Ca2+对骨骼肌钙释放通道的调节

钙释放通道（calcium release channel）又称Ryanodine受体（RyR）,是细胞内质网膜上介导细胞内钙信号转导的离子通道。RyR1在骨骼肌细胞的兴奋-收缩偶联过程中起重要作用,是肌质网快速释放Ca^2＋的通道。许多调节因素,如一些内源性蛋白（FK结合蛋白、钙调素、钙结合蛋白）和一些离子（Ca^2＋、Mg^2＋）,通过不同的作用位点与RyR1结合,调控RyR1的结构与功能。研究表明,Ca^2＋是众多调节RyR1因素中的核心成分和前提条件,其对RyR1的结构与功能有重要的调控作用。     

弱磁场对骨骼肌肌质网钙调控作用的研究

目的：探讨弱磁场对提取的骨骼肌肌质网系（SR）Ca（2＋）转运、钙泵（Ca（2＋）-Mg（2＋）-ATPase）及钙释放通道（RyR）活性的影响,从分子水平和细胞信号系统的角度来解释生物电磁效应。方法：利用动态光谱法检测0.4 mT弱磁场辐照过的SR Ca（2＋）转运、Ca（2＋）-ATPase活性,还原型辅酶（NADH）的氧化初速率和超氧（O_2-）产率,以及用同位素标记方法检测[3H]-Ryanodine与RyR的平衡结合度。结果：弱磁场辐照引起SR的Ca（2＋）摄取功能和Ca（2＋）-ATPase的活性明显下降,Ca（2＋）释放和[3H]-Ryanodine平衡结合度上升,同时上调了NADH的氧化初速率和O_2-的产率。结论：提示0.4 mT弱磁场辐照30 min对SR Ca（2＋）-ATPase活性有明显抑制,对RyR有一定的激活效果。     

钙通道与钙释放通道

1.Ca~(2+)的重要生理作用胞内游离钙浓度([Ca~(2+)])的变化调节着细胞的代谢、基因表达等细胞共有的活动,以及始动兴奋、收缩或出胞分泌以及激活和失活离子通道等细胞不同的反应。[Ca~(2+)]的升高主要依赖于胞外钙经质膜上的钙通道内流或/和胞内储存钙的释放。释放的内钙也是藉细胞器膜的钙释放通道进入胞浆。可见通道启闭活动的正常是维持[Ca~(2+)]正常的一个重要保证。2.离子通道及其分类离子通道是贯穿于质膜或细胞器膜的大分子蛋白质,其中央形成能通过离子的亲水性孔道(pores)。离子的跨膜转运是通过膜上通道蛋白的功能来完     

FK-506结合蛋白对钙释放通道的调控

细胞内自由钙作为一种重要的细胞信使广泛地参与细胞生理功能调控.胞内钙库(内质网系和肌浆网系)对调节细胞内自由钙水平起着重要的作用.钙库膜上的钙释放通道(ryanodine受体和三磷酸肌醇受体)受许多因素调控,其中之一就是新近研究得相当多的FK506结合蛋白.免疫抑制剂FK506能特异地结合钙库上一种分子质量为12 ku左右的蛋白,这种FK506结合蛋白与钙释放通道形成一种紧密连接的复合体,在正常生理情况下对钙释放通道起着十分重要的调控作用.     

解联剂促进BBY膜颗粒电子传递机理的研究

透射电镜结果表明,具有ＰＳⅡ理化特征的ＢＢＹ膜颗粒在结构上不具备完整的类囊体膜特征．９－ＡＡ荧光猝灭与毫秒级Ｃｈｌα延迟发光的测定表明,ＢＢＹ膜颗粒在功能上难以形成．光致跨膜质子浓度差．在ＢＢＹ膜颗粒中,解联剂ｇｒａｍｉｃｉｄｉｎＤ（短杆菌肽〕和ＮＨ４Ｃｌ仅在低ｐＨ值时对ＰＳⅡ电子传递有所促进,ｐＨ６．０时促进尤为显著。两种解联剂促进的数值和对ｐＨ值的依赖特征基本一致,表明两者促进机制相同．综上所述,我们推测,解联剂在ＢＢＹ膜颗粒中并不促进跨越类囊体模的质子运转,而只是加速膜上微区内的质子转移,从而促进相关的电子传递。     

