双孔通道结构及激活机制的研究进展

双孔通道(two-pore channels, TPCs)是一类位于内溶酶体膜上的非选择性阳离子渗透性通道,也是电压门控离子通道(voltage-gated ion channel, VGICs)超家族中进化上的重要成员。双孔通道广泛存在于动植物中,主要以酸性储存的方式表达。该文综述了TPCs的结构、分布以及TPCs激活机制研究进展,并将目前有关TPCs与相关配体激活研究过程中存在的问题加以总结,旨在为今后研究和治疗以TPCs为靶向的相关疾病提供参考。     

TMEM16/ANOCTAMIN:最新的氯通道家族

Cl-通道参与许多生理过程,包括跨上皮细胞的离子吸收与分泌、平滑肌与骨骼肌收缩、神经元兴奋性、器官感知功能及细胞容积调节等.目前对于许多类型Cl-通道的分子构型尚不清楚.新近三个独立的研究小组同时发现Ano1是一种与钙离子激活氯通道(calcium-activated chloridechannels,CaCCs)活性密切相关的膜蛋白.Ano1与其它9个成员共同组成Anoctamin家族.所有Anoctamin蛋白都具有类似结构,推测含8个跨膜结构域以及胞质N-末端和C-末端.Ano1和Ano2的表达都与CaCCs类似,但其它Anoctamin蛋白的作用仍然未知.     

PIP3在中性粒细胞信号通路中的作用及生成调节

趋化性是中性粒细胞参与机体对抗病原体的一个基本的细胞反应。中性粒细胞的趋化过程中涉及一系列信号通路来调节其运动性和极性。信号分子磷酸酰肌醇三磷酸及其参与的信号通路在中性粒细胞趋化过程中起着重要的作用,其自身的生成也受到一系列复杂因素的调节。     

钙信号与细胞分裂

细胞分裂是生命科学的一项重要研究课题。作为一种主要的第二信使，钙离子一直被认为可能参与细胞分裂的调控。因此，世界上许多实验室很早就针对此假说开展了研究。但是，长期以来，一直找不到明确的实验证据。有些研究者称可观察到钙离子浓度在细胞分裂时有轻微的升高，而其它研究报告却指出钙离子浓度有降低或没有明显的变化。     

双孔钾通道TREK-1 结构、分布、调控因素及其抗癫痫作用的研究进展

：钾离子通道是最大最复杂的离子通道家族,迄今为止在人类基因组中共克隆出了70 余种钾离子通道亚型,其中双孔钾离 子通道是近年来新发现的一类钾离子通道亚家族,它们在结构上与电压依赖性钾通道、钙激活钾通道,内向整流型钾通道等传统 的单孔钾离子通道差异很大。双孔钾离子通道,具有4 个跨膜片段,形成独特的2 个孔道结构域,主要介导背景钾电流。由于其介 导背景钾电流而参与并维持静息膜电位形成等重要生理作用而备受关注。近年来研究最多的双孔钾通道TREK-1 几乎表达于机 体的每一个细胞,可被细胞内酸度、膜牵张、多不饱和脂肪酸、温度、受体偶联第二信使系统调控,调节细胞兴奋性,参与一系列生 理、病理过程,与神经系统疾病如癫痫密切相关,本文就此做一综述。     

双孔钾通道TREK-1结构、分布、调控因素及其抗癫痫作用的研究进展

钾离子通道是最大最复杂的离子通道家族,迄今为止在人类基因组中共克隆出了70余种钾离子通道亚型,其中双孔钾离子通道是近年来新发现的一类钾离子通道亚家族,它们在结构上与电压依赖性钾通道、钙激活钾通道,内向整流型钾通道等传统的单孔钾离子通道差异很大。双孔钾离子通道,具有4个跨膜片段,形成独特的2个孔道结构域,主要介导背景钾电流。由于其介导背景钾电流而参与并维持静息膜电位形成等重要生理作用而备受关注。近年来研究最多的双孔钾通道TREK-1几乎表达于机体的每一个细胞,可被细胞内酸度、膜牵张、多不饱和脂肪酸、温度、受体偶联第二信使系统调控,调节细胞兴奋性,参与一系列生理、病理过程,与神经系统疾病如癫痫密切相关,本文就此做一综述。     

