Acetylcholine activation of single muscarinic K+ channels in isolated pacemaker cells of the mammalian heart

Acetylcholine (ACh) released on vagal stimulation reduces the heart rate by increasing K+ conductance of pacemaker cells in the sinoatrial (S-A) node. Fluctuation analysis of ACh-activated currents in pacemaker tissue showed this to be due to opening of a separate class of K+ channels gated by muscarinic ACh receptors (m-AChRs). On the other hand, it has been suggested that m-AChRs may simply regulate the current flow through inward rectifying resting K+ channels (gk1). We report here the measurement of ACh-activated single channel K+ currents and of resting K+ channel currents in isolated cells of the atrioventricular (A-V) and S-A node of rabbit heart. The results show that the ACh-dependent K+ conductance increase in nodal cells is mediated by K+ channels which are different in their gating and conductance properties from the inward rectifying resting K+ channels in atrial and ventricular cells. The resting K+ channels in nodal cells are, however, similar to those activated by ACh.     

Single apamin-blocked Ca-activated K+ channels of small conductance in cultured rat skeletal muscle

Action potentials in many excitable cells are followed by a prolonged afterhyperpolarization that modulates repetitive firing. Although it is established that the afterhyperpolarization is produced by Ca-activated K+ currents, the basis of these currents is not known. The large conductance (250 pS) Ca-activated K+ channel (BK channel) is not a major contributor to the afterhyperpolarization in non-innervated skeletal muscle and some nerve cells, because apamin, a neurotoxic component of bee venom, abolishes the afterhyperpolarization but does not block BK channels, and 5 mM extracellular tetraethylammonium ion (TEA) blocks BK channels but does not reduce the afterhyperpolarization. We now report single-channel currents from small conductance (10-14 pS) Ca-activated K+ channels (SK channels) with the necessary properties to account for the afterhyperpolarization. SK channels are blocked by apamin but not by 5 mM external TEA (TEAo). They are also highly Ca-sensitive at the negative membrane potentials associated with the afterhyperpolarization.     

Characterization of an endo-1,4-β-glucanase from Lilium longiflorum that functioned in pollen germination and pollen tube growth

Endo-β-glucanases play vital roles in the regulation of pollen tube growth. Here, a previously identified endo-l,4-β-glucanase from Lilium Iongiflorum （lily）, named LlpCell, was expressed in Escherichia coli, purified, and further investigated for its physiological function. The recombinant LlpCell protein hydrolyzed carboxy-methylcellulose （CMC） and exhibited activity towards laminarin from Eisenia. arborea and 1,3：l,4-β-glucan of barley. The pH for the optimum activity was 6.0 and the value of Km calculated from CMC was 5.0 mg/mL. Adding EDTA resulted in the total loss of the enzymatic activity, and this effect could be restored by the addition of Ca^2＋. Western blotting analysis showed that LlpCell protein was present in pollen grains and rehydrated pollen grains, and the amount of the protein was increased during pollen germinating, but not in the pollen tube. Consistently, the immunofluorescence labeling study with the antibody against LIpCell also indicated the presence of LIpCell at the begin-ning of germination, but not in the elongating pollen tube. Furthermore, incubation of LlpCell with pollen at the beginning of pollen germination increased the germination percentage and the length of pollen tube. All of these results suggested that LlpCell could play an important role in the regulation of lily pollen germination and the initiation of pollen tube growth.     

Ohmic conductance through the inwardly rectifying K channel and blocking by internal Mg2+

The inwardly rectifying K channel provides the resting K conductance in a variety of cells. This channel acts as a valve or diode, permitting entry of K+ under hyperpolarization, but not its exit under depolarization. This behaviour, termed inward rectification, permits long depolarizing responses which are of physiological significance for the pumping function of the heart and for fertilization of egg cells. Little is known about the outward currents through the inwardly rectifying K channel, despite their great physiological importance, and the mechanism of inward rectification itself is unknown. We have used improved patch clamp techniques to control the intracellular media, and have recorded the outward whole-cell and single-channel currents. We report here that the channel conductance is ohmic and that the well-known inward rectification of the resting K conductance is caused by rapid closure of the channel accompanied by a voltage-dependent block by intracellular Mg2+ ions at physiological concentrations.     