β-肾上腺素能受体调控心肌细胞钙释放的分子机制

β-肾上腺素能受体（βAR）是最经典的G蛋白耦联受体。在心室肌细胞中,βAR可以提高细胞膜L-型钙通道（LCC）介导的钙内流的幅度和同步性,通过钙致钙释放机制触发肌质网（SR）更强的钙释放活动,从而起到调节心脏收缩能力的作用。然而,目前仍不清楚β-蛋白激酶A（PKA）信号通路如何直接调控肌质网钙释放通道ryanodine受体（RyR）的功能,该领域的研究结果存在很大争议。本文使用特殊的单通道钙成像技术,通过去极化方法激活单个LCC产生钙小星,记录触发的RyR钙火花。结果表明,在βAR激动剂异丙肾上腺素（1μM）作用20分钟内,单个LCC触发的钙火花幅度显著上升,且该效应不依赖于LCC单通道钙电流的变化;βAR激动下钙火花的钙释放电流幅度与肌质网钙储量的比值显著提高,表明βAR信号动员了更多的RyR通道参与同步钙释放活动;βAR激动下钙小星触发钙火花的耦联潜伏期时间缩短,成功率上升,表明βAR信号通路增强了LCC—RyR的分子间耦联效率。上述效应不依赖BAR引起的在肌质网钙储量上升,且能够被PKA抑制剂Rp-β-CPT-cAMP（100μM）和H89（10μM）消除。上述结果证明,βAR—PKA信号能够提高RvR对LCC单通道电流的响应速率和同步性。由此揭示的交感神经调节心脏功能的分子机制,将为进一步研究心脏疾病下βhR信号的异常变化奠定基础。     

NO调控下丘脑钙激活钾通道的机制研究

目的 :研究NO对下丘脑神经元钙激活钾通道 (KCa)的作用及其机制。方法 :采用膜片钳技术内面向外式及细胞贴附式。结果 :NO可直接或通过升高cGMP来提高KCa通道的开放概率 (Po) ,这种增强作用是因为通道开放时间延长及开放频率增加。结论 :下丘脑神经元中NO可通过不同机制激活KCa。     

钙离子通道A23187诱导Hela细胞凋亡过程中钙调磷酸酶对乙酰胆碱酯酶表达的调控

2007年1月26日,中科院上海生科院生化细胞所张学军研究组在Biochimicaet Biophysica Acta上在线发表最新学术成果：钙离子通道A23187诱导Hela细胞凋亡过程中钙调磷酸酶对乙酰胆碱酯酶表达的调控。该论文对于深入了解凋亡细胞表达乙酰胆碱酯酶机制,干预细胞凋亡过程,有一定意义。     

化学调控剂对水稻不育系育性恢复效应的研究

１９９２年３月至１１月,我们在海南和长沙两地,先后使用３８种化学调控剂,对水稻两用不育系,三系不育系及普通核不育系进行了喷施和注射试验,筛选出７种较为有效的调控剂,代号为ＣＲ１—ＣＲ７,其中ＣＲ５和ＣＲ７表现较为突出。这些调控剂对两用不育系和三系不育系均表现出一定程度的恢复效应,且ＣＲ１和ＣＲ２还能在可育条件下提高培矮６４Ｓ的结实率,但所有调控剂对普通核不育系均无恢复作用。     

A Procedure for Purification of the Ryanodine Receptor from Skeletal Muscle

In this paper, we describe a simple and reproducible method for purifying large quantities of ryanodine receptor from skeletal muscle membranes. The procedure involves the use of ion exchange chromatography and sucrose gradient centrifugation to purify the protein which has been identified as the calcium release protein of the sarcoplasmic reticulum (Imagawa, T., Smith, J., Coronado, R. and Campbell, K. (1987) J. Biol. Chem. 262:16,636–16,643). Addition of micromolar quantities of unlabeled ryanodine prior to solubilization and throughout the isolation procedure appears to stabilize the tetrameric structure of the ryanodine receptor. The purified receptor, consisting predominantly of a 400K polypeptide on SDS-PAGE, binds [³H]ryanodine with a binding affinity similar to that in membranes. Overall recovery of ryanodine binding activity was 21% of the initial activity with a 30-fold purification of the receptor.     

Unitary Ca2+ Current through Cardiac Ryanodine Receptor Channels under Quasi-Physiological Ionic Conditions

