙连接是细胞间直接进行信息交流的唯一膜通道结构。Cx43是构成间隙连接中分布最广、研究最多的间隙连接分子,目前运用免疫共沉淀、免疫荧光共定位、pull-down以及酵母双杂交等多种方法研究发现了众多的Cx43相关蛋白。这些蛋白通过与Cx43相互作用在间隙连接蛋白的组装、运输、膜定位,间隙连接通道的形成以及对间隙连接通讯的调控等一系列过程中均发挥十分重要的作用。本文就目前已经研究发现的Cx43相关蛋白及其最新的功能研究进展进行综述。     

Connexin31 相互作用蛋白筛选、证实与功能研究

筛选间隙连接蛋白 31 (connexin31 , Cx31) 相互作用蛋白并研究其在 Cx31 运输中的功能 . 运用制备的抗 Cx31 多克隆抗体免疫沉淀, SDS- 聚丙烯酰胺凝胶电泳分离,蛋白质条带回收,蛋白质胶块酶解,电喷雾 - 四极杆 - 飞行时间质谱分析,数据库扫描筛选可能相互作用蛋白,可能互作蛋白经免疫共沉淀、细胞免疫共定位等证实,确定 actin 为 Cx31 相互作用蛋白 . 用药物处理细胞,抑制 actin 的功能,观察 Cx31 定位与间隙连接通道的通透性,确定 actin 在 Cx31 运输中的功能 . 当药物抑制 actin 的功能时, Cx31 很少能到达细胞膜上形成间隙连接通道, Cx31 主要分布在胞质中;当药物抑制 tublin 的功能时, Cx31 能到达细胞膜上形成间隙连接通道,细胞免疫荧光实验显示间隙连接斑有增多的现象,但染料转移实验表明细胞膜上间隙连接通道并没有增加 . Actin 在 Cx31 运输至细胞膜上形成间隙连接通道的过程中具有重要作用 .     

间隙连接功能多样性:连接蛋白基因敲除研究

编码细胞间间隙连接通道结构蛋白的基因是称为连接蛋白(connexin,Cx)基因的多基因家族;间隙连接蛋白基因有20种,且多数细胞同时表达多种连接蛋白,其功能研究较为复杂;小鼠7种连接蛋白基因的敲除为研究连接蛋白功能多样性提供了良好模型,并揭示了不同连接蛋白在维持不同组织正常发育和代谢中的重要作用.     

背景氯离子通道研究进展

综述了目前了解得最为充分的一类电压门控氯通道——背景氯通道,内容涉及选择性、门控和药理学以及通道蛋白的克隆和分子结构.氯通道广泛存在于细胞膜和细胞器膜,作为“总管家”参与细胞pH,体积,静息膜电位和兴奋性等多种细胞过程的调节.由于种种原因,对氯通道的研究起步较晚.目前应用膜片钳和分子生物学技术对氯通道结构功能的研究已经成为一个热点.     

间隙连接蛋白43的功能及温度响应性研究进展

在各种组织和器官中都存在允许相邻细胞的胞质区之间直接通讯的间隙连接,它们在广泛的生理过程中起关键作用。间隙连接是细胞间通道,由间隙连接蛋白组成,其中间隙连接蛋白43(Cx43)在各组织器官中广泛表达。研究发现细胞间隙连接通讯会受到冷热刺激的影响,并与Cx43表达相关。本篇综述主要介绍Cx43转录与翻译水平的调控以及它的降解途径,并对冷热刺激后Cx43表达变化的作用机制进行概述。     

间隙连接蛋白Cx43在人胚肺和肺癌细胞表达的研究

细胞与细胞之间通过细胞膜上的间隙连接通道交换小分子和离子进行细胞间通讯,对细胞增殖分化调控和机体内环境稳定有重要作用。用间隙连接蛋白Ｃｘ４３ｃＤＮＡ探针Ｎｏｒｔｈｅｒｎ印迹杂交,Ｃｘ４３抗体免疫荧光染色和罗氏黄荧光染料传输方法检查,正常人胚肺细胞的Ｃｘ４３在ｍＲＮＡ和蛋白水平有高表达,Ｃｘ４３蛋白免疫荧光分布在间隙连接的部位,细胞间隙连接通讯功能明显。与正常相反,人肺癌ＰＧ系细胞Ｃｋ４３无论在ｍＲＮＡ或蛋白质水平都无表达,细胞通讯功能缺陷。结果表明Ｃｘ４３在培养的人胚肺细胞有功能性表达。人肺癌ＰＧ细胞通讯功能缺陷与Ｃｘ４３基因转录抑制有关。对Ｃｘ基因的抑癌基因性质进行讨论。     

