新型东亚钳蝎小分子成分的生化性质及其对大鼠心室肌细胞Ito的作用

分离纯化获得了两个东亚钳蝎小分子活性多肽ＢｍＰ０２和ＢｍＰ０３。经质谱鉴定二者皆为单一峰,分子量分别为２．９５０ｋＤ和２．９３５ｋＤ。ＢｍＰ０２的氨基酸序列已被测定。用全细胞膜片箝方法记录到ＢｍＰ０２对大鼠心室肌细胞短暂外向钾电流（Ｉｔｏ）具有明显的抑制作用（ｎ＝５）,冲洗后可部分恢复,且其抑制活性具有浓度依赖性和电压非依赖性。进一步研究表明,ＢｍＰ０２可影响短暂外向钾通道的失活化和恢复过程,但却不影响其激活过程     

Simulation analysis of intracellular Na+ and Cl- homeostasis during beta 1-adrenergic stimulation of cardiac myocyte

To quantitatively understand intracellular Na⁺ and Cl⁻ homeostasis as well as roles of Na⁺/K⁺ pump and cystic fibrosis transmembrane conductance regulator Cl⁻ channel (I_CFTR) during the β1-adrenergic stimulation in cardiac myocyte, we constructed a computer model of β1-adrenergic signaling and implemented it into an excitation-contraction coupling model of the guinea-pig ventricular cell, which can reproduce membrane excitation, intracellular ion changes (Na⁺, K⁺, Ca²⁺ and Cl⁻), contraction, cell volume, and oxidative phosphorylation. An application of isoproterenol to the model cell resulted in the shortening of action potential duration (APD) after a transient prolongation, the increases in both Ca²⁺ transient and cell shortening, and the decreases in both Cl⁻ concentration and cell volume. These results are consistent with experimental data. Increasing the density of I_CFTR shortened APD and augmented the peak amplitudes of the L-type Ca²⁺ current (I_CaL) and the Ca²⁺ transient during the β1-adrenergic stimulation. This indirect inotropic effect was elucidated by the increase in the driving force of I_CaL via a decrease in plateau potential. Our model reproduced the experimental data demonstrating the decrease in intracellular Na⁺ during the β-adrenergic stimulation at 0 or 0.5 Hz electrical stimulation. The decrease is attributable to the increase in Na⁺ affinity of Na⁺/K⁺ pump by protein kinase A. However it was predicted that Na⁺ increases at higher beating rate because of larger Na⁺ influx through forward Na⁺/Ca²⁺ exchange. It was demonstrated that dynamic changes in Na⁺ and Cl⁻ fluxes remarkably affect the inotropic action of isoproterenol in the ventricular myocytes.     

Charybdotoxin blocks dendrotoxin-sensitive voltage-activated K channels

Charybdotoxin, a short scorpion venom neurotoxin, which was thought to be specific for the blockade of Ca²⁺-activated K⁺ channels also blocks a class of voltage-sensitive K⁺ channels that are known to be the target of other peptide neurotoxins from snake and bee venoms such as dendrotoxin and MCD peptide. Charybdotoxin also inhibits ¹²⁵-dendrotoxin and ¹²⁵I-MCD peptide binding to their receptors. All these effects are observed with an IC₅₀ of about 30 nM.     

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     

Depletion of intracellular calcium stores activates an outward potassium current in mast and RBL-1 cells that is correlated with CRAC channel activation

Highly Ca²⁺ selective Ca²⁺ channels activated by store depletion have been recently described in several cell types and have been termed CRAC channels (for calcium release-activated calcium). The present study shows that following store depletion in mast and RBL-1 cells, monovalent outward currents could be recorded if the internal solution contained K⁺ but not Cs⁺. The activation of the outward K⁺ current correlated with the activation of I_CRAC, in both time and amplitude, suggesting that the K⁺ current might be carried by CRAC channels. The amplitude of the outward current was increased if external Ca²⁺ was reduced or replaced by external Ba²⁺. The outward K⁺ conductance might have a physiological role in maintaining the driving force for Ca²⁺ entry during the activation of CRAC channels.     