Developmental acquisition of Ca2+-sensitivity by K+ channels in spinal neurones

A developmental change in the ionic basis of the inward current of action potentials has been observed in many excitable cells. In cultured spinal neurones of Xenopus, the timing of the development of the action parallels that seen in vivo. In vitro, as in vivo, neurones initially produce action potentials of long duration which are principally Ca-dependent; after 1 day of development the impulse is brief and primarily Na-dependent. At both ages, however, both inward components are present and the mechanism underlying shortening of the action potential is unknown. One possibility is that the outward currents change during development. Using the patch-clamp technique, we have recorded single K+-channel currents in membrane patches isolated from the cell bodies of cultured embryonic neurones. The unitary conductance of one class of K+ channels was approximately 155 pS and depolarization increased the probability of a channel being open. Neither conductance nor voltage dependence seemed to change with time in culture; in contrast, the Ca2+-sensitivity of this K+ channel increased. In younger neurones, Ca2+-sensitivity was greatly reduced or absent, whereas in more mature neurones, the activity of this channel was Ca-dependent. Such a change could account for the shortening of the action potential duration by increasing the relative contribution of outward currents.     

Single-channel and whole-cell recordings of mitogen-regulated inward currents in human cloned helper T lymphocytes

Cytoplasmic free Ca2+ [( Ca2+]i) appears to be an important signal for DNA synthesis in early stages of lymphocyte activation. In spite of many experimental studies which employ fluorescent Ca2+ indicator dye to demonstrate an early increase of [Ca2+]i in T-lymphocytes after stimulation with lectins, specific antigens, and monoclonal antibodies to T-lymphocyte receptors, the mechanism responsible for the rise of [Ca2+]i is unknown. We have used the extracellular patch clamp technique to investigate this mechanism. Unitary inward currents, mediated by Ca2+ or Ba2+, were recorded in the membrane of T-lymphocytes. The inward current channel was characterized by a conductance of 7 pS and extrapolated reversal potential (Erev) 110 mV positive to resting potential (Vr). While gating kinetic parameters were not affected by membrane potential changes, the probability of channel opening markedly increased upon activation of the T-lymphocyte by the mitogenic lectin, phytohaemagglutinin (PHA). PHA also evoked a cadmium-sensitive, inward Ba2+ current on whole-cell clamp. We suggest that this mitogen-regulated channel introduces Ca2+ into the cytoplasm upon activation and represents a new class of voltage-independent Ca2+ channels.     

Endfeet of retinal glial cells have higher densities of ion channels that mediate K+ buffering

A major function of glial cells in the central nervous system is to buffer the extracellular potassium concentration, [K+]o. A local rise in [K+]o causes potassium ions to enter glial cells, which have membranes that are highly permeable to K+; potassium then leaves the glial cells at other locations where [K+]o has not risen. We report here the first study of the individual ion channels mediating potassium buffering by glial cells. The patch-clamp technique was employed to record single channel currents in Müller cells, the radial glia of the vertebrate retina. Those cells have 94% of their potassium conductance in an endfoot apposed to the vitreous humour, causing K+ released from active retinal neurones to be buffered preferentially to the vitreous. Recordings from patches of endfoot and cell body membrane show that a single type of inward-rectifying K+ channel mediates potassium buffering at both cell locations. The non-uniform density of K+ conductance is due to a non-uniform distribution of one type of K+ channel, rather than to the cell expressing high conductance channels at the endfoot and low conductance channels elsewhere on the cell.     

疏花疏果剂对苹果花粉萌发及花粉管生长的影响

以华美苹果为材料,采集铃铛花的花粉,采用固体培养法研究了TMN-6和6-BA+ NAA对苹果花粉萌发和花粉管生长的影响.结果表明,TMN-6对花粉的萌发及花粉管生长有抑制作用,且抑制程度随TMN-6的浓度增加而增强,浓度为1 mg/L的处理花粉的萌发几乎完全被抑制.NAA+BA各浓度处理对花粉的萌发抑制作用显著,对花粉...     

Regulation of fast inactivation of cloned mammalian IK(A) channels by cysteine oxidation

Modulation of neuronal excitability by regulation of K+ channels potentially plays a part in short-term memory but has not yet been studied at the molecular level. Regulation of K+ channels by protein phosphorylation and oxygen has been described for various tissues and cell types; regulation of fast-inactivating K+ channels mediating IK(A) currents has not yet been described. Functional expression of cloned mammalian K+ channels has provided a tool for studying their regulation at the molecular level. We report here that fast-inactivating K+ currents mediated by cloned K+ channel subunits derived from mammalian brain expressed in Xenopus oocytes are regulated by the reducing agent glutathione. This type of regulation may have a role in vivo to link metabolism to excitability and to regulate excitability in specific membrane areas of mammalian neurons.     

