Dilated and defunct K channels in the absence of K+

Potassium ions are vital for maintaining functionality of K channels. In their absence, many K channel types enter a long-lasting defunct condition characterized by absence of conductance and drastic changes in gating current. We show that channels pass through a dilated condition with altered selectivity as they are becoming defunct. To characterize these abnormalities we examined gating and ionic currents generated by Shaker IR and by three nonconducting mutants, W434F, D447N, and Y445A, in 0 K+. On entering the dilated condition, Shaker IR becomes permeable to Na+ and tetramethylammonium-positive (TMA+), signaling deformation of the selectivity filter. When dilated, nearly normal closing is possible at -140 mV. At -80 mV, however, closing is very slow and channels stray from the dilated into the defunct condition. Restoration from defunct to dilated condition requires tens of seconds at 0 mV and can occur in the absence of K+. W434F and D447N are similar to Shaker IR, showing Na+ and TMA+ permeability when dilated. The defunct gating currents are similar in Shaker IR and these two mutants and are reminiscent of the early transitions of normal gating. Y445A does not become defunct and shows Na+ but not TMA+ permeability on K+ removal.     

Prokaryotic BirA ligase biotinylates K4, K9, K18 and K23 in histone H3

BirA ligase is a prokaryotic ortholog of holocarboxylase synthetase (HCS) that can biotinylate proteins. This study tested the hypothesis that BirA ligase catalyzes the biotinylation of eukaryotic histones. If so, this would mean that recombinant BirA ligase is a useful surrogate for HCS in studies of histone biotinylation. The biological activity of recombinant BirA ligase was confirmed by enzymatic biotinylation of p67. In particular, it was found that BirA ligase biotinylated both calf thymus histone H1 and human bulk histone extracts. Incubation of recombinant BirA ligase with H3-based synthetic peptides showed that lysines 4, 9, 18, and 23 in histone H3 are the targets for the biotinylation by BirA ligase. Modification of the peptides (e.g., serine phosphorylation) affected the subsequent biotinylation by BirA ligase, suggesting crosstalk between modifications. In conclusion, this study suggests that prokaryotic BirA ligase is a promiscuous enzyme and biotinylates eukaryotic histones. Moreover the biotinylation of histones by BirA ligase is consistent with the proposed role of human HCS in chromatin.     

A hetero-dimer model for concerted action of vitamin K carboxylase and vitamin K reductase in vitamin K cycle

Vitamin K carboxylase (VKC) is believed to convert vitamin K, in the vitamin K cycle, to an alkoxide-epoxide form which then reacts with CO₂ and glutamate to generate γ-carboxyglutamic acid (Gla). Subsequently, vitamin K epoxide reductase (VKOR) is thought to convert the alkoxide-epoxide to a hydroquinone form. By recycling vitamin K, the two integral-membrane proteins, VKC and VKOR, maintain vitamin K levels and sustain the blood coagulation cascade. Unfortunately, NMR or X-ray crystal structures of the two proteins have not been characterized. Thus, our understanding of the vitamin K cycle is only partial at the molecular level. In this study, based on prior biochemical experiments on VKC and VKOR, we propose a hetero-dimeric form of VKC and VKOR that may explain the efficient oxidation and reduction of vitamin K during the vitamin K cycle.     

Regulation of apical K and Na channels and Na/K pumps in rat cortical collecting tubule by dietary K

