环核苷酸门控离子通道门控的分子机理

环核苷酸门控离子通道(CNG)最广泛地分布于神经细胞。近年来关于 CNG 通道门控的分子机制的研究取得了很大的进步。研究表明, CNG 通道的组成及组装影响通道的特性及门控。近年来有关 CNG 突变体的研究及半胱氨酸残基亲和性的分析表明, 环核苷酸首先结合到 CNG 通道 C 端的环核苷酸结合域(CNBD)上引起 CNBD 空间构像改变, 然后 4 个亚单元发生空间构像的协调改变, CNG 通道开放。本文详细讨论了 CNG 通道的门控机制、各亚单元之间的相互作用、组装的过程及其空间构想的变化, 为 CNG 通道的进一步研究, 尤其是离子通道疾病方面提供理论指导。     

环核苷酸门控离子通道的结构、功能及活性调节

环核苷酸门控离子通道（cyclic nucleotide-gated ion channels,CNG）是非选择性的阳离子通道,直接被环核苷酸活化.6个不同基因编码CNG离子通道蛋白,4个A亚单元(A1～A4)和2个B亚单元(B1,B3).CNG离子通道是由2个或3个不同的亚单元组成的异四聚体复合物,是Ca2+进入细胞内的主要通道之一.CNG离子通道的活性可被Ca2+ /CaM及磷酸化/去磷酸化作用所调节,从而改变细胞内钙离子浓度,触发一系列生理效应.近年来CNG离子通道的研究进展神速,成为生命科学的一个热点领域.本文对CNG离子通道的结构、功能及活性调节机制进行了综述.     

植物环核苷酸门控离子通道及其功能研究进展

环核苷酸门控离子通道(CNGC)是非选择性的阳离子通道, 可以直接被细胞内信使小分子——环核苷酸(cAMP和cGMP)活化。该通道蛋白包含6个跨膜α-螺旋, C端各具一个交叠的环核苷酸与钙调蛋白结合区。CNGC广泛存在于各种植物细胞中。研究表明, 模式植物拟南芥(Arabidopsis thaliana)的CNGC家族有20个成员, 分为4个亚群, 它们在抗病、花粉管生长、对Ca²⁺响应、抵抗重金属离子毒害和抗盐等多种信号途径中发挥重要作用, 协助植物细胞应对各种生物与非生物胁迫。该文简要介绍了CNGC的结构、表达谱及其调控因子, 并着重总结了近年来CNGC生物学功能的研究进展, 以期为今后系统开展其功能研究提供理论依据。     

电压门控离子通道功能与结构分子模型

离子通道是细胞膜上特殊跨膜蛋白构成的亲水孔道,越来越多的证据表明其与兴奋性,腺体分泌、机体运动、甚至学习和记忆行为等重要生理现象密切相关,由此该领域成为当今年生命学科广为注目的前沿之一。本将简要介绍离子通道的分类和功能,并侧重阐明通道压控原理有压控通道的跨膜拓扑结构和功能分子模型。     

电压依赖性离子通道门控的分子机制

５０年代Ｈｏｄｇｋｉｎ和Ｈｕｘｌｅｙ双通道模型及其激活与失活学说,正逐步被８０年代以来的分子生物学和电生理学研究所证实。Ｎａ＾＋、Ｋ＾＋离子通道的激活主要决定于高度保守的带正电荷氨基酸残基密集的Ｓ４段,由膜内向膜外方向的拧改锥样旋转。Ｎａ＾＋通道的失活主要与其Ⅲ－Ⅳ功能区之间的胞内连结襻的“铰链盖”样运动有关;Ｋ＾＋通的失活分Ｎ－、Ｃ－、Ｐ－三型,分别发生在Ｎ－、Ｃ－末端和Ｐ区,其Ｎ型失活与Ｎ－末     

配体门控离子通道受体别构调节研究

配体门控离子通道(LGIC)在中枢神经系统信息处理的过程中起着极其重要的作用,与多种神经性疾病有着密切联系.与受体正位调节作用相比,别构调节效应具有类内源性生理作用、高选择性及不易过度调节的优点,从而避免了一系列不良反应发生.目前,各种LGIC受体超家族均有别构调节剂发现,部分已在临床上得到应用.在未来的研究中,通过建立及完善针对别构调节剂的筛选策略,别构调节剂的发现效率及生物活性将得到极大地提高,更多的药物将会不断涌现.     