TRP通道与信号转导

TRP(transient receptor potential)通道是一类在外周和中枢神经系统分布很广泛的通道蛋白.到目前为止,有超过30个TRP通道家族成员在哺乳动物中被克隆.TRP通道均为六次跨膜蛋白,其N末端和C末端均在胞内,由第五和第六跨膜结构域共同构成非选择性阳离子孔道.这些通道可被许多种因素调节,包括温度、渗透压、pH值、机械力,以及一些内、外源性配体和细胞内信号分子.TRP通道家族包含七个亚族.目前,它们最公认的功能是介导感觉信号的传递,其他功能包括调节细胞钙平衡和影响发育等.     

影响间隙连接的形成及其通透性的因素

细胞内、外液Ca~(2 )及其他离子成份和pH变化可改变已建立的连接通道的通透性。在激素、维生素、酶、神经递质和细胞内信使中对间隙连接有作用的有数十种。代谢抑制物、药物、化学试剂和肿瘤促进剂中,已观察到有50余种有作用。其他因素,如温度、电压、抗体等,亦对间隙连接有影响。     

间隙连接蛋白43(Cx43)在皮肤伤口愈合中的作用

间隙连接(gap junction,GJ)是细胞膜上的通道结构,其介导的细胞间间隙连接通讯(gap junction intercellular communication,GJIC)对内环境的稳定、细胞生长调控及新陈代谢等起到重要的作用。间隙连接蛋白43(connexin43,Cx43)是哺乳动物细胞中分布最为广泛的间隙连接蛋白,越来越多的研究发现皮肤创伤后Cx43的表达会随着伤口愈合的过程发生动态变化,并影响伤口愈合的速率和质量,人为调控Cx43的表达水平会改善伤口愈合的速率和质量。主要就Cx43结构与功能、Cx43的水平对伤口愈合各阶段的影响及Cx43与慢性伤口的关系进行总结,以期为探索皮肤创伤,尤其是慢性伤口治疗新途径提供参考价值。     

神经系统内水通道蛋白4的功能研究进展

水通道蛋白4 (aquaporin 4,AQP4) 是中枢神经系统内重要的水通道蛋白之一,除了在海马、视上核和室旁核等部位的少数神经元上有分布外,主要表达在星形胶质细胞和室管膜上皮细胞中.近期的研究发现,AQP4除了参与脑脊液(cerebrospinal fluid,CSF)分泌、吸收等中枢神经系统内水代谢平衡的调节外,还有许多令人感兴趣的功能表现.AQP4能够影响星形胶质细胞的迁移和胶质疤痕的愈合;影响神经信号的传导;还能够调节星形胶质细胞对K 和谷氨酸的重摄取;改变神经元神经递质的释放;参与突触以及细胞间隙连接的形成等.上述发现表明AQP4不仅是影响中枢神经系统内水和电解质平衡的关键因素,而且是决定星形胶质细胞结构功能的重要分子基础之一.因此AQP4为众多脑疾病的治疗提供具有重要价值的潜在药物作用靶点,调控AQP4的表达与功能将成为治疗许多神经系统疾病的新策略.     

Chemical gating of gap junction channels

Chemical gating of gap junction channels is a complex phenomenon that may involve intra- and intermolecular interactions among connexin domains and a cytosolic molecule (calmodulin?) that may function as channel plug. This article focuses on the methodology we have employed for studying the molecular basis of chemical gating by lowered cytosolic pH. Our approach has combined molecular genetics and biophysics, using exposure to 100% CO(2) for assaying chemical gating efficiency. Chimeras of connexin 32 (Cx32) and connexin 38 (Cx38) and Cx32 mutants modified at residues of the cytoplasmic loop, the initial C-terminus domain, or both have been expressed in Xenopus oocytes, and channel expression and gating have been tested electrophysiologically by double voltage clamp. In addition, various channel forms, including homotypic, heterotypic, and heteromeric channel combinations, have been evaluated for chemical gating sensitivity.     