The patch-clamp technique was used to study the properties and the density of conducting K and Na channels in the apical membrane of rat cortical collecting tubule. The predominant K channel observed in cell- attached patches (SK channels) had an outward single-channel conductance (with LiCl in the pipette) of 10 pS. The inward conductance (with KCl in the pipette) was 42 pS. The channel had a high open probability that increased with depolarization. Kinetic analysis indicated the presence of a single open state and two closed states. Increasing K intake by maintaining animals on a high K diet for 12-16 d increased the number of SK channels per patch by threefold (0.7- 2.0/patch) over control levels. In addition, conducting Na-selective channels, which were not observed in control animals, were seen at low density (0.5/patch). These channels had properties similar to those observed when the animals were on a low Na diet, except that the mean open probability (0.84) was higher. In other experiments, the whole- cell patch clamp technique was used to measure Na channel activity (as amiloride-sensitive current, INa) and Na pump activity (as ouabain- sensitive current, Ipump). In animals on a high K diet, INa was greater than in controls but much less than in rats on a low Na diet. Ipump was greater after K loading than in controls or Na-depleted animals. These K diet-dependent effects were not accompanied by a significant increase in plasma aldosterone concentrations. To further investigate the relationship between K channel activity and mineralocorticoids, rats were maintained on a low Na diet to increase endogenous aldosterone secretion. Under these conditions, no increase in SK channel density was observed, although there was a large increase in the number of Na channels (to 2.7/patch). Aldosterone was also administered exogenously through osmotic minipumps. As with the low Na diet, there was no change in the density of conducting SK channels, although Na channel activity was induced. These results suggest that SK channels, Na channels and Na/K pumps are regulated during changes in K intake by factors other than aldosterone.     

Molecular cloning and analysis of genes for production of K5, K7, K12, and K92 capsular polysaccharides in Escherichia coli.

With a DNA fragment from within the region encoding the transport functions for K1 production as a hybridization probe in Southern blot experiments, homologous DNA sequences were detected in the DNA from Escherichia coli strains producing K5, K7, K92, and K100 capsular polysaccharides. No homology with the laboratory strain LE392 was detected. The same DNA probe was used to prescreen cosmid libraries in LE392 by colony hybridization, as a rapid method to isolate clones encoding the genes for K5, K7, K12, and K92 antigen production. Clones carrying sequences homologous to the probe that also produced capsular material were identified by using polyclonal and monoclonal antibodies raised against the K antigen in question and K antigen-specific phages. By restriction enzyme mapping of the appropriate cosmid clones it was possible to align the genes for the production of different K antigens in terms of common restriction endonuclease cleavage sites. A DNA fragment encoding the postulated transport functions for K7 antigen production could complement deletion mutations in the transport functions for K1 antigen production. Thus the transport to the cell surface of chemically distinct polysaccharides may be by a common process. Analysis in E. coli of the proteins produced by plasmids carrying the likely transport functions for K1, K5, and K7 antigen production revealed that each region coded for a similar polypeptide.     

Transfer systems of K88 and K99 plasmids

The conjugation systems of three K88-mobilizing plasmids were characterized for the morphology of their pili and type of mating system (surface only or surface + liquid). pREI had a typical IncI1 transfer system with both thick and thin pili. pVIDO determined aggregating thick flexible pili and pPLS nonaggregating thick flexible pili. All three transferred equally well in broth and on plates. pPLS alone was naturally transfer-depressed. pREI and pVIDO were tested for K88 mobilization efficiency, which was greater from their wild-type host strains to Escherichia coli K-12 than between E. coli K-12 strains. The K99 conjugative plasmid from strain B41 was repressed for transfer and determined thick flexible pili that were receptors for the filamentous phage fd.     

烤烟烟叶中N,K营养及N,K平衡的初步研究

研究了滇中地区烤烟中部叶片旺长期（移栽后６０－６５ｄ）Ｎ、Ｋ营养及Ｎ、Ｋ平衡。结果表明：⑴烟叶一般Ｎ含量为２．５％－４％,Ｋ含量为１％－３％,Ｎ／Ｋ比为２．５－３．５。⑵烟叶Ｎ含量＞４％,就有Ｎ过剩的症状产生。⑶烟叶Ｎ含量＜２．５％,Ｋ含量＜１％,就表现出Ｎ、Ｋ缺乏症状。⑷烟叶Ｎ／Ｋ比失调,过高或过低,都会影响烟株正常生长。     

Stereoselective Accumulation of Propranolol Enantiomers in K562 and K562/ADR Cells

The stereoselective uptake of propranolol enantiomers was investigated by using the K562 and K562 adriamycin‐resistant cell line (K562/ADR) as a model. An enantioselective RP‐HPLC method was applied to determine the accumulation of propranolol (PPL) stereoisomers in K562 and K562/ADR cells. The concentration, time and temperature dependent studies showed that the accumulation of S‐(?)‐PPL was higher than R‐(+)‐PPL in K562 cells and uptake of R‐(+)‐PPL was significantly higher than that of S‐(?)‐PPL in K562/ADR cells. The results indicate the enantioselective accumulation of propranolol enantiomers in K562 and K562 / ADR cells. Chirality 25:361–364, 2013. © 2013 Wiley Periodicals, Inc.     