mTOR信号通路与调节

哺乳动物雷帕霉素靶蛋白mTOR是一种非典型丝氨酸／苏氨酸蛋白激酶,可整合细胞外信号,磷酸化下游靶蛋白核糖体p70S6激酶,如S6K1及4E—BP1,影响转录与翻译,从而参与调控细胞生长、增殖等过程。近年来研究发现,调控mTOR通路可以干预某些疾病的病理过程。mTOR研究的新发现,可望为今后相关疾病的治疗提供新的靶点。     

植物体内钙信号及其在调节干旱胁迫中的作用

钙作为植物体内第二信使广泛参与了植物响应的各种非生物和生物胁迫的信号传导。胁迫信号通过激活位于细胞质膜上的钙离子通道,产生胞质内特异性的钙信号,传递至钙信号感受蛋白,如钙调素(calmodulin,CaM)、钙依赖蛋白激酶(Ca2+-dependent protein kinases,CDPK)和类钙调磷酸酶B蛋白(calcineurin B-like protein,CBL)等,进而引起胞内一系列生理生化变化,最终对胁迫做出响应。钙信号在植物响应干旱胁迫信号系统中起枢纽作用,主要通过调节气孔运动,水通道蛋白(aquaporin,AQP)和抗氧化酶活性来减少水分流失,提高水分利用率,最终降低干旱对植物细胞的伤害,并具有一定的生态学功能。该文对近年来国内外有关植物体内钙信号的研究进展以及在干旱逆境中的调节作用进行综述,并对今后的研究做了展望。     

核钙信号与基因表达调节

钙（Ca^2 ）是细胞内重要的第二信使,近年的一些研究证实胞质Ca^2 和核Ca^2 信号通过不同的机制影响基因转录,核Ca^2 通过CaM激酶调节核蛋白磷酸化及cAMP反应元件结合蛋白（CREB）介导转录,胞质Ca^2 信号则触动血清反应元件（SRE）介导的基因转录。另外,核Ca^2 也参与多种核酶和核蛋白转运等核过程的调节。     

低氧预适应下BDNF/TrkB和cAMP/PKA信号通路调控电压依赖性钙离子通道的研究进展

本文探析了低氧预适应对神经系统的保护作用尤其是改善学习记忆能力的相关机制,回顾了电压依赖性钙离子通道在神经系统中的作用以及与学习记忆之间的关系。重点总结了低氧预适应诱导下电压依赖性钙离子通道特性的变化情况,并深入归纳了BDNF/TrkB和cAMP/PKA信号通路对电压依赖性钙离子通道的调节机制以及低氧预适应与这些信号通路之间的关系。通过总结低氧预适应调控BDNF/TrkB和cAMP/PKA信号通路影响电压依赖性钙离子通道相关的最新研究进展,为将来阐明低氧预适应提升认知能力的可能机制奠定理论基础。     

Nicotinic Acid Adenine Dinucleotide Phosphate Plays a Critical Role in Naive and Effector Murine T Cells but Not Natural Regulatory T Cells

