Functional expression of the plant K channel KAT1 in insect cells

Following the biophysical analysis of plant K⁺ channels in their natural environment, three members from the green branch of the evolutionary tree of life KAT1, AKT1 and KST1 have recently been identified on the molecular level. Among them, we focused on the expression and characterization of the Arabidopsis thaliana K⁺ channel KAT1 in the insect cell line Sf9. The infection of Sf9 cells with KAT1-recombinant baculovirus resulted in functional expression of KAT1 channels, which was monitored by inward-rectifying, K⁺-selective (impermeable to Na⁺ and even NH₄⁺) ionic conductance in whole-cell patch-clamp recordings. A voltage threshold as low as −60 to −80 mV for voltage activation compared to other plant inward rectifiers in vivo, and to in vitro expression of KAT1 in Xenopus oocytes or yeast, may be indicative for channel modulation by the expression system. A rise in cytoplasmic Ca²⁺ concentration (up to 1 mM), a regulator of the inward rectifier in Vicia faba guard cells, did not modify the voltage dependence of KAT1 in Sf9 cells. The access to channel function on one side and channel protein on the other make Sf9 cells a suitable heterologous system for studies on the biophysical properties, post-translational modification and assembly of a green inward rectifier.     

Expression of an inwardly rectifying K+ channel from rat basophilic leukemia cell mRNA in Xenopus oocytes

Rat basophilic leukemia cells (RBL-2H3) have previously been shown to contain a single type of voltage-activated channel, namely an inwardly rectifying K⁺ channel, under normal recording conditions. Thus, RBL-2H3 cells seemed like a logical source of mRNA for the expression cloning of inwardly rectifying K⁺ channels. Injection of mRNA isolated from RBL-2H3 cells into Xenopus oocytes resulted in the expression of an inward current which (1) activated at potentials negative to the K⁺ equilibrium potential (E_K), (2)decreased in slope conductance near E_K, (3) was dependent on [K⁺]_o and (4) was blocked by external Ba²⁺ and Cs⁺. These properties were similar to those of the inwardly rectifying K⁺ current recorded from RBL-2H3 cells using whole-cell voltage clamp. Injection of size-fractionated mRNA into Xenopus oocytes revealed that the current was most strongly expressed from the fraction containing mRNA of approximately 4–5 kb. Expression of this channel represents a starting point for the expression cloning of a novel class of K⁺ channels.     

Transient hyperpolarization of yeast by glucose and ethanol

At pH 7, addition of glucose under anaerobic conditions to a suspension of the yeast Saccharomyces cerevisiae causes both a transient hyperpolarization and a transient net efflux of K⁺ from the cells. Hyperpolarization shows a peak at about 3 min and a net K⁺ efflux at 4–5 min. An additional transient hyperpolarization and net K⁺ efflux are found after 60–80 and 100 min, respectively. Addition of 2-deoxyglucose instead of glucose does not lead to hyperpolarization of the cells or K⁺ efflux. At low pH, neither transient hyperpolarization nor a transient K⁺ efflux are found. With ethanol as substrate and applying aerobic conditions, both a transient hyperpolarization and a transient K⁺ efflux are found at pH 7. The fluorescent probe 2-(dimethylaminostyryl)-1-ethylpyridinium appears to be useful for probing changes in the membrane potential of S. cerevisiae. It is hypothesized that the hyperpolarization of the cells is due to opening of K⁺ channels in the plasma membrane. Accordingly, the hyperpolarization of the cells at pH 7 is almost completely abolished by 1.25 mM K⁺, whereas the same amount of Na⁺ does not reduce the hyperpolarization     

Identification and characterization of functional subunits of Clostridium botulinum type A progenitor toxin involved in binding to intestinal microvilli and erythrocytes

Dimethyl adipimidate (DMA) reduces K⁺ loss from, and dehydration of, red cells containing haemoglobin S (HbS cells). Three membrane transporters may contribute to these processes: the deoxygenation-induced cation-selective channel (P_sickle), the Ca²⁺-activated K⁺ channel (or Gardos channel) and the K⁺-Cl⁻ cotransporter (KCC). We show that DMA inhibited all three pathways in deoxygenated HbS cells. The Gardos channel could be activated following Ca²⁺ loading. Considerable KCC activity was present in oxygenated HbS cells, showing a selective action of DMA on the transporter in deoxygenated cells. Inhibition of sickling correlated strongly with that of P_sickle and moderately with that of KCC activity. We conclude that DMA does not inhibit the K⁺ pathways directly, but acts mainly by preventing HbS polymerisation and sickling. These findings are relevant to the development of novel chemotherapeutic agents for amelioration of sickle cell disease.     

