The beta gamma subunits of GTP-binding proteins activate the muscarinic K+ channel in heart

Subunits of guanine nucleotide regulatory proteins purified from bovine cerebral cortex were used to perfuse the intracellular surface of excised patches of chick embryonic atrial cells. Single-channel current measurements unexpectedly indicate that the beta gamma, and not the alpha subunits, are responsible for activating the muscarinic-gated potassium channel.     

Isozyme-selective stimulation of phospholipase C-beta 2 by G protein beta gamma-subunits.

Hydrolysis by phospholipase C (PLC) of phosphatidylinositol 4,5-bisphosphate is a key mechanism by which many extracellular signalling molecules regulate functions of their target cells. At least eight distinct isozymes of PLC are recognized in mammalian cells. Receptor-controlled PLC is often regulated by G proteins, which can be modified by pertussis toxin in some cells but not in others. In the latter cells, PLC-beta 1, but not PLC-gamma 1 or PLC-delta 1, may be activated by members of the alpha q-subfamily of the G protein alpha-subunits. An unidentified PLC in soluble fractions of cultured human HL-60 granulocytes is specifically stimulated by G protein beta gamma subunits purified from retina and brain. Identification of a second PLC-beta complementary DNA (PLC-beta 2) in an HL-60 cell cDNA library prompted us to investigate the effect of purified G protein beta gamma subunits on the activities of PLC-beta 1 and PLC-beta 2 transiently expressed in cultured mammalian cells. We report here that PLC-beta 1 and PLC-beta 2 were stimulated by free beta gamma subunits and that PLC-beta 2 was the most sensitive to beta gamma stimulation. Thus stimulation of PLC by beta gamma subunits is isozyme-selective and PLC-beta 2 is a prime target of beta gamma stimulation. Activation of PLC-beta 2 by beta gamma subunits may be an important mechanism by which pertussis toxin-sensitive G proteins stimulate PLC.     

Subunits beta gamma of heterotrimeric G protein activate beta 2 isoform of phospholipase C.

The activation of heterotrimeric G proteins results in the exchange of GDP bound to the alpha-subunit for GTP and the subsequent dissociation of a complex of the beta- and gamma-subunits (G beta gamma). The alpha-subunits of different G proteins interact with a variety of effectors, but less is known about the function of the free G beta gamma complex. G beta gamma has been implicated in the activation of a cardiac potassium channel, a retinal phospholipase A2 (ref. 9) and a specific receptor kinase, and in vitro reconstitution experiments indicate that the G beta gamma complex can act with G alpha subunit to modulate the activity of different isoforms of adenylyl cyclase. Of two phospholipase activities that can be separated in extracts of HL-60 cells, purified G beta gamma is found to activate one of them. Here we report that in co-transfection assays G beta gamma subunits specifically activate the beta 2 and not the beta 1 isoform of phospholipase, which acts on phosphatidylinositol. We use transfection assays to show also that receptor-mediated release of G beta gamma from G proteins that are sensitive to pertussis toxin can result in activation of the phospholipase. This effect may be the basis of the pertussis-toxin-sensitive phospholipase C activation seen in some cell systems (reviewed in refs 13 and 14).     

Modulation of single Ca2+-dependent K+-channel activity by protein phosphorylation

There is considerable evidence that cyclic AMP can modulate the electrical activity of excitable cells and that protein phosphorylation by the catalytic subunit (CS) of cAMP-dependent protein kinase is a necessary step in these modulatory effects. In analogy to alterations in enzyme activities following phosphorylation, it seems possible that direct phosphorylation of ion-channel proteins may alter their gating properties, giving rise to the observe changes in electrical activity. However, the results obtained so far do not indicate whether it is ion channels themselves that are phosphorylated, or whether phosphorylation is simply an early step in some cascade of events which leads ultimately to modulation of channel activity. The development of single-channel recording techniques has provided a way to investigate this question. Here we describe effects of CS on the activity of individual CA2+-dependent K+ channels from the nervous system of the land snail Helix measured in isolated membrane patches and in artificial phospholipid bilayers. The results demonstrate that cAMP-dependent protein phosphorylation produces long-lasting changes in the activity of individual channels, and indicate that the relevant phosphorylation site is closely associated with the channel.     