生物膜电压门控离子通道的结构和功能性构象

生物膜离子通道具有多种重要的生理功能.近年,已分离、纯化了电压门控的Na~+、Ca~(2+)和K~+通道的蛋白质组分.Na~+和Ca~(2+)通道分别由一个构成离子孔洞的主要亚单位和数目不同的其他亚单位组成,K~+通道是单一的多肽.对Na~+、Ca~(2+)通道主要亚单位和K~+通道的氨基酸序列的测定表明,它们之间有许多相似性.已分别给出了三种通道跨膜排列的二级结构图象.考虑了Na~+通道的功能特性,包括电压敏感性、通道开放动力学、门控电流、神经毒素的作用等,已提出几种Na~+通道功能性构象模型.     

DICE：电压门控离子通道电生理实验数据库

针对现有相关文献中离子通道电生理数据繁多且分散的特点,开发了一套电压门控离子通道电生理实验数据库。数据库中目前主要包括钠离子通道序列数据、调制剂分子结构和序列数据,并收集整理了文献中调制剂和通道相互作用时的电生理学数据和药理学数据。系统实现了数据的收集、录入、存储和查询,为后期进行数据挖掘奠定了基础。用户可以通过网址http：／／biodb．sgst．cn／DICE对数据库进行访问。     

锌离子对配体门控型离子通道的调节作用

在中枢神经系统(central nervous system,CNS)中,锌离子对配体门控型离子通道具有重要的调节作用。锌离子随着神经元的活动从突触前膜的囊泡中释放到突触间隙,对突触内受体进行调控。锌离子抑制N-甲基-D-天冬氨酸(N-methyl-D-aspartate,NMDA)型谷氨酸受体的活性,而对非NMDA型谷氨酸受体的调控具有多样性。由γ氨基丁酸(γ-aminobutyric acid,GABA)受体所介导的抑制性突触传递活动也受到锌离子的抑制;而锌离子对glycine受体则呈现出浓度依赖的双向调节效应。病理条件下,锌离子参与了兴奋性细胞毒作用所触发的神经元凋亡过程。本文主要阐述了在CNS中,锌离子对配体门控型离子通道所介导的突触传递活动的调控作用,以及这些调控作用的生理功能和病理意义。     

植物环核苷酸门控通道(CNGC)基因家族的结构与功能

环核苷酸门控通道(CNGC)是近年来被确认的在动植物细胞中普遍存在的离子通道基因家族。文章就近年来植物中CNGC基因的种类、分子结构、作用机制及其在植物生长发育中的功能的研究进展作了概述。     

Role of Cyclic Nucleotide-Gated Channels in the Modulation of Mouse Hippocampal Neurogenesis

Neural stem cells generate neurons in the hippocampal dentate gyrus in mammals, including humans, throughout adulthood. Adult hippocampal neurogenesis has been the focus of many studies due to its relevance in processes such as learning and memory and its documented impairment in some neurodegenerative diseases. However, we are still far from having a complete picture of the mechanism regulating this process. Our study focused on the possible role of cyclic nucleotide-gated (CNG) channels. These voltage-independent channels activated by cyclic nucleotides, first described in retinal and olfactory receptors, have been receiving increasing attention for their involvement in several brain functions. Here we show that the rod-type, CNGA1, and olfactory-type, CNGA2, subunits are expressed in hippocampal neural stem cells in culture and in situ in the hippocampal neurogenic niche of adult mice. Pharmacological blockade of CNG channels did not affect cultured neural stem cell proliferation but reduced their differentiation towards the neuronal phenotype. The membrane permeant cGMP analogue, 8-Br-cGMP, enhanced neural stem cell differentiation to neurons and this effect was prevented by CNG channel blockade. In addition, patch-clamp recording from neuron-like differentiating neural stem cells revealed cGMP-activated currents attributable to ion flow through CNG channels. The current work provides novel insights into the role of CNG channels in promoting hippocampal neurogenesis, which may prove to be relevant for stem cell-based treatment of cognitive impairment and brain damage.     