Nicotinic acid adenine dinucleotide phosphate (NAADP), the most potent Ca²⁺ mobilizing second messenger discovered to date, has been implicated in Ca²⁺ signaling in some lymphomas and T cell clones. In contrast, the role of NAADP in Ca²⁺ signaling or the identity of the Ca²⁺ stores targeted by NAADP in conventional naive T cells is less clear. In the current study, we demonstrate the importance of NAADP in the generation of Ca²⁺ signals in murine naive T cells. Combining live-cell imaging methods and a pharmacological approach using the NAADP antagonist Ned-19, we addressed the involvement of NAADP in the generation of Ca²⁺ signals evoked by TCR stimulation and the role of this signal in downstream physiological end points such as proliferation, cytokine production, and other responses to stimulation. We demonstrated that acidic compartments in addition to the endoplasmic reticulum were the Ca²⁺ stores that were sensitive to NAADP in naive T cells. NAADP was shown to evoke functionally relevant Ca²⁺ signals in both naive CD4 and naive CD8 T cells. Furthermore, we examined the role of this signal in the activation, proliferation, and secretion of effector cytokines by Th1, Th2, Th17, and CD8 effector T cells. Overall, NAADP exhibited a similar profile in mediating Ca²⁺ release in effector T cells as in their counterpart naive T cells and seemed to be equally important for the function of these different subsets of effector T cells. This profile was not observed for natural T regulatory cells.     

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) and Endolysosomal Two-pore Channels Modulate Membrane Excitability and Stimulus-Secretion Coupling in Mouse Pancreatic β Cells

Pancreatic β cells are electrically excitable and respond to elevated glucose concentrations with bursts of Ca²⁺ action potentials due to the activation of voltage-dependent Ca²⁺ channels (VDCCs), which leads to the exocytosis of insulin granules. We have examined the possible role of nicotinic acid adenine dinucleotide phosphate (NAADP)-mediated Ca²⁺ release from intracellular stores during stimulus-secretion coupling in primary mouse pancreatic β cells. NAADP-regulated Ca²⁺ release channels, likely two-pore channels (TPCs), have recently been shown to be a major mechanism for mobilizing Ca²⁺ from the endolysosomal system, resulting in localized Ca²⁺ signals. We show here that NAADP-mediated Ca²⁺ release from endolysosomal Ca²⁺ stores activates inward membrane currents and depolarizes the β cell to the threshold for VDCC activation and thereby contributes to glucose-evoked depolarization of the membrane potential during stimulus-response coupling. Selective pharmacological inhibition of NAADP-evoked Ca²⁺ release or genetic ablation of endolysosomal TPC1 or TPC2 channels attenuates glucose- and sulfonylurea-induced membrane currents, depolarization, cytoplasmic Ca²⁺ signals, and insulin secretion. Our findings implicate NAADP-evoked Ca²⁺ release from acidic Ca²⁺ storage organelles in stimulus-secretion coupling in β cells.     

Two-pore Channels (TPC2s) and Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) at Lysosomal-Sarcoplasmic Reticular Junctions Contribute to Acute and Chronic β-Adrenoceptor Signaling in the Heart

Ca²⁺-permeable type 2 two-pore channels (TPC2) are lysosomal proteins required for nicotinic acid adenine dinucleotide phosphate (NAADP)-evoked Ca²⁺ release in many diverse cell types. Here, we investigate the importance of TPC2 proteins for the physiology and pathophysiology of the heart. NAADP-AM failed to enhance Ca²⁺ responses in cardiac myocytes from Tpcn2^−/− mice, unlike myocytes from wild-type (WT) mice. Ca²⁺/calmodulin-dependent protein kinase II inhibitors suppressed actions of NAADP in myocytes. Ca²⁺ transients and contractions accompanying action potentials were increased by isoproterenol in myocytes from WT mice, but these effects of β-adrenoreceptor stimulation were reduced in myocytes from Tpcn2^−/− mice. Increases in amplitude of L-type Ca²⁺ currents evoked by isoproterenol remained unchanged in myocytes from Tpcn2^−/− mice showing no loss of β-adrenoceptors or coupling mechanisms. Whole hearts from Tpcn2^−/− mice also showed reduced inotropic effects of isoproterenol and a reduced tendency for arrhythmias following acute β-adrenoreceptor stimulation. Hearts from Tpcn2^−/− mice chronically exposed to isoproterenol showed less cardiac hypertrophy and increased threshold for arrhythmogenesis compared with WT controls. Electron microscopy showed that lysosomes form close contacts with the sarcoplasmic reticulum (separation ∼25 nm). We propose that Ca²⁺-signaling nanodomains between lysosomes and sarcoplasmic reticulum dependent on NAADP and TPC2 comprise an important element in β-adrenoreceptor signal transduction in cardiac myocytes. In summary, our observations define a role for NAADP and TPC2 at lysosomal/sarcoplasmic reticulum junctions as unexpected but major contributors in the acute actions of β-adrenergic signaling in the heart and also in stress pathways linking chronic stimulation of β-adrenoceptors to hypertrophy and associated arrhythmias.     

