植物保卫细胞离子通道在气孔运动中的作用

介绍保卫细胞质膜和液泡膜上的离子通道活性变化及其在气孔运动中的作用,同时对各种刺激引发气孔运动过程中的信使Ca2 、H2O2和pH等对离子通道的调节作用作了概述.     

The zinc binding site of the Shaker channel KDC1 from Daucus carota

KDC1 is a voltage-dependent Shaker-like potassium channel subunit cloned from Daucus carota which produces conductive channels in Xenopus oocytes only when coexpressed with other plant Shaker potassium subunits, such as KAT1 from Arabidopsis thaliana. External Zn²⁺ determines a potentiation of the current mediated by the dimeric construct KDC1-KAT1, which has been ascribed to zinc binding at a site comprising three histidines located at the S3-S4 (H161, H162) and S5-S6 (H224) linkers of KDC1. Here we demonstrate that also glutamate 164, located in close proximity of the KDC1 S4 segment, is an essential component of the zinc-binding site. On the contrary, glutamate 159, located in symmetrical position with respect to E164 in the sequence E₁₅₉XHHXE₁₆₄ but more distant from the voltage sensor, does not play any role in zinc binding. The effects of Zn²⁺ can be expressed as a “shift” of the gating parameters along the voltage axis. Kinetic modeling shows that Zn²⁺ slows the closing kinetics of KDC1-KAT1 without affecting the opening kinetics. Possibly, zinc affects the movement of the voltage sensor in and out of the membrane phase through electrostatic modification of a site close to the voltage sensor.     

AP2功能基因在植物花发育中的重要作用

AP2基因作为调控植物花发育的功能基因,参与花分生特性建立、花器官的特性特化以及形成调控。所编码的AP2/EREBP转录因子的主要特征是都至少含有一个由60到70个左右的氨基酸组成高度保守的DNA结合区,称作AP2结合域。按其所含的AP2结构域的数目分为3个亚族,即AP2亚家族、EREBP亚家族和RAV亚家族,每个亚家族都有各自的作用。AP2基因不但自身调控着花、胚珠的发育,而且与其他因子相互协作,参与到复杂的花发育调控网络。将对AP2基因的特征和分类及其在花发育中的作用进行概述。     

Structural and Functional Properties of Chicken Lysozyme Fused Serine-rich Heptapeptides at the C-Terminus

Two serine-rich heptapeptides, Ser-Ser-Ser-Lys-Ser-Ser-Ser (S6K) and Ser-Ser-Ser-Ser-Ser-Ser-Ser (S7), were fused to the C-terminus of chicken lysozyme (Lz) by genetic modification to improve the functional properties of lysozyme. The cDNAs of S6K-lysozyme (S6K-Lz) and S7-lysozyme (S7-Lz) were inserted into the expression vector of Pichia pastoris and secreted in yeast cultivation medium. The secretion amounts of S6K-Lz and S7-Lz were about 60% of that of wild-type lysozyme (Wt-Lz). The CD spectra showed that the conformation of S6K-Lz and S7-Lz was conserved regardless of the attachment of serine-rich peptides. The denaturation curves of S6K-Lz and S7-Lz also showed that the conformational changes were very small. The lytic activity of S6K-Lz and S7-Lz was almost the same as that of Wt-Lz, while the bactericidal activity against Escherichia coli of S6K-Lz and S7-Lz was greatly increased. The acetic acid-urea PAGE of phosphatase-treated S6K-Lz and S7-Lz indicated the possibility of phosphorylation of the fused serine-rich heptapeptides.     

The C-Terminus of ClpC1 of Mycobacterium tuberculosis Is Crucial for Its Oligomerization and Function

Mycobacterium tuberculosis ClpC1 is a member of the Hsp100/Clp AAA+ family of ATPases. The primary sequence of ClpC1 contains two N-terminal domains and two nucleotide binding domains (NBD). The second NBD has a long C-terminal sub-domain containing several motifs important for substrate interaction. Generally, ClpC proteins are highly conserved, however presence of C-terminal domains of variable lengths is a remarkable difference in ClpC from different species. In this study, we constructed deletion mutants at the C-terminus of M. tuberculosis ClpC1 to determine its role in the structure and function of the protein. In addition, a deletion mutant having the two conserved N-terminal domains deleted was also constructed to investigate the role of these domains in M. tuberculosis ClpC1 function. The N-terminal domains were found to be dispensable for the formation of oligomeric structure, and ATPase and chaperone activities. However, deletions beyond a specific region in the C-terminus of the ClpC1 resulted in oligomerization defects and loss of chaperonic activity of the protein without affecting its ATPase activity. The truncated mutants, defective in oligomerization were also found to have lost the chaperonic activity, showing the formation of oligomer to be required for the chaperonic activity of M. tuberculosis ClpC1. The current study has identified a region in the C-terminus of M. tuberculosis ClpC1 which is essential for its oligomerization and in turn its function.     