Single canine cardiac ryanodine receptor channels were incorporated into planar lipid bilayers. Single-channel currents were sampled at 1–5 kHz and filtered at 0.2–1.0 kHz. Channel incorporations were obtained in symmetrical solutions (20 mM HEPES-Tris, pH 7.4, and pCa 5). Unitary Ca²⁺ currents were monitored when 2–30 mM Ca²⁺ was added to the lumenal side of the channel. The relationship between the amplitude of unitary Ca²⁺ current (at 0 mV holding potential) and lumenal [Ca²⁺] was hyperbolic and saturated at ∼4 pA. This relationship was then defined in the presence of different symmetrical CsCH₃SO₃ concentrations (5, 50, and 150 mM). Under these conditions, unitary current amplitude was 1.2 ± 0.1, 0.65 ± 0.1, and 0.35 ± 0.1 pA in 2 mM lumenal Ca²⁺; and 3.3 ± 0.4, 2.4 ± 0.2, and 1.63 ± 0.2 pA in 10 mM lumenal Ca²⁺ (n > 6). Unitary Ca²⁺ current was also defined in the presence of symmetrical [Mg²⁺] (1 mM) and low [Cs⁺] (5 mM). Under these conditions, unitary Ca²⁺ current in 2 and 10 mM lumenal Ca²⁺ was 0.66 ± 0.1 and 1.52 ± 0.06 pA, respectively. In the presence of higher symmetrical [Cs⁺] (50 mM), Mg²⁺ (1 mM), and lumenal [Ca²⁺] (10 mM), unitary Ca²⁺ current exhibited an amplitude of 0.9 ± 0.2 pA (n = 3). This result indicates that the actions of Cs⁺ and Mg²⁺ on unitary Ca²⁺ current were additive. These data demonstrate that physiological levels of monovalent cation and Mg²⁺ effectively compete with Ca²⁺ as charge carrier in cardiac ryanodine receptor channels. If lumenal free Ca²⁺ is 2 mM, then our results indicate that unitary Ca²⁺ current under physiological conditions should be <0.6 pA.     

Ca2+-induced Ca2+ Release in Chinese Hamster Ovary (CHO) Cells Co-expressing Dihydropyridine and Ryanodine Receptors

Combined patch-clamp and Fura-2 measurements were performed on chinese hamster ovary (CHO) cells co-expressing two channel proteins involved in skeletal muscle excitation-contraction (E-C) coupling, the ryanodine receptor (RyR)-Ca²⁺ release channel (in the membrane of internal Ca²⁺ stores) and the dihydropyridine receptor (DHPR)-Ca²⁺ channel (in the plasma membrane). To ensure expression of functional L-type Ca²⁺ channels, we expressed α₂, β, and γ DHPR subunits and a chimeric DHPR α₁ subunit in which the putative cytoplasmic loop between repeats II and III is of skeletal origin and the remainder is cardiac. There was no clear indication of skeletal-type coupling between the DHPR and the RyR; depolarization failed to induce a Ca²⁺ transient (CaT) in the absence of extracellular Ca²⁺ ([Ca²⁺]_o). However, in the presence of [Ca²⁺]_o, depolarization evoked CaTs with a bell-shaped voltage dependence. About 30% of the cells tested exhibited two kinetic components: a fast transient increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) (the first component; reaching 95% of its peak <0.6 s after depolarization) followed by a second increase in [Ca²⁺]_i which lasted for 5–10 s (the second component). Our results suggest that the first component primarily reflected Ca²⁺ influx through Ca²⁺ channels, whereas the second component resulted from Ca²⁺ release through the RyR expressed in the membrane of internal Ca²⁺ stores. However, the onset and the rate of Ca²⁺ release appeared to be much slower than in native cardiac myocytes, despite a similar activation rate of Ca²⁺ current. These results suggest that the skeletal muscle RyR isoform supports Ca²⁺-induced Ca²⁺ release but that the distance between the DHPRs and the RyRs is, on average, much larger in the cotransfected CHO cells than in cardiac myocytes. We conclude that morphological properties of T-tubules and/or proteins other than the DHPR and the RyR are required for functional “close coupling” like that observed in skeletal or cardiac muscle. Nevertheless, some of our results imply that these two channels are potentially able to directly interact with each other.     

Comparison of the Effects Exerted by Luminal Ca2+ on the Sensitivity of the Cardiac Ryanodine Receptor to Caffeine and Cytosolic Ca2+

Ca²⁺ released from the sarcoplasmic reticulum (SR) via ryanodine receptor type 2 (RYR2) is the key determinant of cardiac contractility. Although activity of RYR2 channels is primary controlled by Ca²⁺ entry through the plasma membrane, there is growing evidence that Ca²⁺ in the lumen of the SR can also be effectively involved in the regulation of RYR2 channel function. In the present study, we investigated the effect of luminal Ca²⁺ on the response of RYR2 channels reconstituted into a planar lipid membrane to caffeine and Ca²⁺ added to the cytosolic side of the channel. We performed two sets of experiments when the channel was exposed to either luminal Ba²⁺ or Ca²⁺. The given ion served also as a charge carrier. Luminal Ca²⁺ effectively shifted the EC₅₀ for caffeine sensitivity to a lower concentration but did not modify the response of RYR2 channels to cytosolic Ca²⁺. Importantly, luminal Ca²⁺ exerted an effect on channel gating kinetics. Both the open and closed dwell times were considerably prolonged over the whole range (response to caffeine) or the partial range (response to cytosolic Ca²⁺) of open probability. Our results provide strong evidence that an alteration of the gating kinetics is the result of the interaction of luminal Ca²⁺ with the luminally located Ca²⁺ regulatory sites on the RYR2 channel complex.     