K+ Channel in the Chora Plasmalemma: Estimations of K+ Channel Density and Single K+ Channel Conductance

The electromotive force E and the conductance G of the Characorallina plasmalemma were measured under voltage clamp conditions.In the depolarized voltage range less negative than –60mV, E changed according to the Nerhst equation for K⁺, and Gincreased with the external K⁺ concentration [K⁺]_o and alsowith the depolarization of the membrane potential. This is attributedto the voltage-dependent opening of the K⁺ channels in the largelydepolarized voltage region. The voltage-dependent increase ofG was due to the increase of the number of open K⁺ channelsper unit area. The density of the total K⁺ channels in the C. corallina plasmalemmawas estimated to be about 6.50/(10 µm)2. The single K⁺channel conductance _K changed with the external [K⁺]_o; it was79.3, 86.1, 105.9, 119.0 pS for external [K⁺]_o of 0.2, 0.5,2.0 and 5.0 mu respectively. (Received May 22, 1986; Accepted August 22, 1986)     

Genetic Mapping of the Antigenic Determinants of Two Polysaccharide K Antigens, K10 and K54, in Escherichia coli

The genes controlling synthesis of the Escherichia coli acidic polysaccharide capsular antigens K10 and K54 were transferred by conjugation to E. coli strains of other serotypes. The genes concerned with these K antigen determinants showed genetic linkage with the serA locus. We propose to name the K antigen-controlling gene kpsA. The genetic determinants of the two K antigens could also be transferred to enteropathogenic serotypes, even though such strains have never been found in nature with special acidic polysaccharide K antigens. A noncapsulated derivative, K(-), of the K10 strain can transfer the genetic determinant of the K antigen, demonstrating the probable existence of another chromosomal locus involved in the production of such acidic polysaccharide K antigens.     

过表达钾转运蛋白基因trkH提高玉米的钾营养

钾是植物生长发育必须的营养元素,参与多种生理生化过程。土壤缺钾严重影响了玉米的产量和品质,通过遗传改良的方式提高玉米的钾营养是解决这个问题的有效途径。本实验室前期从海洋微生物宏基因组DNA中克隆得到细菌钾转运蛋白基因trkH,在酵母和烟草中验证了功能。为进一步分析trkH基因在玉米中的功能,以Hi-Ⅱ基因型玉米为转化受体,采用农杆菌介导法将trkH基因转入玉米,获得具有Baster抗性的独立转化苗21株。PCR检测结果表明trkH基因已成功转入到玉米基因组中。Bar试纸条检测结果表明Bar基因在蛋白水平上成功表达。将部分T₀代转基因植株与玉米骨干自交系PH6WC杂交,获得8个T₁代株系,半定量RT-PCR检测结果表明转基因植株在转录水平上均能表达。三叶一心期喷洒除草剂进行筛选,选择比例接近1：1的L3、L5和L7转基因株系进行PCR检测,统计检测结果并进行χ²测验,结果表明符合孟德尔分离定律。L3、L5和L7转基因株系玉米苗期的钾耗竭实验结果表明过表达trkH基因能够提高转基因玉米的K⁺吸收,为培育钾营养高效玉米新品种奠定基础。     

K8 and K12 are biotinylated in human histone H4.