Histidines Are Responsible for Zinc Potentiation of the Current in KDC1 Carrot Channels

Unlike all plant inward-rectifying potassium channels, the carrot channel KDC1 has two histidine pairs (H161,H162) in the S3–S4 and (H224,H225) in the S5–S6 linkers. When coinjected with KAT1 in Xenopus oocytes, KDC1 participates in the formation of heteromultimeric KDC1:KAT1 channels and the ionic current is potentiated by extracellular Zn²⁺. To investigate the potential interactions between KDC1 and zinc, a KDC1-KAT1 dimer was constructed. The dimeric and heteromeric channels displayed similar characteristics and the same sensitivity to zinc and other metals; this result suggests that zinc binding is mediated by residues in a single channel subunit. The KDC1:KAT1 currents were also potentiated by external Pb²⁺ and Cd²⁺ and inhibited by Ni²⁺. To investigate further the role of KDC1-histidines, these amino acids were mutated into alanines. The single mutations H225A, H161A, and H162A did not affect the response of the heteromeric channels to zinc. Conversely, the single mutant H224A and the double mutants (H224A,H225A) and (H161A,H162A) abolished zinc potentiation, but not that induced by Pb²⁺ or Cd²⁺. These results suggest that Zn²⁺ potentiation cannot be ascribed to simple electrostatic interactions between zinc and channel residues and that histidine 224 is crucial for zinc but not for lead potentiation of the current.     

Evidence for a Multi-ion Pore Behavior in the Plant Potassium Channel KAT1

KAT1 is a cloned voltage-gated K⁺ channel from the plant Arabidopsis thaliana L., which displays an inward rectification reminiscent of `anomalous' rectification of the i <sub>f</sub> pacemaker current recorded in animal cells. Macroscopic conductance of KAT1 expressed in Xenopus oocytes was 5-fold less in pure Rb<sup>+</sup> solution than in pure K<sup>+</sup> solution, and negligible in pure Na<sup>+</sup> solution. Experiments in different K<sup>+</sup>/Na<sup>+</sup> or K<sup>+</sup>/Rb<sup>+</sup> mixtures revealed deviations from the principle of independence and notably two anomalous effects of the K<sup>+</sup>/Rb<sup>+</sup> mole fraction (i.e., the ratio [K<sup>+</sup>]/([K<sup>+</sup>]+[Rb<sup>+</sup>])). First, the KAT1 deactivation time constant was both voltage- and mole fraction-dependent (a so-called `foot in the door' effect was thus observed in KAT1 channel). Second, when plotted against the K⁺/Rb⁺ mole fraction, KAT1 conductance values passed through a minimum. This minimum is more important for two pore mutants of KAT1 (T259S and T260S) that displayed an increase in P_Rb/P_K. These results are consistent with the idea that KAT1 conduction requires several ions to be present simultaneously within the pore. Therefore, this atypical `green' member of the Shaker superfamily of K⁺ channels further shows itself to be an interesting model as well for permeation as for gating mechanism studies. Received: 9 February 1998/Revised: 28 July 1998     