Expression of Cyclic Nucleotide-Gated Cation Channels in Airway Epithelial Cells

Using the whole-cell patch-clamp technique, the selectivity and pharmacology of 8-Br-cGMP-stimulated currents in the human alveolar cell line A549 was compared to 8-Br-cGMP-stimulated currents in HK293 cells transfected with hαCNC1. Whole cell currents stimulated by 8-Br-cGMP in HK293 cells transfected with hαCNC1 or A549 cells are carried by inward sodium and outward potassium with nearly the same selectivity. The whole-cell inward currents that are stimulated by 8-Br-cGMP in HK293 cells transfected with hαCNC1 are inhibited by l-cis-diltiazem with an IC₅₀ of 154 μm, by 2′,4′-dichlorobenzamil with an IC₅₀ of 50 μm and by amiloride with an IC₅₀ of 133 μm. The whole-cell inward currents in A549 cells that are stimulated by 8-Br-cGMP, are inhibited by l-cis-diltiazem with an IC₅₀ of 87 μm, by 2′4′-dichlorobenzamil with an IC₅₀ of 38 μm and by amiloride with an IC₅₀ of 32 μm suggesting that these airway cells contain cyclic nucleotide-gated cation channels. RT-PCR data suggest that mRNA of both αCNC1 and βCNC subunits are present in A549 cells and the presence of the βCNC subunit, may as previously reported, increase the affinity of these channel blockers compared to the hαCNC1 subunit alone. The mRNA of two other isoforms of this channel, CNC2 and CNC3, are also expressed in the A549 cell line. This study documents the IC₅₀ of externally applied channel blockers that can be used for in vitro or in vivo experiments to document sodium absorption via cyclic nucleotide-gated cation channels in airway cells. Received: 24 February/Revised: 28 May 1999     

Cyclic Nucleotide-Gated Channels Contribute to Thromboxane A2-Induced Contraction of Rat Small Mesenteric Arteries

Background

Thromboxane A₂ (TxA₂)-induced smooth muscle contraction has been implicated in cardiovascular, renal and respiratory diseases. This contraction can be partly attributed to TxA₂-induced Ca²⁺ influx, which resulted in vascular contraction via Ca²⁺-calmodulin-MLCK pathway. This study aims to identify the channels that mediate TxA₂-induced Ca²⁺ influx in vascular smooth muscle cells.

Methodology/Principal Findings

Application of U-46619, a thromboxane A₂ mimic, resulted in a constriction in endothelium-denuded small mesenteric artery segments. The constriction relies on the presence of extracellular Ca²⁺, because removal of extracellular Ca²⁺ abolished the constriction. This constriction was partially inhibited by an L-type Ca²⁺ channel inhibitor nifedipine (0.5–1 µM). The remaining component was inhibited by L-cis-diltiazem, a selective inhibitor for CNG channels, in a dose-dependent manner. Another CNG channel blocker LY83583 [6-(phenylamino)-5,8-quinolinedione] had similar effect. In the primary cultured smooth muscle cells derived from rat aorta, application of U46619 (100 nM) induced a rise in cytosolic Ca²⁺ ([Ca²⁺]_i), which was inhibited by L-cis-diltiazem. Immunoblot experiments confirmed the presence of CNGA2 protein in vascular smooth muscle cells.

Conclusions/Significance

These data suggest a functional role of CNG channels in U-46619-induced Ca²⁺ influx and contraction of smooth muscle cells.     