Electrophysiology of mammalian Schwann cells

Schwann cells are the satellite cell of the peripheral nervous system, and they surround axons and motor nerve terminals. The review summarises evidence for the ion channels expressed by mammalian Schwann cells, their molecular nature and known or speculated functions. In addition, the recent evidence for gap junctions and cytoplasmic diffusion pathways within the myelin and the functional consequences of a lower-resistance myelin sheath are discussed.

The main types of ion channel expressed by Schwann cells are K⁺ channels, Cl⁻ channels, Na⁺ channels and Ca²⁺ channels. Each is represented by a variety of sub-types. The molecular and biophysical characteristics of the cation channels expressed by Schwann cells are closely similar or identical to those of channels expressed in peripheral axons and elsewhere. In addition, Schwann cells express P₂X ligand-gated ion channels. Possible in vivo roles for each ion channel type are discussed. Ion channel expression in culture could have a special function in driving or controlling cell proliferation and recent evidence indicates that some Ca²⁺ channel and Kir channel expression in culture is dependent upon the presence of neurones and local electrical activity.     

植物K+通道AKT1的研究进展

钾(K)是植物生长发育必需的大量营养元素之一, 主要通过根细胞的K⁺通道及转运蛋白介导吸收。AKT1是Shaker型K⁺通道家族的重要成员, 在植物根吸收K⁺和体内跨膜转运中发挥重要作用。该文综述了植物AKT1的分子结构、组织特异性表达、调控机制及生物学功能等方面的研究进展, 并对该通道今后的研究方向进行了展望。     

Development of renal function

The mammalian metanephric kidney develops following a general principle of organogenesis of epithelial organs, i.e., along the tree-like structure of an arborizing ductal system (the ureteric bud and cortical collecting duct). In parallel, the proximal portions of the uriniferous tubule develop by mesenchymal-to-epithelial transition of the neighbouring mesenchyme. On one hand, vectorial transport systems in nephrogenesis should be functional at the onset of glomerular filtration in any of the newly formed nephron generations to prevent loss of salt, water and metabolites. On the other hand, developing nephron epithelia must serve the needs of organ-formation such as cell proliferation and fluid-secretion for morphogenic purposes. This review intends to summarize current data and concepts on the development of renal epithelial functions with an emphasis on ion channels. Current model systems are introduced, such as ureteric bud cell monolayer culture, in vitro nephron culture, HEK293 cell culture, and the dissection of tubular cells for direct analysis. The current data on the developmental expression and functions of ENaC Na⁺ channels, the CFTR, ClC-2 Cl^ndash; channels, L-type Ca²⁺ channels, P2 purinoceptors, and the Kir6.1/SUR2, ROMK (Kir1.1), and Kv K⁺ channels are presented.     

Functional regulation of reconstituted Na, K-ATPase by protein kinase A phosphorylation

Reconstituted Na⁺,K⁺-ATPase from either pig kidney or shark rectal glands was phosphorylated by cAMP dependent protein kinase, PKA. The stoichiometry was 0.9 mole P_i/mole -subunit in the pig kidney enzyme and 0.2 mol P_i/mol -subunit in the shark enzyme. In shark Na⁺,K⁺-ATPase PKA phosphorylation increased the maximum hydrolytic activity for cytoplasmic Na⁺ activation and extracellular K⁺ activation without affecting the apparent K_m values. In contrast, no significant functional effect after PKA phosphorylation was observed in pig kidney Na⁺,K⁺-ATPase.     

Development of a cell-free translation system from Cucumis melo: preparation, optimization and evaluation of sensitivity to some translational inhibitors

A very active cell-free translation system was prepared from 4–5-day-old embryonic axes of melon (Cucumis melo L.), a species whose dry seeds contain a powerful translational inhibitor. The system was optimized for Mg²⁺, K⁺, NH₄⁺, high speed supernatant, total wheat germ tRNA, time and temperature. Using a 30 000 × g supernatant, the system translates endogenous messengers and polyuridylic acid very efficiently. Melon ribosomes were inhibited in vitro by several well-known eukaryotic inhibitors including melonin, the protein inhibitor present in the dry seeds of C. melo. Our results suggest that the protein inhibitor does not affect the activity of melon ribosomes neither in vivo nor during their isolation.     