Regulation of Ca2+-dependent K+-channel activity in tracheal myocytes by phosphorylation

Isoprenaline is a beta-adrenergic agonist of clinical importance as a remedy for asthma. In airway smooth muscle its relaxant action is accompanied by hyperpolarization of the membrane and elevation of the level of intracellular cyclic AMP. Hyperpolarization and relaxation are also induced by drugs such as forskolin, theophylline and dibutyryl cAMP, indicating that cAMP-dependent phosphorylation is involved in producing the electrical response. Cyclic AMP-dependent protein kinase (protein kinase A) has been reported to activate Ca2+-dependent K+ channels in cultured aortic smooth muscle cells and snail neurons. The membrane of tracheal smooth-muscle cells is characterized by a dense distribution of Ca2+-dependent K+-channels. We have now examined the effect of isoprenaline and protein kinase A on Ca2+-dependent K+-channels in isolated smooth muscle cells of rabbit trachea, using the patch-clamp technique. Our results show that the open-state probability of Ca2+-dependent K+-channel of tracheal myocytes is reversibly increased by either extracellular application of isoprenaline or intracellar application of protein kinase A. We also show that this effect is significantly enhanced and prolonged in the presence of a potent protein phosphatase inhibitor, okadaic acid.     

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.     

(K 3,K2+Tn)的Ramsey数

Zhou Huai-lu给出了当m≥1，n≥5m 3时，r(Bm，Wn)=2n 1；当m=1，n≥9或m≥2，n≥(m-1)(16m^3-16m^2-24m-10) 1时r(Bm，K2 Cn)=2n 3．这里Bm表示：Kz Kc/m，w。表示n个辐条的轮．Gu H给出了当n≥3时，r(K3，K1 Tn)=2n 1;当m≥1，n≥5m 2时r(Bm，K1 Tn)=2n 1．在此启发下，该首先用组合的方法证明了r(K3，K2 T4)=11．     

Open structure of the Ca2+ gating ring in the high-conductance Ca2+-activated K+ channel

High-conductance voltage- and Ca(2+)-activated K(+) channels function in many physiological processes that link cell membrane voltage and intracellular Ca(2+) concentration, including neuronal electrical activity, skeletal and smooth muscle contraction, and hair cell tuning. Like other voltage-dependent K(+) channels, Ca(2+)-activated K(+) channels open when the cell membrane depolarizes, but in contrast to other voltage-dependent K(+) channels, they also open when intracellular Ca(2+) concentrations rise. Channel opening by Ca(2+) is made possible by a structure called the gating ring, which is located in the cytoplasm. Recent structural studies have defined the Ca(2+)-free, closed, conformation of the gating ring, but the Ca(2+)-bound, open, conformation is not yet known. Here we present the Ca(2+)-bound conformation of the gating ring. This structure shows how one layer of the gating ring, in response to the binding of Ca(2+), opens like the petals of a flower. The degree to which it opens explains how Ca(2+) binding can open the transmembrane pore. These findings present a molecular basis for Ca(2+) activation of K(+) channels and suggest new possibilities for targeting the gating ring to treat conditions such as asthma and hypertension.     

Arachidonic acid metabolites as intracellular modulators of the G protein-gated cardiac K+ channel

Arachidonic acid is released from cell membranes in response to receptor-dependent as well as receptor-independent stimulation in various cells, including cardiac myocytes. Arachidonic acid is converted to prostaglandins by cyclooxygenase and to leukotrienes by 5-lipoxygenase, metabolites which are very biologically active and modulate cellular functions such as platelet aggregation, smooth muscle contraction and neural excitation. The molecular mechanisms underlying their modulations are, however, still badly understood. Here, we report that the 5-lipoxygenase metabolites of arachidonic acid activate the pertussis toxin-sensitive G protein-gated muscarinic K+ channel (IK.ACh): arachidonic acid activation of IK.ACh was prevented by the lipoxygenase inhibitors, nordihydroguaiaretic acid and AA-861; leukotriene A4 and C4 activated IK.ACh. The activation occurred in pertussis toxin-treated atrial cells and ceased when inside-out patches were formed but the patches were still susceptible to stimulation by GTP and to inhibition by GDP-beta-S. These results indicate that arachidonic acid metabolites may stimulate the G-protein in a receptor-independent way.     

Regulation of Ca2+ channel expression at the cell surface by the small G-protein kir/Gem

Voltage-dependent calcium (Ca2+) channels are involved in many specialized cellular functions, and are controlled by intracellular signals such as heterotrimeric G-proteins, protein kinases and calmodulin (CaM). However, the direct role of small G-proteins in the regulation of Ca2+ channels is unclear. We report here that the GTP-bound form of kir/Gem, identified originally as a Ras-related small G-protein that binds CaM, inhibits high-voltage-activated Ca2+ channel activities by interacting directly with the beta-subunit. The reduced channel activities are due to a decrease in alpha1-subunit expression at the plasma membrane. The binding of Ca2+/CaM to kir/Gem is required for this inhibitory effect by promoting the cytoplasmic localization of kir/Gem. Inhibition of L-type Ca2+ channels by kir/Gem prevents Ca2+-triggered exocytosis in hormone-secreting cells. We propose that the small G-protein kir/Gem, interacting with beta-subunits, regulates Ca2+ channel expression at the cell surface.     

