Identification and functional characterization of cation-chloride cotransporters in plants

Chloride (Cl(-)) is an essential nutrient and one of the most abundant inorganic anions in plant tissues. We have cloned an Arabidopsis thaliana cDNA encoding for a member of the cation-Cl(-) cotransporter (CCC) family. Deduced plant CCC proteins are highly conserved, and phylogenetic analyses revealed their relationships to the sub-family of animal K(+):Cl(-) cotransporters. In Xenopus laevis oocytes, the A. thaliana CCC protein (At CCC) catalysed the co-ordinated symport of K(+), Na(+) and Cl(-), and this transport activity was inhibited by the 'loop' diuretic bumetanide, a specific inhibitor of vertebrate Na(+):K(+):Cl(-) cotransporters, indicating that At CCC encodes for a bona fide Na(+):K(+):Cl(-) cotransporter. Analysis of At CCC promoter-beta-glucuronidase transgenic Arabidopsis plants revealed preferential expression in the root and shoot vasculature at the xylem/symplast boundary, root tips, trichomes, leaf hydathodes, leaf stipules and anthers. Plants homozygous for two independent T-DNA insertions in the CCC gene exhibited shorter organs such as inflorescence stems, roots, leaves and siliques. The elongation zone of the inflorescence stem of ccc plants often necrosed during bolt emergence, while seed production was strongly impaired. In addition, ccc plants exhibited defective Cl(-) homeostasis under high salinity, as they accumulated higher and lower Cl(-) amounts in shoots and roots, respectively, than the treated wild type, suggesting At CCC involvement in long-distance Cl(-) transport. Compelling evidence is provided on the occurrence of cation-chloride cotransporters in the plant kingdom and their significant role in major plant developmental processes and Cl(-) homeostasis.     

反义寡核苷酸片段可抑制鲫鱼视网膜电压依赖性钾离子通道的表达

爪蟾卵母细胞经注射鲫鱼视网总ＲＮＡ后,可表达大量电压依赖性钾离子通道。在此基础上,我们进一步发现,一个特定序列的寡核苷酸能专一地抑制该通道的表达。由于我们设计与合成的该片段与果蝇、小鼠中已克隆的钾通道中编码Ｎ端的一个多肽的ｍＲＮＡ完全互补,因此推测：鲫鱼视网膜中的Ｋ这个区域与其它物种的Ｋ通道有高度同源性,这为克隆该基因和研究它的功能提供了重要资料。     

Mechanism of inhibition of net ion transport across frog corneal epithelium by calcium channel antagonists

Summary In the isolated bullfrog cornea, three calcium channel antagonists had dose-dependent inhibitory effects on the Cl-originated short-circuit current (SCC). Their order of decreasing potency was bepridil, verapamil and diltiazem. One millimolar diltiazem inhibited the SCC by 98% and subsequent incubation with the calcium ionophore A23187 had no restorative effect. Increasing the bathing solution Ca concentration from 0.05 to 15mm, however, decreased diltiazem's inhibitory efficacy. This antagonist depolarized the intracellular potential differenceV _m from –54 to –18 mV (tear: reference) and the voltage divider ratioFR ₀ decreased from 0.58 to 0.30, suggesting an increase in basolateral membrane electrical resistance. Additional indication of a basolateral membrane effect by the drug was that preincubation with 10⁵ m amphotericin B in Cl-free Ringer's did not eliminate the inhibitory effect of the drug on the Na- and K-elicited SCC. In the absence of amphotericin B in Cl-free Ringer's (SCC=0), 1 ×10³ m diltiazem depolarized theV _m from –78 to –9 mV suggesting that the increase in basolateral membrane resistance was due to K channel blockade. Diltiazem (1×10³ m) significantly decreased cyclic AMP content; however, isoproterenol in the presence of the drug increased cyclic AMP fourfold without having any restorative effect on the inhibited SCC. Therefore, the inhibition of the Cl-originated SCC resulting from an increase in basolateral membrane K resistance is not caused by a decline in cyclic AMP content. In plasma membrane-enriched fractions prepared from broken cell preparations of bovine corneal epithelium, 1×10³ m diltiazem had no inhibitory effects on either Na,K-ATPase or Ca,Mg-ATPase activities. These latter effects further point to the selectivity of diltiazem as an inhibitor of K-channel activity, but do not preclude a Ca-channel blocker effect by the drug in the micromolar range.     