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) and Ca2+ Mobilization

Many physiological processes are controlled by a great diversity of Ca^{2 +} signals that depend on Ca^{2 +} entry into the cell and/or Ca^{2 +} release from internal Ca^{2 +} stores. Ca^{2 +} mobilization from intracellular stores is gated by a family of messengers including inositol-1,4,5-trisphosphate (InsP₃), cyclic ADP-ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP). There is increasing evidence for a novel intracellular Ca^{2 +} release channel that may be targeted by NAADP and that displays properties distinctly different from the well-characterized InsP₃ and ryanodine receptors. These channels appear to localize on a wider range of intracellular organelles, including the acidic Ca^{2 +} stores. Activation of the NAADP-sensitive Ca^{2 +} channels evokes complex changes in cytoplasmic Ca^{2 +} levels by means of channel chatter with other intracellular Ca^{2 +} channels. The recent demonstration of changes in intracellular NAADP levels in response to physiologically relevant extracellular stimuli highlights the significance of NAADP as an important regulator of intracellular Ca^{2 +} signaling.     

Fluorescent analogs of NAADP with calcium mobilizing activity

Nicotinic acid adenine dinucleotide phosphate (NAADP) mobilizes Ca²⁺ through a mechanism totally independent of cyclic ADP-ribose or inositol trisphosphate. Fluorescent analogs of NAADP were synthesized in this study to facilitate further characterization of this novel Ca²⁺ release mechanism. The base-exchange reaction catalyzed by ADP-ribosyl cyclase was utilized to convert nicotinamide 1,N⁶-ethenoadenine dinucleotide phosphate to a fluorescent product, nicotinic acid 1,N⁶-ethenoadenine dinucleotide phosphate (etheno-NAADP). The excitation spectrum of the product showed two maxima at 275 nm and 300 nm and an emission maximum at 410 nm. An aza derivative of etheno-NAADP was also synthesized by sequential treatments with NaOH and nitrite. The product, nicotinic acid 1,N⁶-etheno-2-aza-adenine dinucleotide phosphate (etheno-aza-NAADP) had excitation maxima at 280 nm and 360 nm and an emission maximum at 470 nm. The fluorescence of both analogs was sensitive to polarity and exhibited a 3–4-fold enhancement going from an aqueous buffer to an organic solvent. Proton-NMR measurements confirmed the presence of the etheno ring in both analogs. In the aza derivative the proton at the 2-position of the adenine ring was absent, consistent with the conversion of the 2-carbon to a nitrogen. Both analogs could activate Ca²⁺ release from sea urchin egg homogenates and the half-maximal concentrations for etheno-aza-NAADP and etheno-NAADP were at about 2.5 μM and 5 μM, respectively. At sub-threshold concentrations, both analogs could also function as antagonists, inactivating the NAADP-sensitive Ca²⁺ release with a half-maximal concentration of 60–80 nM. Microinjection of etheno-aza-NAADP into live eggs activated Ca²⁺ increase and triggered a cortical exocytotic reaction confirming its effectiveness in vivo. These fluorescent analogs are potentially useful for visualizing the novel Ca²⁺ stores that are sensitive to NAADP in live cells.     