Solution structure of BmP02, a new potassium channel blocker from the venom of the Chinese scorpion Buthus martensi Karsch

BmP02 is a 28-amino acid residue peptide purified from the venom of the Chinese scorpion Buthus martensi Karsch, which had been demonstrated to be a weak blocker of apamin-sensitive calcium-activated potassium channels. Two-dimensional NMR spectroscopy techniques were used to determine the solution structure of BmP02. The results show that BmP02 formed a alpha/beta scorpion fold, the typical three-dimensional structure adopted by most short chain scorpion toxins whose structures have been determined. However, in BmP02 this alpha/beta fold was largely distorted. The alpha-helix was shortened to only one turn, and the loop connecting the helix to the first beta-strand exhibited conformational heterogeneity. The instability of BmP02 could be attributed to a proline at position 17, which is usually a glycine. Because the residue at this position makes intense contact with the alpha-helix, it was supposed that the bulky side chain of proline had pushed the helix away from the beta-sheet. This had a significant influence on the structure and function of BmP02. The alpha-helix rotated by about 40 degrees to avoid Pro17 while forming two disulfides with the second beta-strand. The rotation further caused both ends of the helix to be unwound due to covalent restrictions. According to its structure, BmP02 was supposed to interact with its target via the side chains of Lys11 and Lys13.     

Purification and partial characterization of K+ channel blockers from the venom of Dendroaspis angusticeps.

Two polypeptides (designated DTX-A and DTX-B) were purified from crude snake venom of Dendroaspis angusticeps using gel filtration, cation exchange colum chromatography and cation exchange high performance liquid chromatography, and their blocking actions of K⁺ channels were investigated in rat brain synaptosomes. Both DTX-A and DTX-B inhibited the voltage-dependent ⁴²K efflux from the synaptosomes. DTX-A blocked ⁴²K efflux of both the rapidly inactivating phase (component T) and the slowly inactivating phase (component S). The inhibitory effect of DTX-A on component T was pronounced compared with that on component S. However, DTX-B selectively blocked ⁴²K efflux of component S. The molecular weights of DTX-A and DTX-B were estimated to be ca 10,000 by SDS-polyacrylamide gel electrophoresis. The amino acid composition of these toxins is different from that of polypeptide purified from the venom of D. angusticeps (-, β, γ- and δ-DTX). These results suggest that DTX-A and DTX-B are new polypeptides which block voltage-dependent K⁺ channels selectively, and that they are useful tools for investigating the K⁺ channel.     

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.     

Actions of emigrated neutrophils on Na(+) and K(+) currents in rat ventricular myocytes

Interactions between neutrophils and the ventricular myocardium can contribute to tissue injury, contractile dysfunction and generation of arrhythmias in acute cardiac inflammation. Many of the molecular events responsible for neutrophil adhesion to ventricular myocytes are well defined; in contrast, the resulting electrophysiological effects and changes in excitation–contraction coupling have not been studied in detail. In the present experiments, rat ventricular myocytes were superfused with either circulating or emigrated neutrophils and whole-cell currents and action potential waveforms were recorded using the nystatin-perforated patch method. Almost immediately after adhering to ventricular myocytes, emigrated neutrophils caused a depolarization of the resting membrane potential and a marked prolongation of myocyte action potential. Voltage clamp experiments demonstrated that following neutrophil adhesion, there was (i) a slowing of the inactivation of a TTX-sensitive Na⁺ current, and (ii) a decrease in an inwardly rectifying K⁺ current.

One cytotoxic effect of neutrophils appears to be initiated by enhanced Na⁺ entry into the myocytes. Thus, manoeuvres that precluded activation of Na⁺ channels, for example holding the membrane potential at −80 mV, significantly increased the time to cell death or prevented contracture entirely. A mathematical model for the action potential of rat ventricular myocytes has been modified and then utilized to integrate these findings. These simulations demonstrate the marked effects of (50-fold) slowing of the inactivation of 2–4% of the available Na⁺ channels on action potential duration and the corresponding intracellular Ca²⁺ transient. In ongoing studies using this combination of approaches, are providing significant new insights into some of the fundamental processes that modulate myocyte damage in acute inflammation.     