Size and selectivity of gap junction channels formed from different connexins

Gap junction channels have long been viewed as static structures containing a large-diameter, aqueous pore. This pore has a high permeability to hydrophilic molecules of 900 daltons in molecular weight and a weak ionic selectivity. The evidence leading to these conclusions is reviewed in the context of more recent observations primarily coming from unitary channel recordings from transfected connexin channels expressed in communication-deficient cell lines. What is emerging is a more diverse view of connexin-specific gap junction channel structure and function where electrical conductance, ionic selectivity, and dye permeability vary by one full order of magnitude or more. Furthermore, the often held contention that channel conductance and ionic or molecular selectivity are inversely proportional is refuted by recent evidence from five distinct connexin channels. The molecular basis for this diversity of channel function remains to be identified for the connexin family of gap junction proteins.     

Gating properties of connexin32 cell-cell channels and their mutants expressed in Xenopus oocytes.

Carboxyl-terminal deletion mutants of the gap junction protein connexin32 were tested in the oocyte cell-cell channel assay. Oocytes expressing a mutant lacking 58 carboxyl terminal amino acids were found to exhibit junctional conductances of the same magnitude as oocytes expressing wild-type connexin32. The gating properties of the channels formed by this mutant of connexin32 with respect to transjunctional voltage and cytoplasmic acidification are indistinguishable from those found with wild-type connexin32 channels. This includes a novel pH-dependent voltage gate. In another mutant, two carboxyl terminal serine residues, Ser233 and Ser240, were replaced by Asn residues. This double mutant has properties indistinguishable from wild-type connexin32, suggesting that phosphorylation of either of these serines is not required for channel opening.     

Structure and closure of connexin gap junction channels

Connexin gap junctions comprise assembled channels penetrating two plasma membranes for which gating regulation is associated with a variety of factors, including voltage, pH, Ca²⁺, and phosphorylation. Functional studies have established that various parts of the connexin peptides are related to channel closure and electrophysiology studies have provided several working models for channel gating. The corresponding structural models supporting these findings, however, are not sufficient because only small numbers of closed connexin structures have been reported. To fully understand the gating mechanisms, the channels should be visualized in both the open and closed states. Electron crystallography and X-ray crystallography studies recently revealed three-dimensional structures of connexin channels in a couple of states in which the main difference is the conformation of the N-terminal domain, which have helped to clarify the structure in regard to channel closure. Here the closure models for connexin gap junction channels inferred from structural and functional studies are described in the context of each domain of the connexin protein associated with gating modulation.     

Two-dimensional kinetics of inter-connexin interactions from single-molecule force spectroscopy

Gap junction channels are intercellular channels that form by docking the extracellular loops of connexin protein subunits. While the structure and function of gap junctions as intercellular channels have been characterized using different techniques, the physics of the inter-connexin interaction remain unknown. Moreover, as far as we know, the capacity of gap junction channels to work as adhesion complexes supporting pulling forces has not yet been quantitatively addressed. We report the first quantitative characterization of the kinetics and binding strength of the interaction of a short peptide mimicking extracellular loop 2 of Cx26 with membrane-reconstituted Cx26, combining the imaging and force spectroscopy capabilities of atomic force microscopy. The fast dissociation rate inferred a dynamic bond, while the slow association rate reflected the reduced flexibility and small size of extracellular loops. Our results propose the gap junction channel as an adhesion complex that associates slowly and dissociates fast at low force but is able to support important pulling forces in its native, hexameric form.     