A Suppressor Screen of the Chimeric AtCNGC11/12 Reveals Residues Important for Intersubunit Interactions of Cyclic Nucleotide-Gated Ion Channels

To investigate the structure-function relationship of plant cyclic nucleotide-gated ion channels (CNGCs), we identified a total of 29 mutant alleles of the chimeric AtCNGC11/12 gene that induces multiple defense responses in the Arabidopsis (Arabidopsis thaliana) mutant, constitutive expresser of PR genes22 (cpr22). Based on computational modeling, two new alleles, S100 (AtCNGC11/12:G459R) and S137 (AtCNGC11/12:R381H), were identified as counterparts of human CNGA3 (a human CNGC) mutants. Both mutants lost all cpr22-mediated phenotypes. Transient expression in Nicotiana benthamiana as well as functional complementation in yeast (Saccharomyces cerevisiae) showed that both AtCNGC11/12:G459R and AtCNGC11/12:R381H have alterations in their channel function. Site-directed mutagenesis coupled with fast-protein liquid chromatography using recombinantly expressed C-terminal peptides indicated that both mutations significantly influence subunit stoichiometry to form multimeric channels. This observation was confirmed by bimolecular fluorescence complementation in planta. Taken together, we have identified two residues that are likely important for subunit interaction for plant CNGCs and likely for animal CNGCs as well.Cyclic nucleotide-gated ion channels (CNGCs) were first discovered in retinal photoreceptors and olfactory sensory neurons (Zagotta and Siegelbaum, 1996; Kaupp and Seifert, 2002). CNGCs play crucial roles for the signal transduction in these neurons that are excited by photons and odorants, respectively. In mammalian genomes, six CNGC genes have been found and named CNGA1 to CNGA4, CNGB1, and CNGB3 (Kaupp and Seifert, 2002). It has been reported that in mammalian cells, CNGCs function as heterotetramers that are composed of A and B subunits with cell-specific stoichiometry (Kaupp and Seifert, 2002; Cukkemane et al., 2011). For example, CNGCs in rod photoreceptors are composed of three A1 subunits and one B1a subunit, whereas in cone photoreceptors, they are believed to be composed of two A3 and two B3 subunits (Zhong et al., 2002; Peng et al., 2004). The structure of each subunit is similar to that of the voltage-gated K⁺-selective ion channel (Shaker) proteins, including a cytoplasmic N terminus, six membrane-spanning regions (S1–S6), a pore domain located between S5 and S6, and a cytoplasmic C terminus (Zagotta and Siegelbaum, 1996). However, CNGCs are only weakly voltage dependent and are opened by the direct binding of cyclic nucleotides (cAMP and cGMP), which are universally important secondary messengers that control diverse cellular responses (Fesenko et al., 1985). The cytoplasmic C terminus contains a cyclic nucleotide-binding domain (CNBD) and a C-linker region that connects the CNBD to the S6 domain. CNGC activity is also regulated by feedback inhibitory mechanisms involving the Ca²⁺ sensor protein, calmodulin (CaM). CaM-binding sites in animal CNGCs have been found in various regions of both the C- and N-terminal domains (Ungerer et al., 2011). It has been reported that the subunit composition has significant influence on the mode of CaM-mediated regulation (Kramer and Siegelbaum, 1992; Bradley et al., 2004; Song et al., 2008).On the other hand, plant CNGCs have only been investigated much more recently. The first plant CNGC, HvCBT1, was identified as a CaM-binding transporter protein in barley (Hordeum vulgare; Schuurink et al., 1998). Subsequently, several CNGCs were identified from Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum; Arazi et al., 1999; Köhler and Neuhaus, 1998; Köhler et al., 1999). Interestingly, the Arabidopsis genome sequencing project identified a large family comprising 20 members (AtCNGC1–AtCNGC20), indicating a significant expansion of Arabidopsis CNGCs that suggests a higher level of diversity and functional importance in plants (Mäser et al., 2001). To date, possible biological functions of Arabidopsis CNGCs in development, ion homeostasis, thermal sensing, as well as pathogen resistance have been reported (Kaplan et al., 2007; Chin et al., 2009; Dietrich et al., 2010; Moeder et al., 2011; Finka et al., 2012). With respect to structure, plant CNGCs are believed to have a similar architecture to their animal counterparts (Chin et al., 2009). However, only a handful of studies on the structure-function analysis of plant CNGCs have been published so far, and this field is still very much in its infancy (Hua et al., 2003; Bridges et al., 2005; Kaplan et al., 2007; Baxter et al., 2008; Chin et al., 2010).Previously, we have reported two functionally important residues in plant CNGCs (Baxter et al., 2008; Chin et al., 2010). These residues were discovered using a suppressor screen of the rare gain-of-function Arabidopsis mutant constitutive expresser of PR genes22 (cpr22; Yoshioka et al., 2006). The cpr22 mutant, which has a deletion between AtCNGC11 and AtCNGC12 resulting in a novel but functional chimeric CNGC (AtCNGC11/12), exhibits multiple resistance responses without pathogen infection in the hemizygous state and conditional lethality in the homozygous state (Yoshioka et al., 2001, 2006; Moeder et al., 2011). It has been reported that the cpr22 phenotype is attributable to the expression of AtCNGC11/12 and its channel activity (Yoshioka et al., 2006; Baxter et al., 2008), thereby making the suppressor screen an invaluable tool for identifying intragenic mutants to further elucidate the structure-function relationship of plant CNGCs (Baxter et al., 2008; Chin et al., 2010).In this study, we describe a total of 29 mutant alleles of AtCNGC11/12, including the three previously published alleles (Baxter et al., 2008; Chin at al., 2010), and compare their predicted three-dimensional structural positions with equivalent mutations of a human CNGC, CNGA3. In this analysis, two AtCNGC11/12 mutations emerged as counterparts of human mutations (Wissinger et al., 2001). Both the AtCNGC11/12 as well as the human CNGA3 mutations were computationally predicted to affect intersubunit interactions. This prediction was experimentally validated by size-exclusion chromatography (FPLC) as well as bimolecular fluorescence complementation (BiFC) in combination with site-direct mutagenesis using recombinant C-terminal peptides.     