阿诺碱受体及其亚型2调控因子研究进展

阿诺碱受体(RyR)是心肌细胞等可兴奋细胞中重要的Ca2+释放受体,在维持细胞的兴奋性和生理功能方面起重要作用.研究发现,RyR存在3个亚型,每个亚型都是由4个单体组成的四聚体,后者构成Ca2+释放通道.RyR的结构中有调控因子的结合位点,一些内源性调控因子可影响RyR的构型和Ca2+释放.结合作者的研究,就RyR的结构功能、RyR2的一些重要内源性调控因子及其调控机制做一简要综述.     

Structural Uniformity of Pullulan Produced by Several Strains of Pullularia pullulans

Extracellular polysaccharides produced by 3 strains of Pullularia pullulans were fractionated by treating with cetyl trimethyl ammonium hydroxide into soluble and insoluble fractions, and the structure of the former fraction, i.e., pullulan, was studied. The yield and the ratio of 2 fractions varied widely according to the strains. But the structure of pullulan was found to be uniform irrespective of the strains used. All 3 samples of pullulan gave only glucose on complete acid hydrolysis, and 93~95% maltotriose and 5~7% maltotetraose after isoamylase (pullulanase) action. The ratio of α-1,4- to α-1,6-glucosidic linkages calculated from periodate oxidation data coincided very well with the value expected from the ratio of maltotriose to maltotetraose units. An evidence for the complete absence of branch structure in pullulan was presented from the results of hydrolysis by pullulan 4-glucanohydrolase.     

Pore Hydration States of KcsA Potassium Channels in Membranes

Water-filled hydrophobic cavities in channel proteins serve as gateways for transfer of ions across membranes, but their properties are largely unknown. We determined water distributions along the conduction pores in two tetrameric channels embedded in lipid bilayers using neutron diffraction: potassium channel KcsA and the transmembrane domain of M2 protein of influenza A virus. For the KcsA channel in the closed state, the distribution of water is peaked in the middle of the membrane, showing water in the central cavity adjacent to the selectivity filter. This water is displaced by the channel blocker tetrabutyl-ammonium. The amount of water associated with the channel was quantified, using neutron diffraction and solid state NMR. In contrast, the M2 proton channel shows a V-shaped water profile across the membrane, with a narrow constriction at the center, like the hourglass shape of its internal surface. These two types of water distribution are therefore very different in their connectivity to the bulk water. The water and protein profiles determined here provide important evidence concerning conformation and hydration of channels in membranes and the potential role of pore hydration in channel gating.     

Sirt1与生物钟的相互调控

生物钟调控机制广泛存在于各种类型的细胞中,控制着细胞代谢的节律性变化.最近的研究发现,NAD+依赖的组蛋白去乙酰化酶Sirt1参与了生物钟调控过程,对维持正常的生物钟节律具有重要作用;另一方面,Sirt1的表达也受到生物钟系统的调控,呈现出昼夜节律性的表达.因此Sirt1能与生物钟进行相互调控,并且这一作用机制很可能广泛参与了不同类型细胞内的信号转导和能量代谢过程.本文总结了Sirt1与生物钟之间相互调控的一些研究进展,对它们之间的分子调控机制进行了概述.     

烟酰胺腺嘌呤二核苷酸合成相关酶有助于1,4-丁二醇转化为4-羟基丁酸

4-羟基丁酸(4-HB)不仅具有医学应用价值,而且是合成生物材料P3HB4HB的重要前体.在烟酰胺腺嘌呤二核苷酸(NAD)参与情况下,大肠杆菌Escherichia coli S17-1(pZL-dhaT-aldD)可以把1,4-丁二醇(1,4-BD)转化为4HB.为提高4HB产率,通过过表达烟酸磷酸核糖转移酶(PncB)和烟酰胺腺嘌呤二核苷酸合成酶(NadE)增加胞内NAD含量,从而加速1,4-BD转化反应的进行.结果表明,PncB-NadE的表达使1,4-BD转化率比对照组增加13.03％,由10g/L的1,4-BD得到4.87 g/L的4HB,单位细胞的4HB产量由1.32 g/g提高40.91％至1.86 g/g.因此PncB和NadE可用于促进1,4-BD转化为4HB.