转铁蛋白在低钾刺激Madin Darby狗肾细胞钠-钾ATP酶中的作用

体外低钾培养肾细胞能刺激细胞膜钠-钾ATP酶。本研究利用Madin Darby狗肾细胞能在无血清培养液中健康生存48h这一特征,研究体外低钾刺激细胞膜钠-钾ATP酶所依赖的血清中的活性因子,观察了表皮生长因子(EGF)、胰岛素样生长因子(IGF1)、前列腺素1(PGE1)和转铁蛋白(tranderrin)在这一过程中的作用。结果表明,在无血清培养液中低钾并不能刺激细胞膜钠—钾ATP酶,而添加转铁蛋白可模拟血清的作用。转铁蛋白能剂量依赖性地增加ouabain结合位点,对细胞膜钠-钾ATP酶作用呈良好的时间效应关系。在低钾无血清培养液中,细胞膜钠-钾ATP酶α1亚基启动子活性增强,α1与β1亚基蛋白质表达的增加依赖于转铁蛋白的存在。进一步研究结果表明,低钾在转铁蛋白的无血清培养液环境中能增加细胞对铁的摄取(^59Fe),该作用可被铁螯合剂(deferoxamine,DFO;35 μmol/L)所阻断。DFO也可阻断转铁蛋白依赖性低钾刺激细胞膜钠-钾ATP酶数目的增多,α1亚基启动子活性增强,α1与β1亚基蛋白质表达增加。以上结果表明,低钾对细胞膜钠-钾ATP酶活性的刺激作用依赖于转铁蛋白所调节的铁的摄取。     

Nuclear magnetic resonance studies of the location and function of plant nutrients in vivo

The cytoplasmic and vacuolar pools of ammonium, inorganic phosphate and potassium can be studied non-invasively in plant tissues using high resolution nuclear magnetic resonance spectroscopy. The techniques that allow these pools to be discriminated in vivo are described and their application to plants is reviewed with reference to the phosphorus, nitrogen and potassium nutrition of root tissues.     

检测钾离子通道的小分子荧光探针的研究进展

钾离子通道是分布最为广泛、种类繁多的一类离子通道,因其生理功能的多样性,已成为许多疾病的药物作用靶点。近年来,许 多化学结构不同的药物均因钾离子通道阻滞引起的严重心肌毒性而被撤出市场,使得小分子药物的钾通道抑制活性筛选面临重大挑战。 介绍检测钾离子通道的小分子荧光探针的研究进展,并总结小分子荧光探针的作用机制,为今后小分子荧光探针的设计提供思路,使得 小分子荧光探针可以广泛应用于候选药物的高通量筛选、钾离子通道的活体成像与检测。     

Cross talk between mitochondria and NADPH oxidases

Reactive oxygen species (ROS) play an important role in physiological and pathological processes. In recent years, a feed-forward regulation of the ROS sources has been reported. The interactions between the main cellular sources of ROS, such as mitochondria and NADPH oxidases, however, remain obscure. This work summarizes the latest findings on the role of cross talk between mitochondria and NADPH oxidases in pathophysiological processes. Mitochondria have the highest levels of antioxidants in the cell and play an important role in the maintenance of cellular redox status, thereby acting as an ROS and redox sink and limiting NADPH oxidase activity. Mitochondria, however, are not only a target for ROS produced by NADPH oxidase but also a significant source of ROS, which under certain conditions may stimulate NADPH oxidases. This cross talk between mitochondria and NADPH oxidases, therefore, may represent a feed-forward vicious cycle of ROS production, which can be pharmacologically targeted under conditions of oxidative stress. It has been demonstrated that mitochondria-targeted antioxidants break this vicious cycle, inhibiting ROS production by mitochondria and reducing NADPH oxidase activity. This may provide a novel strategy for treatment of many pathological conditions including aging, atherosclerosis, diabetes, hypertension, and degenerative neurological disorders in which mitochondrial oxidative stress seems to play a role. It is conceivable that the use of mitochondria-targeted treatments would be effective in these conditions.     