Conformational Changes in the Orai1 C-Terminus Evoked by STIM1 Binding

Store-operated CRAC channels regulate a wide range of cellular functions including gene expression, chemotaxis, and proliferation. CRAC channels consist of two components: the Orai proteins (Orai1-3), which form the ion-selective pore, and STIM proteins (STIM1-2), which form the endoplasmic reticulum (ER) Ca²⁺ sensors. Activation of CRAC channels is initiated by the migration of STIM1 to the ER-plasma membrane (PM) junctions, where it directly interacts with Orai1 to open the Ca²⁺-selective pores of the CRAC channels. The recent elucidation of the Drosophila Orai structure revealed a hexameric channel wherein the C-terminal helices of adjacent Orai subunits associate in an anti-parallel orientation. This association is maintained by hydrophobic interactions between the Drosophila equivalents of human Orai1 residues L273 and L276. Here, we used mutagenesis and chemical cross-linking to assess the nature and extent of conformational changes in the self-associated Orai1 C-termini during STIM1 binding. We find that linking the anti-parallel coiled-coils of the adjacent Orai1 C-termini through disulfide cross-links diminishes STIM1-Orai1 interaction, as assessed by FRET. Conversely, prior binding of STIM1 to the Orai1 C-terminus impairs cross-linking of the Orai1 C-termini. Mutational analysis indicated that a bend of the Orai1 helix located upstream of the self-associated coils (formed by the amino acid sequence SHK) establishes an appropriate orientation of the Orai1 C-termini that is required for STIM1 binding. Together, our results support a model wherein the self-associated Orai1 C-termini rearrange modestly to accommodate STIM1 binding.     

Role for the TRPV1 Channel in Insulin Secretion from Pancreatic Beta Cells

Transient receptor potential channels have been put forward as regulators of insulin secretion. A role for the TRPV1 ion channel in insulin secretion has been suggested in pancreatic beta cell lines. We explored whether TRPV1 is functionally expressed in RINm5F and primary beta cells from neonate and adult rats. We examined if capsaicin could activate cationic non-selective currents. Our results show that TRPV1 channels are not functional in insulin-secreting cells, since capsaicin did not produce current activation, not even under culture conditions known to induce the expression of other ion channels in these cells. Although TRPV1 channels seem to be irrelevant for the physiology of isolated beta cells, they may play a role in glucose homeostasis acting through the nerve fibers that regulate islet function. At the physiological level, we observed that Trpv1 ^?/? mice presented lower fasting insulin levels than their wild-type littermates, however, we did not find differences between these experimental groups nor in the glucose tolerance test or in the insulin secretion. However, we did find that the Trpv1 ^?/? mice exhibited a higher insulin sensitivity compared to their wild-type counterparts. Our results demonstrate that TRPV1 does not contribute to glucose-induced insulin secretion in beta cells as was previously thought, but it is possible that it may control insulin sensitivity.     

Functional Characterization and Determination of the Physiological Role of a Calcium-Dependent Potassium Channel from Cyanobacteria

Despite the important achievement of the high-resolution structures of several prokaryotic channels, current understanding of their physiological roles in bacteria themselves is still far from complete. We have identified a putative two transmembrane domain-containing channel, SynCaK, in the genome of the freshwater cyanobacterium Synechocystis sp. PCC 6803, a model photosynthetic organism. SynCaK displays significant sequence homology to MthK, a calcium-dependent potassium channel isolated from Methanobacterium thermoautotrophicum. Expression of SynCaK in fusion with enhanced GFP in mammalian Chinese hamster ovary cells’ plasma membrane gave rise to a calcium-activated, potassium-selective activity in patch clamp experiments. In cyanobacteria, Western blotting of isolated membrane fractions located SynCaK mainly to the plasma membrane. To understand its physiological function, a SynCaK-deficient mutant of Synechocystis sp. PCC 6803, ΔSynCaK, has been obtained. Although the potassium content in the mutant organisms was comparable to that observed in the wild type, ΔSynCaK was characterized by a depolarized resting membrane potential, as determined by a potential-sensitive fluorescent probe. Growth of the mutant under various conditions revealed that lack of SynCaK does not impair growth under osmotic or salt stress and that SynCaK is not involved in the regulation of photosynthesis. Instead, its lack conferred an increased resistance to the heavy metal zinc, an environmental pollutant. A similar result was obtained using barium, a general potassium channel inhibitor that also caused depolarization. Our findings thus indicate that SynCaK is a functional channel and identify the physiological consequences of its deletion in cyanobacteria.Detailed structural and mechanistic data now exist for many prokaryotic channels, but their physiological roles remain largely unclear (Martinac et al., 2008). This is especially true for potassium channels. K⁺ is the most abundant cation in organisms, and in general, it plays a crucial role in the survival and development of cells by regulating enzyme activity and tuning electrochemical membrane potential. Potassium channels in prokaryotes have been hypothesized to contribute to the setting of membrane potential rather than to high-affinity potassium uptake normally achieved thanks to specific ATP-dependent potassium transporters (Kuo et al., 2005). K⁺ channel genes are found in almost every prokaryotic genome that has been sequenced, but in most of the few studies where their deletion was obtained, no specific phenotype has been observed, suggesting either functional redundancy or that these channels are only required in case of rather specific environmental stresses. Gain-of-function potassium channel (Kch) mutants of Escherichia coli failed to grow in millimolar-added K⁺ but not Na⁺ (Kuo et al., 2003), and external H⁺ suppressed the gain-of-function phenotype, supporting the hypothesis that KCh might function to regulate membrane potential. However, a clear-cut role of prokaryotic potassium channels by genetic deletion was demonstrated only in a few cases.The model organism Synechocystis sp. PCC 6803 harbors an intracellular membrane system, the thylakoids, where both photosynthesis and respiration take place. In this work, we have identified in Synechocystis sp. PCC 6803 a so-far uncharacterized putative potassium channel, NP_440478, encoded by the open reading frame (ORF) sll0993, with sequence homology to MthK, a Ca²⁺-activated K⁺ channel of the archaeon Methanobacterium thermoautotrophicum. The structure of MthK in an open conformation has been determined (Jiang et al., 2002). The MthK subunit has two transmembrane segments and one pore region, followed by an extension of approximately 200 amino acid residues, which contains a region called the regulator of the conductance of K⁺ (RCK) domain. RCK of MthK binds divalent cations, such as Ca²⁺ or Cd²⁺ (Jiang et al., 2001, 2002). The physiological meaning of the activation of MthK with millimolar Ca²⁺ (Jiang et al., 2002) is, however, unclear, because Ca²⁺ as a second messenger operates at micromolar concentrations in eukaryotes, and the possible signaling roles of Ca²⁺ in prokaryotes are still unclear. Another RCK-containing 160-picoSiemens (pS) K⁺ channel from the archaeon Thermoplasma volcanium, TvoK, was also found to be activated by millimolar Ca²⁺ (Parfenova et al., 2007). Here, we report evidence that, similar to MthK, SynCaK can also be activated by calcium. Furthermore, we show localization of the protein in cyanobacteria and describe a phenotype associated with the lack of the channel in SynCaK-less mutant Synechocystis sp. PCC 6803 cells.     