Folding of DNA into chromatin is mediated by binding to histones such as H4; association of DNA with histones is regulated by covalent histone modifications, e.g. acetylation, methylation, and biotinylation. We sought to identify amino-acid residues that are biotinylated in histone H4, and to determine whether acetylation and methylation of histones affect biotinylation. Synthetic peptides spanning fragments of human histone H4 were biotinylated enzymatically using biotinidase. Peptide-bound biotin was probed with streptavidin-peroxidase. Peptides based on the N-terminal sequence of histone H4 were effectively recognized by biotinidase as substrates for biotinylation; in contrast, peptides based on the C-terminal sequences were not biotinylated. Substitution of K8 or K12 with alanine or arginine decreased biotinylation, suggesting that these lysines are targets for biotinylation; K8 and K12 are also known targets for acetylation. Chemical acetylation or methylation of a given lysine decreased subsequent enzymatic biotinylation of neighboring lysines, consistent with cross-talk among histone modifications. Substitution of a given lysine (positive charge) with glutamate (negative charge) abolished biotinylation of neighboring lysines, providing evidence that the net charge of histones has a role in biotinylation. An antibody was generated that specifically recognized histone H4 biotinylated at K12. This antibody was used to detect biotinylated histone H4 in nuclear extracts from human cells. These studies suggest that K8 and K12 in histone H4 are targets for biotinylation, that acetylation and biotinylation compete for the same binding sites, and that acetylation and methylation of histones affect biotinylation of neighboring lysines.     

K4, K9 and K18 in human histone H3 are targets for biotinylation by biotinidase

Histones are modified post-translationally, e.g. by methylation of lysine and arginine residues, and by phosphorylation of serine residues. These modifications regulate processes such as gene expression, DNA repair, and mitosis and meiosis. Recently, evidence has been provided that histones are also modified by covalent binding of the vitamin biotin. The aims of this study were to identify biotinylation sites in histone H3, and to investigate the crosstalk among histone biotinylation, methylation and phosphorylation. Synthetic peptides based on the sequence of human histone H3 were used as substrates for enzymatic biotinylation by biotinidase; biotin in peptides was probed using streptavidin peroxidase. These studies provided evidence that K4, K9 and K18 in histone H3 are good targets for biotinylation; K14 and K23 are relatively poor targets. Antibodies were generated to histone H3, biotinylated either at K4, K9 or K18. These antibodies localized to nuclei in human placental cells in immunocytochemistry and immunoblotting experiments, suggesting that lysines in histone H3 are biotinylated in vivo. Dimethylation of R2, R8 and R17 increased biotinylation of K4, K9 and K18, respectively, by biotinidase; phosphorylation of S10 abolished biotinylation of K9. These observations are consistent with crosstalk between biotinylation of histones and other known modifications of histones. We speculate that this crosstalk provides a link to known roles for biotin in gene expression and cell proliferation.     

Voltage-dependent K+ currents and underlying single K+ channels in pheochromocytoma cells

Properties of the whole-cell K+ currents and voltage-dependent activation and inactivation properties of single K+ channels in clonal pheochromocytoma (PC-12) cells were studied using the patch-clamp recording technique. Depolarizing pulses elicited slowly inactivating whole-cell K+ currents, which were blocked by external application of tetraethylammonium+, 4-aminopyridine, and quinidine. The amplitudes and time courses of these K+ currents were largely independent of the prepulse voltage. Although pharmacological agents and manipulation of the voltage-clamp pulse protocol failed to reveal any additional separable whole-cell currents in a majority of the cells examined, single-channel recordings showed that, in addition to the large Ca++-dependent K+ channels described previously in many other preparations, PC-12 cells had at least four distinct types of K+ channels activated by depolarization. These four types of K+ channels differed in the open-channel current-voltage relation, time course of activation and inactivation, and voltage dependence of activation and inactivation. These K+ channels were designated the Kw, Kz, Ky, and Kx channels. The typical chord conductances of these channels were 18, 12, 7, and 7 pS in the excised configuration using Na+-free saline solutions. These four types of K+ channels opened in the presence of low concentrations of internal Ca++ (1 nM). Their voltage-dependent gating properties can account for the properties of the whole-cell K+ currents in PC-12 cells.