Function,Modulation, and Pharmacology of Inhibitory Glycine Receptor Channels

Ion channels of the glycine receptors (GlyR) provide an inhibitory drive in the nervous system of vertebrates. Four agonist-binding subunits and one subunit were cloned from the CNS of mammals (human, rat, and mouse), a bird (chicken), and a fish (zebrafish). The subunit composition determines the pharmacological properties of the GlyR and the function of the glycinergic synapses during development. Dysfunction of these receptors results in motor disorders (hyperekplexia, spasticity, and/or oscillatory phenomena), and mutations in the gene encoding the subunit of GlyR underlie the molecular basis of these heritable diseases. Extra- and intracellular pharmacological tools can modulate the activity of GlyR channels. A recently discovered phenomenon of Ca-induced modulation of the GlyR suggests an important way of modulation of the activity of glycinergic synapses.     

Rectification of the Olfactory Cyclic Nucleotide-Gated Channel by Intracellular Polyamines

Polyamine-induced inward rectification of cyclic nucleotide-gated channels was studied in inside-out patches from rat olfactory neurons. The polyamines, spermine, spermidine and putrescine, induced an `instantaneous' voltage-dependent inhibition with K <sub>d</sub> values at 0 mV of 39, 121 μm and 2.7 mm, respectively. Hill coefficients for inhibition were significantly < 1, suggesting an allosteric inhibitory mechanism. The Woodhull model for voltage-dependent block predicted that all 3 polyamines bound to a site 1/3 of the electrical distance through the membrane from the internal side. Instantaneous inhibition was relieved at positive potentials, implying significant polyamine permeation. Spermine also induced exponential current relaxations to a `steady-state' impermeant level. This inhibition was also mediated by a binding site 1/3 of the electrical distance through the pore, but with a K _d of 2.6 mm. Spermine inhibition was explained by postulating two spermine binding sites at a similar depth. Occupation of the first site occurs rapidly and with high affinity, but once a spermine molecule has bound, it inhibits spermine occupation of the second binding site via electrostatic repulsion. This repulsion is overcome at higher membrane potentials, but results in a lower apparent binding affinity for the second spermine molecule. The on-rate constant for the second spermine binding saturated at a low rate (∼200 sec⁻¹ at +120 mV), providing further evidence for an allosteric mechanism. Polyamine-induced inward rectification was significant at physiological concentrations. Received: 17 February 1999/Revised: 27 April 1999     