A binding-block ion selective mechanism revealed by a Na/K selective channel

Mechanosensitive (MS) channels are extensively studied membrane protein for maintaining intracellular homeostasis through translocating solutes and ions across the membrane, but its mechanisms of channel gating and ion selectivity are largely unknown. Here, we identified the YnaI channel as the Na⁺/K⁺ cation-selective MS channel and solved its structure at 3.8 Å by cryo-EM single-particle method. YnaI exhibits low conductance among the family of MS channels in E. coli, and shares a similar overall heptamer structure fold with previously studied MscS channels. By combining structural based mutagenesis, quantum mechanical and electrophysiological characterizations, we revealed that ion selective filter formed by seven hydrophobic methionine (YnaI^Met158) in the transmembrane pore determined ion selectivity, and both ion selectivity and gating of YnaI channel were affected by accompanying anions in solution. Further quantum simulation and functional validation support that the distinct binding energies with various anions to YnaI^Met158 facilitate Na⁺/K⁺ pass through, which was defined as bindingblock mechanism. Our structural and functional studies provided a new perspective for understanding the mechanism of how MS channels select ions driven by mechanical force.     

Effects of Tetraethylammoniumchloride (TEA), Vanadate, and Alkali Ions on the Lateral Leaflet Movement Rhythm of Desmodium motorium (Houtt.) Merr

The period (∼3-5 min) of the ultradian rhythm of the lateral leaflet movement of Desmodium motorium is strongly lengthened (≤30-40%) by the K⁺ channel blocker tetraethylammoniumchloride (20, 30, and 40 mM) and vanadate (0.5 and 1 mM), which is an effective inhibitor of the plasma membrane-bound H⁺ pump. The alkali ions K⁺, Na⁺, Rb⁺, and Cs⁺ (10-40 mM) shorten the period only slightly (≤ 10-15%). Li⁺ (5-30 mM), however, increases the period of the leaflet rhythm drastically (≤80%). We concluded that the plasmalemma-H⁺-ATP-ase-driven K⁺ transport through K⁺ channels is an essential component of the ultradian oscillator of Desmodium, as has been proposed for the circadian oscillator.     

Influence of ATPase activity on PPi dependent H-transport in tonoplast vesicles of Acer pseudoplatanus

Tonoplast H⁺-ATPase and H⁺-pyrophosphatase (H⁺-PPase) were previously characterized in Acer pseudoplatanus cells (A. Pugin et al., Plant Sci., 73 (1991) 23–34; A. Fraichard et al., Plant Physiol. Biochem., 31 (1993) 349–359). The present study concerns the relationships between these two enzymes in vitro. ATP and PPi hydrolysis were additive and the inhibition of one did not affect the activity of the second one. ATP and PPi H⁺-transports were also additive. The H⁺ -PPase inhibition did not change ATP-dependent H⁺-transport but H⁺-ATPase inhibition inhibited the PPi dependent H⁺-transport. Because H⁺-PPase was reported to transport H⁺ and K⁺ into the vacuole (Davies et al., Proc. Natl. Acad. Sci. USA, 89 (1992) 11701–11705), these results led us to suggest that the inhibition of the H⁺-ATPase activity could modify the H⁺/K⁺ stoichiometry for the benefit of K⁺-transport.     

The blocking effect of BmP02, one novel short-chain scorpion peptide on transient outward K(+) channel of adult rat ventricular myocyte