氮、磷、钾肥配施对桂花品种'金玉台阁'开花性状及叶片中叶绿素和营养元素含量的影响

以桂花品种'金玉台阁'(Osmanthus fragrans'Jinyu Taige')盆栽苗为供试材料,采用"3414"肥料效应试验设计方案,研究了不同单株施用量CO(NH2)2(0.0、1.0、2.0和3.0 g)、NH4H2PO4(0.0、1.5、3.0和4.5 g)和KCl(0.0、0.5、1.0、1.5 g)对秋冬生长期(2014年10月至2015年2月)'金玉台阁'开花性状及叶片中叶绿素和营养元素的影响进行了比较和分析,并筛选出适宜的氮、磷、钾肥施用量.结果表明:与CK〔CO(NH2)2、NH4H2PO4和KCl的单株施用量均为0.0 g〕组相比,各氮、磷、钾肥配施处理组'金玉台阁'的单株产花量、花瓣相对含水量、花径以及叶片中叶绿素、全氮、全磷和全钾的含量总体上显著升高.CO(NH2)2单株施用量对'金玉台阁'单株产花量、叶片中叶绿素含量和全氮含量的影响最大,NH4H2PO4单株施用量对这3个指标的影响次之;NH4H2PO4单株施用量对'金玉台阁'叶片中全磷含量的影响最大;KCl单株施用量对'金玉台阁'花瓣相对含水量、花径及叶片中全钾含量的影响最大.在秋冬生长期内叶片中叶绿素含量呈先降低后升高的趋势,均在12月份降至最低值.通过建立'金玉台阁'单株产花量(y)与CO(NH2)2、NH4 H2 PO4和KCl的单株施用量(分别为xN、xP和xK)间的肥料效应模型,得到的三元二次肥料效应模型为y=1.2027+0.7561×xN+0.2632×xP+0.4590×xK-0.2761×xN2-0.1201×xP2-0.5007×xK2+0.1901×xN×xP-0.0960×xN×xK+0.1185×xP×xK.以单株产花量为目标,'金玉台阁'的CO(NH2)2、NH4 H2 PO4和KCl的最佳单株施用量分别为2.58、3.56和0.86 g.     

Structural insight into the transmembrane segments 3 and 4 of the hERG potassium channel

The hERG (human ether‐a‐go‐go related gene) potassium channel is a voltage‐gated potassium channel containing an N‐terminal domain, a voltage‐sensor domain, a pore domain and a C‐terminal domain. The transmembrane segment 4 (S4) is important for sensing changes of membrane potentials through positively charge residues. A construct containing partial S2–S3 linker, S3, S4 and the S4–S5 linker of the hERG channel was purified into detergent micelles. This construct exhibits good quality NMR spectrum when it was purified in lyso‐myristoyl phosphatidylglycerol (LMPG) micelles. Structural study showed that S3 contains two short helices with a negatively charged surface. The S4 and S4–S5 linker adopt helical structures. The six positively charged residues in S4 localize at different sides, suggesting that they may have different functions in channel gating. Relaxation studies indicated that S3 is more flexible than S4. The boundaries of S3–S4 and S4–S4–S5 linker were identified. Our results provided structural information of the S3 and S4, which will be helpful to understand their roles in channel gating. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.