Quantification of Ca2+-activated K+ channels under hormonal control in pig pancreas acinar cells

Ca2+- and voltage-activated K+ channels are found in many electrically excitable cells and have an important role in regulating electrical activity. Recently, the large K+ channel has been found in the baso-lateral plasma membranes of salivary gland acinar cells, where it may be important in the regulation of salt transport. Using patch-clamp methods to record single-channel currents from excised fragments of baso-lateral acinar cell membranes in combination with current recordings from isolated single acinar cells and two- and three-cell clusters, we have now for the first time characterized the K+ channels quantitatively. In pig pancreatic acini there are 25-60 K+ channels per cell with a maximal single channel conductance of about 200 pS. We have quantified the relationship between internal ionized Ca2+ concentration [( Ca2+]i) membrane potential and open-state probability (p) of the K+ channel. By comparing curves obtained from excised patches relating membrane potential to p, at different levels of [Ca2+]i, with similar curves obtained from intact cells, [Ca2+]i in resting acinar cells was found to be between 10(-8) and 10(-7) M. In microelectrode experiments acetylcholine (ACh), gastrin-cholecystokinin (CCK) as well as bombesin peptides evoked Ca2+-dependent opening of the K+ conductance pathway, resulting in membrane hyperpolarization. The large K+ channel, which is under strict dual control by internal Ca2+ and voltage, may provide a crucial link between hormone-evoked increase in internal Ca2+ concentration and the resulting NaCl-rich fluid secretion.     

ATP-sensitive K+ channel in the mitochondrial inner membrane.

Mitochondria take up and extrude various inorganic and organic ions, as well as larger substances such as proteins. The technique of patch clamping should provide real-time information on such transport and on energy transduction in oxidative phosphorylation. It has been applied to detect microscopic currents from mitochondrial membranes and conductances of ion channels in the 5-1,000 pS range in the outer and inner membranes. These pores are not, however, selective for particular ions. Here we use fused giant mitoplasts prepared from rat liver mitochondria to identify a small conductance channel highly selective for K+ in the inner mitochondrial membrane. This channel can be reversibly inactivated by ATP applied to the matrix side under inside-out patch configuration; it is also inhibited by 4-aminopyridine and by glybenclamide. The slope conductance of the unitary currents measured at negative membrane potentials was 9.7 +/- 1.0 pS (mean +/- s.d., n = 6) when the pipette solution contained 100 mM K+ and the bathing solution 33.3 mM K+. Our results indicate that mitochondria depolarize by generating a K+ conductance when ATP in the matrix is deficient.     

盐胁迫对含笑花粉萌发的影响

以含笑(Micheliafigo)花粉为试验材料,采用花粉人工培养法研究了不同浓度NaCl对含笑花粉萌发特性的影响。结果表明:NaCl对含笑花粉萌发有抑制作用,且抑制程度随培养基中NaCl浓度的增大而加强,2 mmol·L-1NaCl就能显著(P<0.05)抑制含笑花粉萌发。耐盐程度分析表明,含笑花粉萌发的耐盐浓度、耐盐半致死浓度、耐盐极限浓度分别为1.86、15.80、38.11 mmol·L-1。     

不同贮藏温度对4种杨树花粉生活力的影响

采用花粉离体培养的方法对不同温度条件下贮藏的4个杨树无性系花粉活力进行了研究,并对超低温贮藏1年后杨树花粉用于杂交育种的可行性进行了研究。结果表明,杨树花粉在离体培养28 h萌发率达到最大。贮藏3个月后,-20℃贮藏条件下4个杨树无性系花粉的萌发率最高,分别是:白杨84 K为36%,白杨毛新杨×银灰杨为35.2%,中菏1号为39.2%,钻天杨为41.9%。-80℃贮藏1年后的花粉仍具有生活力,能够正常授粉并获得正常发育的种子。     

电子束辐照对黄瓜、丝瓜和花生花粉萌发率和果实的影响

对黄瓜、丝瓜和花生的花粉分别进行剂量为300、400、500、600和800Gy的电子束辐照,在液体培养基中进行了花粉离体培养和观察,并以黄瓜的辐照后花粉进行人工授粉.研究结果表明,电子束辐照剂量在300～500Gy范围内明显提高了黄瓜花粉的萌发率;300～800Gy辐照剂量均大大提高了花生花粉的萌发率;300～400Gy的辐照剂量对丝瓜花粉萌发率无显著影响;高于500Gy的辐照剂量使花粉萌发率明显降低;600和800Gy辐照后的黄瓜花粉授粉产生的果实均发生了诱变效应,外形较短粗,质量增加,但种子的胚全都不发育.此研究结果对探讨应用电子束辐照花粉进行诱变育种的研究具有重要参考意义.     