Effects of Metal-Binding Properties of Human Kv Channel-Interacting Proteins on their Molecular Structure and Binding with Kv4.2 Channel

The goal of the present study is to explore whether Ca²⁺ and Mg²⁺-binding properties of isomeric Kv channel-interacting proteins (KChIPs) have different effects on their molecular structure and the binding with Kv channel. 8-Anilinonaphthalene- 1-sulfonate fluorescence measurement showed that KChIP4.1 and KChIP2.2 possessed one and two types of Ca²⁺-binding sites, respectively, and only one type of Mg²⁺-binding site was noted in the two KChIP proteins. Removal of EF-hand 4 (EF-4) caused a marked drop in their high affinities for Ca²⁺, but the binding affinity for Mg²⁺ remained mostly the same. Unlike KChIP4.1, the intact EF-4 was essential for the Kv channel-binding ability of KChIP2.2 in a metal-free buffer. Nevertheless, the interaction of wild-type KChIPs and EF-4-truncated mutants with Kv channel was enhanced by the addition of Mg²⁺ and Ca²⁺. In contrast to KChIP4.1, the thermal stability of KChIP2.2 was decreased by the binding of Mg²⁺ and Ca²⁺. These results suggest that the conformational change with metal-bound KChIP4.1 is crucial for its interaction with Kv channel but not for KChIP2.2, and that the Mg²⁺- and Ca²⁺-binding properties of KChIP2.2 and KChIP4.1 have different effects on their molecular structure.     

Location of the permeation pathway in the recombinant type 1 inositol 1,4,5-trisphosphate receptor.

The inositol 1,4,5-trisphosphate receptor (InsP(3)R) forms ligand-regulated intracellular Ca(2+) release channels in the endoplasmic reticulum of all mammalian cells. The InsP(3)R has been suggested to have six transmembrane regions (TMRs) near its carboxyl terminus. A TMR-deletion mutation strategy was applied to define the location of the InsP(3)R pore. Mutant InsP(3)Rs were expressed in COS-1 cells and single channel function was defined in planar lipid bilayers. Mutants having the fifth and sixth TMR (and the interceding lumenal loop), but missing all other TMRs, formed channels with permeation properties similar to wild-type channels (gCs = 284; gCa = 60 pS; P(Ca)/P(Cs) = 6.3). These mutant channels bound InsP(3), but ligand occupancy did not regulate the constitutively open pore (P(o) > 0.80). We propose that a region of 191 amino acids (including the fifth and sixth TMR, residues 2398-2589) near the COOH terminus of the protein forms the InsP(3)R pore. Further, we have produced a constitutively open InsP(3)R pore mutant that is ideal for future site-directed mutagenesis studies of the structure-function relationships that define Ca(2+) permeation through the InsP(3)R channel.     

Ryanodine as a functional probe of the skeletal muscle sarcoplasmic reticulum Ca2+ release channel

Ryanodine is a neutral plant alkaloid which functions as a probe for an intracellular Ca²⁺ release channel (ryanodine receptor) in excitable tissues. Using [³H]ryanodine, a 30 S protein complex comprised of four polypeptides of M_r 565,000 has been isolated and functionally reconstituted into planar lipid bilayers. The effects of salt concentration and divalent cations on skeletal muscle sarcoplasmic reticulum [³H]ryanodine binding and Ca²⁺ release channel activity have been compared. These studies suggest that ryanodine is a good probe for investigating the function of the release channel.     

Ion Channel Selectivity through Stepwise Changes in Binding Affinity

Voltage-gated Ca²⁺ channels select Ca²⁺ over competing, more abundant ions by means of a high affinity binding site in the pore. The maximum off rate from this site is ∼1,000× slower than observed Ca²⁺ current. Various theories that explain how high Ca²⁺ current can pass through such a sticky pore all assume that flux occurs from a condition in which the pore''s affinity for Ca²⁺ transiently decreases because of ion interactions. Here, we use rate theory calculations to demonstrate a different mechanism that requires no transient changes in affinity to quantitatively reproduce observed Ca²⁺ channel behavior. The model pore has a single high affinity Ca²⁺ binding site flanked by a low affinity site on either side; ions permeate in single file without repulsive interactions. The low affinity sites provide steps of potential energy that speed the exit of a Ca²⁺ ion off the selectivity site, just as potential energy steps accelerate other chemical reactions. The steps could be provided by weak binding in the nonselective vestibules that appear to be a general feature of ion channels, by specific protein structures in a long pore, or by stepwise rehydration of a permeating ion. The previous ion-interaction models and this stepwise permeation model demonstrate two general mechanisms, which might well work together, to simultaneously generate high flux and high selectivity in single file pores.     

Calcium and inositol trisphosphate receptors

Work from the authors' laboratory is supported by the Wellcome Trust, and the Medical, and Agricultural and Food Research Councils. CWT is a Lister Institute Research Fellow.