植物内生细菌对植物健康的作用

植物内生菌是一个多样性十分丰富的微生物类群,存在于没有外在感染症状的健康植物组织内,并与宿主植物协同进化.随着研究领域的不断拓宽和研究方法的不断更新,植物内生菌与植物健康的关系以及应用逐渐成为研究热点.本文主要综述了内生细菌的多样性、进入植物组织内的机制以及内生细菌的主要功能及应用,提出了现阶段内生菌研究存在的问题,并展望了内生菌研究的前景.     

Evidence for Functional Diversity between the Voltage-Gated Proton Channel Hv1 and Its Closest Related Protein HVRP1

The Hv1 channel and voltage-sensitive phosphatases share with voltage-gated sodium, potassium, and calcium channels the ability to detect changes in membrane potential through voltage-sensing domains (VSDs). However, they lack the pore domain typical of these other channels. Na_V, K_V, and Ca_V proteins can be found in neurons and muscles, where they play important roles in electrical excitability. In contrast, VSD-containing proteins lacking a pore domain are found in non-excitable cells and are not involved in neuronal signaling. Here, we report the identification of HVRP1, a protein related to the Hv1 channel (from which the name Hv1 Related Protein 1 is derived), which we find to be expressed primarily in the central nervous system, and particularly in the cerebellum. Within the cerebellar tissue, HVRP1 is specifically expressed in granule neurons, as determined by in situ hybridization and immunohistochemistry. Analysis of subcellular distribution via electron microscopy and immunogold labeling reveals that the protein localizes on the post-synaptic side of contacts between glutamatergic mossy fibers and the granule cells. We also find that, despite the similarities in amino acid sequence and structural organization between Hv1 and HVRP1, the two proteins have distinct functional properties. The high conservation of HVRP1 in vertebrates and its cellular and subcellular localizations suggest an important function in the nervous system.     