Effects of extracellular calmodulin on pollen germination and tube growth

The effects of calmoddin (CaM) antagonist W7-agarose, anti-CaM serum and exogenous purified CaM on pollen germination and tube growth ofForsythia suspensu were studied. The pollen germination and tube growth were inhibited or completely stopped by CaM antagonist W7-agarose. The addition of exogenous purified CaM stimulated pollen germination and tube growth, whereas the same amount of bovine serum albumin (BSA) had no effect. The inhibitory effects caused by W7-agarose and anti-CaM serum could be reversed completely by the addition of exogenous purified CaM. The tube growth of germinated pollen was also inhibited or completely stopped by W7-agarose. The results suggest that endogenous extracellular CaM initiates pollen germination and tube growth, whereas exogenous CaM enhances the above processes.     

Mechanism of calcium channel blockade by verapamil, D600, diltiazem and nitrendipine in single dialysed heart cells

Organic inhibitors of calcium influx prevent outward as well as inward current through cardiac calcium channels but do not slow current activation. Although block is antagonized by raising external calcium or barium concentrations, the competitive effect of permeant cations does not occur at the same cation binding site at which inorganic blockers act. Organic drugs show varying degrees of use-dependent block, due in part to blockade of open channels. Nitrendipine blockade of calcium currents requires doses greater than 100-fold higher than expected from radioligand binding to isolated membranes.     

红花罂粟花粉萌发的研究

以新鲜红花罂粟花粉为试材,比较研究了培养基中蔗糖浓度及硼酸和Ca^2＋、赤霉素浓度对红花罂粟花粉萌发和花粉管生长的影响。结果表明：过高过低的蔗糖、硼酸浓度抑制红花罂粟花粉萌发。低浓度Ca^2＋和赤霉素促进了花粉萌发和花粉管的生长,高浓度Ca、赤霉素抑制了花粉萌发和花粉管的生长。红花罂粟花粉萌发的最佳培养基是琼脂20g＋蔗糖110g＋硼酸110mg／L＋200mg／LCa^2＋＋80mg／LGA3。     

芍药花粉离体萌发培养基组分的优化

以芍药花粉为材料,采用正交设计L^9（34）法研究蔗糖、硝酸钙、硼酸对芍药花粉离体萌发和花粉管生长的影响,筛选芍药花粉离体萌发最适宜的培养基,并研究了不同pH值对芍药花粉萌发的影响。结果表明,蔗糖对芍药花粉萌发率和花粉管生长影响最大,硼酸次之,硝酸钙影响最小;芍药花粉离体萌发最适宜培养基为,蔗糖20g·L^-1＋硼酸20mg·L^-1＋硝酸钙60mg·L^-1;其萌发率和花粉管长度分别为63．12％和183．33μm;适合芍药花粉萌发的pH为6～7。     

20个荷花品种花粉活力测定及贮藏研究

为研究荷花花粉的生活力,选择了20个荷花品种分别用TTC染色法、亚甲基蓝染色法和离体萌发法测定了花粉的染色率和萌发率,并研究了不同温度贮藏条件对花粉生活力的影响.结果表明,亚甲基蓝染色法不能作为荷花花粉生活力的测定方法,离体萌发法结果更直观和准确;20个荷花品种间的离体萌发率差异明显,最高为13.1%,最低为0;不同品种在三种贮藏温度下萌发率变化趋势相同,4℃条件下贮藏较有利于荷花花粉生活力的保持.     

Sodium current of neurosecretory cells in the eyestalk from the shrimp Penaeus japonicus

The properties of the inward current of medulla terminalis-X-organ (MTXO) cells isolated from the Penaeus japonicus eyestalk were studied with the whole-cell clamp technique in the presence of Ca²⁺ and K⁺ channel blockers. The inward currents had a threshold at about −50 mV and peaked at −10 mV. The reversed potential (V_rev) was very close to V_Na, the theoretical Nernst equilibrium potential for Na⁺. V_rev followed V_Na when the external Na⁺ concentration was varied and the currents were entirely suppressed by 30 nM tetrodotoxin (TTX), indicating that it was carried by Na⁺. The smooth line of concentration-dependent inhibition of sodium currents by TTX represented the best fit with the Hill equation, yielding an IC₅₀ of 2.1 ± 0.1 nM. The values of the half-maximal activation voltage V_h were −20.6 ± 0.5 and −19.3 ± 0.5 mV, respectively, in the absence and presence of 2 nM TTX. TTX had no significant effect on the voltage dependence of steady-state activation and inactivation of I_Na. Taken together, the results suggest that the inward current recorded under our experimental conditions was carried by sodium ions flowing through fast voltage-dependent Na⁺ channels.