Solution structure of BmP01 from the venom of scorpion Buthus martensii Karsch

From the venom of scorpion Buthus martensii Karsch,a short peptide (BmP01, 29 amino acid residues) was isolated and characterized as previously reported (Lebren, R. R., et al. (1997) Eur. J. Biochem. 245, 457-464). It was shown to reduce 33% outward K(+) channel (hippocampal neurons) currents at 10 microM. The solution structure of BmP01 was determined by 2D (1)H NMR spectroscopy. The NOEs, coupling constants, and H-D exchange obtained from NMR spectroscopy were used in structural calculations. The conformation of BmP01 is composed of a short alpha-helix (Cys 3-Thr 12) and a two-stranded antiparallel beta-sheet (Ala 15-Asp 20 and Lys 23-Pro 28). There are three disulfide bridges (Cys 3-Cys 19, Cys 6-Cys 24 and Cys 10-Cys 26) connecting the alpha-helix and beta-sheet. Asp 20 to Lys 23 form a type II turn linking the two strands. Structural and electrostatic potential comparison between BmP01 and its analogues are also presented.     

Dynamic properties of stretch-activated K+ channels in adult rat atrial myocytes

The effect of mechanical stress on the heart's electrical activity has been termed mechanoelectric feedback. The response to stretch depends upon the magnitude and the waveform of the stimulus, and upon the timing relative to the cardiac cycle. Stretch-activated ion channels (SACs) have been regarded as the most likely candidates for serving as the primary transducers of mechanical stress. We explored the steady state and dynamic responses of single channels in adult rat atrial cells using the patch clamp with a pressure clamp. Surprisingly, we only observed K⁺-selective SACs, probably of the 2P domain family. The channels were weakly outward rectifying with flickery bursts. In cell attached mode, the mean conductance was 74±14 and 65±16 pS for +60 and −60 mV, respectively (140 mM [K⁺]_out, 2 mM [Mg²⁺]_out and 0 mM [Ca²⁺]_out). The latency of the response to pressure steps was 50–100 ms and the time to peak 400 ms. About half of the channels in cell-attached patches showed adaptation/inactivation where channel activity declined to a plateau of 20–30% of peak in 1 s. The time dependent behavior of these SACs is generally consistent with whole-cell currents observed in chick and rat ventricular cells, although the net current was outward rather than inward.     

Physiological modulation of voltage-dependent inactivation in the cardiac muscle L-type calcium channel: a modelling study

The inactivation of the L-type Ca²⁺ current is composed of voltage-dependent and calcium-dependent mechanisms. The relative contribution of these processes is still under dispute and the idea that the voltage-dependent inactivation could be subject to further modulation by other physiological processes had been ignored. This study sought to model physiological modulation of inactivation of the current in cardiac ventricular myocytes, based upon the recent detailed experimental data that separated total and voltage-dependent inactivation (VDI) by replacing extracellular Ca²⁺ with Mg²⁺ and monitoring L-type Ca²⁺ channel behaviour by outward K⁺ current flowing through the channel in the absence of inward current flow. Calcium-dependent inactivation (CDI) was based upon Ca²⁺ influx and formulated from data that was recorded during β-adrenergic stimulation of the myocytes. Ca²⁺ influx and its competition with non-selective monovalent cation permeation were also incorporated into channel permeation in the model. The constructed model could closely reproduce the experimental Ba²⁺ and Ca²⁺ current results under basal condition where no β-stimulation was added after a slight reduction of the development of fast voltage-dependent inactivation with depolarization. The model also predicted that under β-adrenergic stimulation voltage-dependent inactivation is lost and calcium-dependent inactivation largely compensates it. The developed model thus will be useful to estimate the respective roles of VDI and CDI of L-type Ca²⁺ channels in various physiological and pathological conditions of the heart which would otherwise be difficult to show experimentally.     