The Functional Regulation of TRPV1 and Its Role in Pain Sensitization

Transient receptor potential V1 (TRPV1) is specifically expressed in the nociceptive receptors and can detect a variety of noxious stimuli, thus potentiating pain sensitization. While peripheral delivery of capsaicin causes the desensitization of sensory neurons, thus alleviating pain. Therefore capsaicin is used in the clinical treatment of various types of pain; however, these treatments will bring many side effects, such as a strong burning pain in the early stages of treatment which hampers the further use of capsaicin. Thus, the studies of the functional regulation of TRPV1 are mainly focused on two aspects: to develop more potent analogues of capsaicin with less side effects; or to elucidate the mechanisms of TRPV1 in pain sensitivity, especially of that TRPV1 as a target of various protein kinases such as PKD1 and Cdk5 is involved pain hypersensitivity. Thus we would summarize the progress of these two aspects in this mini review. Special issue article in honor of Dr. Ji-Sheng Han.     

植物固醇合成途径关键基因CPI1的功能进化

固醇是真核生物膜的重要组分, 在生长发育中具有重要作用。CPI1 (CYCLOPROPYL STEROL ISOMERASE1)基因是植物特有的固醇合成途径基因, 其编码产物为环丙基固醇异构酶。目前只有拟南芥(Arabidopsis thaliana)的CPI1基因被克隆并解析。研究发现, 从藻类到高等开花植物中均存在单一拷贝的CPI1基因。陆生植物CPI1的基因结构及其所编码的氨基酸序列均高度保守, 蛋白质序列相似性范围为48%–90%, 但陆生植物CPI1与绿藻CPI1的蛋白序列之间存在显著差异。蛋白质结构预测发现CPI1具有非常相似的拓扑结构, 均具有7个跨膜结构域和6个亲水环。组织表达模式分析显示, 陆生植物CPI1在不同组织中均表达, 是组成型表达基因。为了验证CPI1基因的功能, 克隆了二穗短柄草(Brachypodium distachyon)BdCPI1基因, 并转化拟南芥cpi1-1突变体。结果表明, BdCPI1能完全回补cpi1-1突变体的表型。基于单拷贝基因数目、保守的基因结构和蛋白质拓扑结构及基因表达模式, 推测CPI1基因的功能可能在陆生植物中高度保守。     

Structural Insights into a Plant Mechanosensitive Ion Channel MSL1

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Stoichiometry studies reveal functional properties of KDC1 in plant shaker potassium channels

Functional heteromeric plant Shaker potassium channels can be formed by the assembly of subunits from different tissues, as well as from diverse plant species. KDC1 (K(+) Daucus carota 1) produces inward-rectifying currents in Xenopus oocytes when coexpressed with KAT1 and other subunits appertaining to different plant Shaker subfamilies. Owing to the presence of KDC1, resulting heteromeric channels display slower activation kinetics, a shift of the activation threshold toward more negative membrane potentials and current potentiation upon the addition of external zinc. Despite available information on heteromerization of plant Shaker channels, very little is known to date on the properties of the various stoichiometric configurations formed by different subunits. To investigate the functional properties of heteromeric nKDC1/mKAT1 configurations, we realized a series of dimeric constructs combining KDC1 and KAT1 alpha-subunits. We found that homomeric channels, formed by monomeric or dimeric alpha-subunit constructs, show identical biophysical characteristics. Coinjections of diverse tandem constructs, instead, displayed significantly different currents proving that KDC1 has high affinity for KAT1 and participates in the formation of functional channels with at most two KDC1 subunits, whereas three KDC1 subunits prevented the formation of functional channels. This article brings a contribution to the understanding of the molecular mechanisms regulating plant Shaker channel functionality by association of modulatory subunits.     

The Microtubule Binding Properties of CENP-E's C-Terminus and CENP-F

CENP-E (centromere protein E) and CENP-F (centromere protein F), also known as mitosin, are large, multi-functional proteins associated with the outer kinetochore. CENP-E features a well-characterized kinesin motor domain at its N-terminus and a second microtubule-binding domain at its C-terminus of unknown function. CENP-F is important for the formation of proper kinetochore–microtubule attachment and, similar to CENP-E, contains two microtubule-binding domains at its termini. While the importance of these proteins is known, the details of their interactions with microtubules have not yet been investigated. We have biochemically and structurally characterized the microtubule-binding properties of the amino- and carboxyl-terminal domains of CENP-F as well as the carboxyl-terminal (non-kinesin) domain of CENP-E. CENP-E's C-terminus and CENP-F's N-terminus bind microtubules with similar affinity to the well-characterized Ndc80 complex, while CENP-F's C-terminus shows much lower affinity. Electron microscopy analysis reveals that all of these domains engage the microtubule surface in a disordered manner, suggesting that these factors have no favored binding geometry and may allow for initial side-on attachments early in mitosis.