Two components F-2-7-4 and F-2-7-5, each composed of 28 amino acid residues, were purified from the venom of Buthus martensi Karsch by an opportune procedure with cation-exchange column chromatography and repeated HPLC. Both components were totally accounted to about 0.88% dry weight of the crude venom.The molecular weights of both components were determined to be 2950 and 2935 by mass spectrometry, which were fully coincidence with that of the known novel short-chain peptides BmP02 and BmP03, respectively [Romi-Lebrun R, Martin-Eauclaire M-F, Escoubas P, Wu FQ, Lebrun B, Hisada M, Nakajima T. Characterization of four toxins from Buthus martensi scorpion venom, which act on apamin-sensitive Ca²⁺-activated K⁺ channels. Eur J Biochem 1997;145:457–464]. In addition, the sequence of component F-2-7-4 was analyzed to be the same as that of BmP02. The components F-2-7-4 and F-2-7-5 purified in this study were, thus, finally distinguished to be BmP02 and BmP03 from the same venom. Using whole cell patch-clamp recording, it was found that BmP02 diminished the current of transient outward K⁺ channel in adult rat ventricular myocyte in a concentration-dependent manner. The inhibitory effect was reversible. Dynamic studies showed that the activation, inactivation and recovery processes of the transient outward K⁺ channel were not changed significantly after applying of BmP02. In addition, when BmP02 was applied to guinea pig ventricular myocyte, both delayed and inward rectified K⁺ currents showed no change compared with the control. The results suggest strongly that BmP02 or -like peptides from scorpion venom may provide a useful probe for the studying of transient outward K⁺ channel in rat ventricular myocyte.     

Novel ion channels in the protists, Mougeotia and Saprolegnia, using sub-gigaseals.

Protoplasts of the filamentous alga, Mougeotia, and the filamentous fungal oomycete, Saprolegnia ferax, exhibit two K⁺ ion channels (2–6 pA) using the patch-clamp technique when the seals are less than 1 GΩ (about 100 MΩ). The membrane potential of the protoplasts was near 0 mV as measured intracellularly with double-barreled micropipettes; thus, inward K⁺ flux is due solely to concentration differences. Although conductances are in the range expected for K⁺ channels, the activity at 0 mV is not seen in other organisms under gigaseal conditions. This paper draws attention to the usefulness of this subsidiary patch-clamp technique and the novel characteristics of ion channels in Mougeotia and Saprolegnia.     

The viral potassium channel Kcv: structural and functional features

The chlorella virus PBCV-1 was the first virus found to encode a functional potassium channel protein (Kcv). Kcv is small (94 aa) and basically consists of the M1-P-M2 (membrane-pore-membrane) module typical of the pore regions of all known potassium channels. Kcv forms functional channels in three heterologous systems. This brief review discusses the gating, permeability and modulation properties of Kcv and compares them to the properties of bacterial and mammalian K+ channels.     

外源喜树碱对植物叶片气孔导度的影响

前人研究表明,保卫细胞微管系统在气孔运动中起到重要作用:保卫细胞质膜上内向K⁺通道的正常活性有赖于微管的正常解聚/聚合的动态变化,微管系统可能通过调节保卫细胞K⁺通道而控制气孔运动,即微管解聚导致内向K⁺通道关闭,保卫细胞无法因膨压调节吸收水分而抑制气孔开放。有学者认为,喜树碱或其类似物能够与微管蛋白结合,并降低微管结构稳定性,其机制则可能是抑制细胞高分子量微管结合蛋白对微管组装的辅助作用,但这方面的实验证据相对匮乏。因此,为了进一步研究喜树碱的生态生物化学功能,我们采取叶面喷施喜树碱和PEG模拟干旱诱导气孔关闭的方法,研究了外源喜树碱对喜树幼苗气孔导度的影响,同时以没有内源喜树碱的烟草为实验材料进行了对照研究。研究结果表明:0.0115mmol·L^-1的喜树碱水饱和溶液对喜树和烟草幼苗叶片气孔导度的影响规律一致,均表现出明显的抑制气孔开放的效果,这为喜树碱与微管蛋白结合提供了部分证据。     

Sweet taste receptor interacting protein CIB1 is a general inhibitor of InsP3-dependent Ca2+ release in vivo

In a search for sweet taste receptor interacting proteins, we have identified the calcium- and integrin-binding protein 1 (CIB1) as specific binding partner of the intracellular carboxyterminal domain of the rat sweet taste receptor subunit Tas1r2. In heterologous human embryonic kidney 293 (HEK293) cells, the G protein chimeras Gα_16gust44 and Gα_15i3 link the sweet taste receptor dimer TAS1R2/TAS1R3 to an inositol 1,4,5-trisphosphate (InsP₃)-dependent Ca²⁺ release pathway. To demonstrate the influence of CIB1 on the cytosolic Ca²⁺ concentration, we used sweet and umami compounds as well as other InsP₃-generating ligands in FURA-2-based Ca²⁺ assays in wild-type HEK293 cells and HEK293 cells expressing functional human sweet and umami taste receptor dimers. Stable and transient depletion of CIB1 by short-hairpin RNA increased the Ca²⁺ response of HEK293 cells to the InsP₃-generating ligands ATP, UTP and carbachol. Over-expression of CIB1 had the opposite effect as shown for the sweet ligand saccharin, the umami receptor ligand monosodium glutamate and UTP. The CIB1 effect was dependent on the thapsigargin-sensitive Ca²⁺ store of the endoplasmic reticulum (ER) and independent of extracellular Ca²⁺. The function of CIB1 on InsP₃-evoked Ca²⁺ release from the ER is most likely mediated by its interaction with the InsP₃ receptor. Thus, CIB1 seems to be an inhibitor of InsP₃-dependent Ca²⁺ release in vivo .     