Multiple transport modes of the cardiac Na+/Ca2+ exchanger

The cardiac Na+/Ca2+ exchanger (NCX1; ref. 2) is a bi-directional Ca2+ transporter that contributes to the electrical activity of the heart. When, and if, Ca2+ is exported or imported depends on the Na+/Ca2+ exchange ratio. Whereas a ratio of 3:1 (Na+:Ca2+) has been indicated by Ca2+ flux equilibrium studies, a ratio closer to 4:1 has been indicated by exchange current reversal potentials. Here we show, using an ion-selective electrode technique to quantify ion fluxes in giant patches, that ion flux ratios are approximately 3.2 for maximal transport in either direction. With Na+ and Ca2+ on both sides of the membrane, net current and Ca2+ flux can reverse at different membrane potentials, and inward current can be generated in the absence of cytoplasmic Ca2+, but not Na+. We propose that NCX1 can transport not only 1 Ca2+ or 3 Na+ ions, but also 1 Ca2+ with 1 Na+ ion at a low rate. Therefore, in addition to the major 3:1 transport mode, import of 1 Na+ with 1 Ca2+ defines a Na+-conducting mode that exports 1 Ca2+, and an electroneutral Ca2+ influx mode that exports 3 Na+. The two minor transport modes can potentially determine resting free Ca2+ and background inward current in heart.     

Structure of the gating domain of a Ca2+-activated K+ channel complexed with Ca2+/calmodulin

Small-conductance Ca2+-activated K+ channels (SK channels) are independent of voltage and gated solely by intracellular Ca2+. These membrane channels are heteromeric complexes that comprise pore-forming alpha-subunits and the Ca2+-binding protein calmodulin (CaM). CaM binds to the SK channel through the CaM-binding domain (CaMBD), which is located in an intracellular region of the alpha-subunit immediately carboxy-terminal to the pore. Channel opening is triggered when Ca2+ binds the EF hands in the N-lobe of CaM. Here we report the 1.60 A crystal structure of the SK channel CaMBD/Ca2+/CaM complex. The CaMBD forms an elongated dimer with a CaM molecule bound at each end; each CaM wraps around three alpha-helices, two from one CaMBD subunit and one from the other. As only the CaM N-lobe has bound Ca2+, the structure provides a view of both calcium-dependent and -independent CaM/protein interactions. Together with biochemical data, the structure suggests a possible gating mechanism for the SK channel.     

一类k步2k＋1阶刚性稳定的二阶导数方法

本文导出新的二阶导数线性多步法,这些方法适合于求解刚性方程组,方程的稳定域由根轨迹法给出,数值试验显示方法是行之有效的．     

Na~+、K~+、Ca~(2+)对柞蚕幼虫在体胸神经节自发放电活动的影响

以柞蚕五龄幼虫为材料 ,运用微电极电生理技术 ,以胞外记录的方法 ,记录了柞蚕幼虫在体胸神经节的自发放电活动 .并观察了胞外Na+、K+、Ca2 +的浓度变化对放电活动的影响 .结果表明 ,柞蚕幼虫胸神经节胞外自发放电有 3种形式 ,即慢节律单个放电、簇状放电及连续放电 ,其离子机制可能既有Na+动作电位 ,又有Ca2 +动作电位     

K2MgF4∶Mn2+体系局域结构的理论研究

采用对角化四角晶体场中d5组态离子的完全能量矩阵的方法,研究了K2MgF4∶Mn2 体系的EPR谱与局域晶格结构间的关系,通过拟和EPR谱的低对称参量b02,b04的实验值计算给出了过渡金属Mn2 离子在K2MgF4∶Mn2 体系中局域结构参量R1=0.203 82 nm,R2=0.205 58 nm,以及晶格畸变量ΔR1=0.004 75 nm,ΔR2=0.007 08 nm.     

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.     

Use of chlorotetracycline fluorescence to demonstrate Ca2+-induced release of Ca2+ from the sarcoplasmic reticulum of skinned cardiac cells.

It has been proposed that the trans-sarcolemmal influx of Ca2+ occurring during the plateau of the mammalian cardiac action potentials is insufficient in itself to activate the myofilaments, but can trigger a release of Ca2+ from the sarcoplasmic reticulum (SR) which is sufficient for activation. The demonstration of this Ca2+-induced release of Ca2+ relied entirely on experiments in which the tension developed by the myofilaments was used as a sensor of the changes of myoplasmic free Ca2+ concentration ([free Ca2+]) in segments of single cardiac cells from which the sarcolemma had been removed by microdissection (skinned cardiac cells). The small size of these preparations has previously prevented the use of more direct methods for the detection of myoplasmic Ca2+ movements. The present study is a direct demonstration of Ca2+-induced release of Ca2+ from the SR of skinned cardiac cells treated with chlorotetracycline (CTC), a fluorescent chelate probe which enables changes in the amount of Ca2+ bound to a variety of biological membranes or micelles to be monitored. The fluorescence increases when more Ca2+ is bound.