Nitric Oxide Inhibition of the Rat Olfactory Cyclic Nucleotide-Gated Cation Channel

The effects of nitric oxide (NO) and other cysteine modifying agents were examined on cyclic nucleotide-gated (CNG) cation channels from rat olfactory receptor neurons. The NO compounds, S-nitroso-cysteine (SNC) and 3-morpholino-sydnonomine (SIN-1), did not activate the channels when applied for up to 10 min. The cysteine alkylating agent, N-ethylmaleimide (NEM), and the oxidising agent, dithionitrobensoate (DTNB), were also without agonist efficacy. Neither SNC nor DTNB altered the cAMP sensitivity of the channels. However, 2-min applications of SIN-1, SNC and DTNB inhibited the cAMP-gated current to approximately 50% of the control current level. This inhibition showed no spontaneous reversal for 5 min but was completely reversed by a 2-min exposure to DTT. The presence of cAMP protected the channels against NO-induced inhibition. These results indicate that inhibition is caused by S-nitrosylation of neighboring sulfhydryl groups leading to sulfhydryl bond formation. This reaction is favored in the closed channel state. Since recombinantly expressed rat olfactory α and β CNG channel homomers and α/β heteromers are activated and not inhibited by cysteine modification, the results of this study imply the existence of a novel subunit or tightly bound factor which dominates the effect of cysteine modification in the native channels. As CNG channels provide a pathway for calcum influx, the results may also have important implications for the physiological role of NO in mammalian olfactory receptor neurons. Received: 30 March 1998/Revised: 17 June 1998     

Structure,Dynamics and Implied Gating Mechanism of a Human Cyclic Nucleotide-Gated Channel

Cyclic nucleotide-gated (CNG) ion channels are nonselective cation channels, essential for visual and olfactory sensory transduction. Although the channels include voltage-sensor domains (VSDs), their conductance is thought to be independent of the membrane potential, and their gating regulated by cytosolic cyclic nucleotide–binding domains. Mutations in these channels result in severe, degenerative retinal diseases, which remain untreatable. The lack of structural information on CNG channels has prevented mechanistic understanding of disease-causing mutations, precluded structure-based drug design, and hampered in silico investigation of the gating mechanism. To address this, we built a 3D model of the cone tetrameric CNG channel, based on homology to two distinct templates with known structures: the transmembrane (TM) domain of a bacterial channel, and the cyclic nucleotide-binding domain of the mouse HCN2 channel. Since the TM-domain template had low sequence-similarity to the TM domains of the CNG channels, and to reconcile conflicts between the two templates, we developed a novel, hybrid approach, combining homology modeling with evolutionary coupling constraints. Next, we used elastic network analysis of the model structure to investigate global motions of the channel and to elucidate its gating mechanism. We found the following: (i) In the main mode of motion, the TM and cytosolic domains counter-rotated around the membrane normal. We related this motion to gating, a proposition that is supported by previous experimental data, and by comparison to the known gating mechanism of the bacterial KirBac channel. (ii) The VSDs could facilitate gating (supplementing the pore gate), explaining their presence in such ‘voltage-insensitive’ channels. (iii) Our elastic network model analysis of the CNGA3 channel supports a modular model of allosteric gating, according to which protein domains are quasi-independent: they can move independently, but are coupled to each other allosterically.