Ion dependent efflux from neuronal and glial cell cultures

A spontaneous efflux of choline originating from the cytoplasmic free choline compartment and, partly, from metabolized form was measured from neurons and glial cells in culture. The efflux was stimulated by an excess of K⁺ and by the absence of Ca²⁺ ions from the incubation medium in both types of culture. The two effects did not appear to be synergistic.

The stimulation produced by an excess of K⁺ (100 mM) was blocked in neurons by 0.5 μM BaCl₂ and in glia cells by 0.1 μM BaCl₂ (in the presence of 30 mM K⁺). The stimulation produced by the absence of Ca²⁺ instead was not blocked by Ba²⁺ ions in either of the two types of culture. The results suggest that the stimulation induced by K⁺ (high concentration and long time of incubation) might be of biochemical rather than physiological nature and that choline may be driven out of the cells in correlation with the K⁺ gradient. The greater sensitivity of glial cells to K⁺ ions may also suggest a supportive role of these cells with respect to neurons, as they seem capable of furnishing choline for neuronal needs during depolarization.     

Structure-activity relationships of an inhibitory nematode FMRFamide-related peptide,SDPNFLRFamide (PF1), on Ascaris suum muscle

FMRFamide-related peptides are widespread among the Nematoda. Among them is a family of extended PNFLRFamide peptides encoded on the flp-1 peptide precursor gene in Caenorhabditis elegans. The most studied peptide from this series is SDPNFLRFamide (PF1). Each residue in this peptide was sequentially substituted with either alanine or the corresponding d-isomer of the native amino acid in order to define structure–function relationships in this peptide using an Ascaris suum muscle tension assay. In general, substitutions in the N-terminal tetrapeptide had only minor consequences for efficacy, while substitutions in the C-terminal tetrapeptide caused more dramatic changes. Such substitutions typically markedly diminished efficacy, but d-isomer substitution at either position 5 (Phe) or 6 (Leu) converted the inhibitory activity of the prototype into excitation. In addition, it has been evident that KPNFLRFamide and SDPNFLRFamide, though encoded on flp-1 and sharing a PNFLRFamide hexapeptide, act through different receptors. KPNFLRFamide directly gates a chloride channel in A. suum muscle cells, while SDPNFLRFamide acts through nitric oxide synthase to open K⁺ channels in the same tissue. The use of K⁺ channel blockers and nitric oxide synthase inhibitors in electrophysiological experiments employing A. suum muscle membranes allowed the unambiguous conclusion that the N-terminal lysine is absolutely required for activation of the chloride channel and excludes interaction with the SDPNFLRFamide receptor.     

Inhibition of cell growth by K channel modulators is due to interference with agonist-induced Ca release

The effects of K⁺ channel modulators, tetraethylammonium, 4-aminopyridine and diazoxide, and high extracellular K⁺ on cell growth and agonist-induced intracellular Ca²⁺ mobilization were investigated. Two human brain tumour cell lines, U-373 MG astrocytoma and SK-N-MC neuroblastoma, were used as model cellular systems. K⁺ channel modulators and increased extracellular K⁺ concentration inhibited tumour cell growth in a dose-related fashion in both cell lines. In addition, agonist (carbachol or serum)-induced intracellular Ca²⁺ mobilization was also blocked by the pretreatment of growth-inhibitory concentrations of K⁺ channel modulators and high extracellular K⁺. Thus, these results suggest that K⁺ channel modulators are effective inhibitors of brain tumour cell growth and that their growth regulation may be due to the interference with the intracellular Ca²⁺ signalling mechanisms.