Responses of apical ion fluxes to NaCl stress in Elaeagnus angustifolia seedlings

Aims Elaeagnus angustifolia is one of the most salt-tolerant species. The objective of this study was to understand the mechanisms of ion transporation in E. angustifolia exposed to different salt concentrations through manipulations of K⁺/Na⁺ homeostasis.
Methods Seedlings of two variants of the species, Yinchuan provenance (YC, salt-sensitive type) and the Alaer provenance (ALE, salt-tolerant type), were treated with three different NaCl application modes, and the ion fluxes in the apical regions were measured using non-invasive micro-test technology (NMT). In mode 1, Na⁺ and K⁺ fluxes were measured after 150 mmol·L^-1 NaCl stress lasted for 24 h. In mode 2, K⁺ and H⁺ fluxes were quantified with a transient stimulation of NaCl solution. In mode 3, Amiloride (Na⁺/H⁺ antiporters inhibitor) and tetraethylammonium (TEA, K⁺ channel inhibitor) was used to treat apical regions of E. angustifolia seedlings after NaCl stress for 24 h, respectively.
Important findings Under NaCl stress for 24 h, net effluxes of Na⁺ and K⁺ were increased significantly. The net Na⁺ effluxes of YC provenance seedlings (720 pmol·cm^-2•s^-1) were lower than that of ALE provenance (912 pmol·cm^-2·s^-1), but the net K⁺ efflux was higher in YC provenance. Under the instantaneous NaCl stimulation, net K⁺ efflux was remarkably increased, with the net K⁺ efflux of YC provenance always higher than that of ALE provenance. Interestingly, H⁺ at the apical regions was found from influx to efflux, with the net H⁺ efflux of ALE provenance greater than that of the YC provenance. Under the NaCl and NaCl + Amiloride treatment, the net Na⁺ efflux of ALE provenance seedlings was higher than that of YC provenance, while the net K⁺ efflux was less in ALE provenance seedlings. On the other hand, the differences in net Na⁺ and K⁺ effluxes were insignificant between the two provenances under the control group and NaCl + TEA treatment. In conclusion, NaCl stress caused Na⁺ accumulation and K⁺ outflows of E. angustifolia seedlings; The E. angustifolia seedlings utilize Na⁺/H⁺ antiporters to reduce Na⁺ accumulation by excretion; and the maintenance of K⁺/Na⁺ homeostasis in salt-tolerant E. angustifolia provenance seedlings roots accounted for a greater Na⁺ extrusion and a lower K⁺ efflux under NaCl stress. Results from this study provide a theoretical basis for further exploring salt-tolerant E. angustifolia germplasm resource.     

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.     

Molecular cloning and sequencing of two 'short chain' and two 'long chain' K(+) channel-blocking peptides from the Chinese scorpion Buthus martensii Karsch.

Five full-length cDNAs encoding the precursors of two 'short chain' scorpion non-toxic peptides active on Ca(2+)-activated K(+) channels (BmP02 and BmP03) and two novel putative long chain K(+) channel-blocking peptides (named BmTXKbeta and BmTXKbeta2) were first isolated from the venom gland cDNA library of the Chinese scorpion Buthus martensii Karsch (BmK). BmTXKbeta2 showed a high similarity with AaTXKbeta, while BmTXKbeta was completely different in the deduced primary structure from the long chain and short chain scorpion toxins already characterized. Thus, BmTXKbeta expands the scorpion long chain K(+) channel-blocking peptide family. Although little sequence similarity exists between the above two short and two long peptides, they are similar at the positions of six cysteines, suggesting that they should all share a similar scaffold composed of an alpha-helix and a three-stranded beta-sheet.     

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。     

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.     

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.     

Effect of glutamate on extracellular potassium activity in isolated retina of cyprinid fish

The effect of ( -)glutamate on extracellular K⁺ activity of the isolated retina of the cyprinid fish, roach, was investigated using double-barrelled K⁺-sensitive micro-electrodes. Application of μM-mM glutamate to the retina as a “puff” from an atomizer induced a transient rise in extracellular K⁺ activity, which was maximal 50–100 μm below the photoreceptor surface. The effect was concentration-dependent, but not related to the state of light adaptation of the retina. In the presence of dinitrophenol or ouabain, the glutamate-induced increase in extracellular K⁺ activity was maintained.

The following conclusions are made. (1) The most likely cellular origin(s) of the glutamate-induced rise in extracellular K⁺ activity are the photoreceptors and/or the Muller (glial) cells. (2) The mechanism responsible for returning the extracellular K⁺ activity to normal depends strongly on metabolic, Na⁺, K⁺ pump activity. (3) The effect parallels the action of endogenous glutamate, and may be important for modulation of neurotransmission in the intact retina.