Connexin phosphorylation as a regulatory event linked to gap junction internalization and degradation

Gap junction proteins, connexins, are dynamic polytopic membrane proteins that exhibit unprecedented short half-lives of only a few hours. Consequently, it is well accepted that in addition to channel gating, gap junctional intercellular communication is regulated by connexin biosynthesis, transport and assembly as well as the formation and removal of gap junctions from the cell surface. At least nine members of the 20-member connexin family are known to be phosphorylated en route or during their assembly into gap junctions. For some connexins, notably Cx43, evidence exists that phosphorylation may trigger its internalization and degradation. In recent years it has become apparent that the mechanisms underlying the regulation of connexin turnover are quite complex with the identification of many connexin binding molecules, a multiplicity of protein kinases that phosphorylate connexins and the involvement of both lysosomal and proteasomal pathways in degrading connexins. This paper will review the evidence that connexin phosphorylation regulates, stimulates or triggers gap junction disassembly, internalization and degradation.     

Gap junction channels: distinct voltage-sensitive and -insensitive conductance states.

All mammalian gap junction channels are sensitive to the voltage difference imposed across the junctional membrane, and parameters of voltage sensitivity have been shown to vary according to the gap junction protein that is expressed. For connexin43, the major gap junction protein in the cardiovascular system, in the uterus, and between glial cells in brain, voltage clamp studies have shown that transjunctional voltages (Vj) exceeding +/- 50 mV reduce junctional conductance (gj). However, substantial gj remains at even very large Vj values; this residual voltage-insensitive conductance has been termed gmin. We have explored the mechanism underlying gmin using several cell types in which connexin43 is endogenously expressed as well as in communication-deficient hepatoma cells transfected with cDNA encoding human connexin43. For pairs of transfectants exhibiting series resistance-corrected maximal gj (gmax) values ranging from < 2 to > 90 nS, the ratio gmin/gmax was found to be relatively constant (about 0.4-0.5), indicating that the channels responsible for the voltage-sensitive and -insensitive components of gj are not independent. Single channel studies further revealed that different channel sizes comprise the voltage-sensitive and -insensitive components, and that the open times of the larger, more voltage-sensitive conductance events declined to values near zero at large voltages, despite the high gmin. We conclude that the voltage-insensitive component of gj is ascribable to a voltage-insensitive substate of connexin43 channels rather than to the presence of multiple types of channels in the junctional membrane. These studies thus demonstrate that for certain gap junction channels, closure in response to specific stimuli may be graded, rather than all-or-none.     

Connexin phosphorylation as a regulatory event linked to gap junction internalization and degradation

Gap junction proteins, connexins, are dynamic polytopic membrane proteins that exhibit unprecedented short half-lives of only a few hours. Consequently, it is well accepted that in addition to channel gating, gap junctional intercellular communication is regulated by connexin biosynthesis, transport and assembly as well as the formation and removal of gap junctions from the cell surface. At least nine members of the 20-member connexin family are known to be phosphorylated en route or during their assembly into gap junctions. For some connexins, notably Cx43, evidence exists that phosphorylation may trigger its internalization and degradation. In recent years it has become apparent that the mechanisms underlying the regulation of connexin turnover are quite complex with the identification of many connexin binding molecules, a multiplicity of protein kinases that phosphorylate connexins and the involvement of both lysosomal and proteasomal pathways in degrading connexins. This paper will review the evidence that connexin phosphorylation regulates, stimulates or triggers gap junction disassembly, internalization and degradation.     

Calmodulin directly gates gap junction channels

Cytosolic changes control gap junction channel gating via poorly understood mechanisms. In the past two decades calmodulin participation in gating has been suggested, but compelling evidence for it has been lacking. Here we show that calmodulin indeed is associated with gap junctions and plays a direct role in chemical gating. Expression of a calmodulin mutant with the N-terminal EF hand pair replaced by a copy of the C-terminal pair dramatically increases the chemical gating sensitivity of gap junction channels composed of connexin 32 and decreases their sensitivity to transjunctional voltage. The increased chemical gating sensitivity, most likely because of the higher overall Ca(2+) binding affinity of this mutant as compared with native calmodulin, and the decreased voltage sensitivity are only observed when the mutant is expressed before connexin 32. This indicates that the mutant, and by extension native calmodulin, must interact with connexin 32 before gap junctions are formed. Immunofluorescence data suggest further that this interaction leads to incorporation of native or mutant calmodulin into the connexon as an integral regulatory subunit.