The CRAC channel activator STIM1 binds and inhibits L-type voltage-gated calcium channels

Voltage- and store-operated calcium (Ca(2+)) channels are the major routes of Ca(2+) entry in mammalian cells, but little is known about how cells coordinate the activity of these channels to generate coherent calcium signals. We found that STIM1 (stromal interaction molecule 1), the main activator of store-operated Ca(2+) channels, directly suppresses depolarization-induced opening of the voltage-gated Ca(2+) channel Ca(V)1.2. STIM1 binds to the C terminus of Ca(V)1.2 through its Ca(2+) release-activated Ca(2+) activation domain, acutely inhibits gating, and causes long-term internalization of the channel from the membrane. This establishes a previously unknown function for STIM1 and provides a molecular mechanism to explain the reciprocal regulation of these two channels in cells.     

BKCa-Cav channel complexes mediate rapid and localized Ca2+-activated K+ signaling

Large-conductance calcium- and voltage-activated potassium channels (BKCa) are dually activated by membrane depolarization and elevation of cytosolic calcium ions (Ca2+). Under normal cellular conditions, BKCa channel activation requires Ca2+ concentrations that typically occur in close proximity to Ca2+ sources. We show that BKCa channels affinity-purified from rat brain are assembled into macromolecular complexes with the voltage-gated calcium channels Cav1.2 (L-type), Cav2.1 (P/Q-type), and Cav2.2 (N-type). Heterologously expressed BKCa-Cav complexes reconstitute a functional "Ca2+ nanodomain" where Ca2+ influx through the Cav channel activates BKCa in the physiological voltage range with submillisecond kinetics. Complex formation with distinct Cav channels enables BKCa-mediated membrane hyperpolarization that controls neuronal firing pattern and release of hormones and transmitters in the central nervous system.     

猪BMSCs成脂分化中细胞膜钙离子通道、钙敏感受体及成脂定向相关基因表达研究

【目的】研究猪骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs)定向分化为脂肪细胞过程中细胞膜上钙离子通道、钙敏感受体(Calcium-sensing receptor,Ca SR)基因及成脂定向相关基因的表达。【方法】从5~7日龄仔猪骨髓中分离纯化出猪BMSCs,诱导猪BMSCs成脂分化。油红O法和三酰甘油法检测细胞分化聚酯状况。在成脂分化不同时间(0、1、2、5和10 d)收集细胞,利用荧光定量PCR检测锌指蛋白423(Zinc finger protein423,Zfp423)、脂肪前体细胞因子(Preadipocyte factor 1,Pref-1)、骨形态发生蛋白2(Bone morphogenetic protein 2,BMP2)、骨形态发生蛋白4(Bone morphogenetic protein 4,BMP4)、细胞膜钙离子通道及Ca SR基因的mRNA表达变化。【结果】油红O染色和三酰甘油检测结果表明,成功诱导猪BMSCs成脂分化;定量PCR结果显示,在猪BMSCs成脂分化第5天,成脂定向标志基因Zfp423、脂肪前体细胞标志基因Pref-1及促进成脂分化基因BMP2、BMP4的mRNA相对表达量显著提高(P0.05),说明第5天是猪BMSCs成脂定向形成脂肪前体细胞的关键时期;同时,细胞膜上的电压门控钙离子通道亚基电压依赖型α/δ亚型1(Voltage-dependentalpha-2/delta subunit 1,CACNA2D1)、钙释放激活钙通道调节分子1(Calciumr elease-activated calcium channel modulator 1,Orai1)、瞬时受体电位通道传统型1(Transient receptor potential canonical type 1,TRPC1)、瞬时受体电位通道M型7(Transient receptor potential melastatin 7,TRPM7)、瞬时受体电位通道香草素受体亚型1(Transient receptor potential vanilloid receptor1,TRPV1)基因和Ca SR基因在诱导成脂第5天mRNA相对表达量也显著提高(P0.05),提示细胞膜钙离子通道及Ca SR基因可能参与了猪BMSCs成脂分化过程。【结论】揭示了猪BMSCs成脂分化过程中细胞膜钙离子通道、钙敏感受体及成脂定向相关基因的表达模式。     

Odor stimuli trigger influx of calcium into olfactory neurons of the channel catfish

Olfactory transduction is thought to be mediated by a G protein-coupled increase in intracellular adenosine 3',5'-monophosphate (cAMP) that triggers the opening of cAMP-gated cation channels and results in depolarization of the plasma membrane of olfactory neurons. In olfactory neurons isolated from the channel catfish, Ictalurus punctatus, stimulation with olfactory stimuli (amino acids) elicits an influx of calcium that leads to a rapid increase in intracellular calcium. In addition, in a reconstitution assay a plasma membrane calcium channel has been identified that is gated by inositol-1,4,5-trisphosphate (IP3), which could mediate this calcium influx. Together with previous studies indicating that stimulation with olfactory stimuli leads to stimulation of phosphoinositide turnover in olfactory cilia, these data suggest that an influx of calcium triggered by odor stimulation of phosphoinositide turnover may be an alternate or additional mechanism of olfactory transduction.     

Glutamate receptor-like genes form Ca2+ channels in pollen tubes and are regulated by pistil D-serine

Elevations in cytosolic free calcium concentration ([Ca(2+)](cyt)) constitute a fundamental signal transduction mechanism in eukaryotic cells, but the molecular identity of Ca(2+) channels initiating this signal in plants is still under debate. Here, we show by pharmacology and loss-of-function mutants that in tobacco and Arabidopsis, glutamate receptor-like channels (GLRs) facilitate Ca(2+) influx across the plasma membrane, modulate apical [Ca(2+)](cyt) gradient, and consequently affect pollen tube growth and morphogenesis. Additionally, wild-type pollen tubes grown in pistils of knock-out mutants for serine-racemase (SR1) displayed growth defects consistent with a decrease in GLR activity. Our findings reveal a novel plant signaling mechanism between male gametophyte and pistil tissue similar to amino acid-mediated communication commonly observed in animal nervous systems.     

NaCl对泌盐红树和非泌盐红树Cd吸收和积累的影响

为研究NaCl对红树植物Cd吸收和转运的影响,本文以非泌盐红树秋茄和泌盐红树桐花树幼苗为实验材料,研究了不同浓度NaCl和CdCl2处理下地上和地下各器官中Na+、Ca2+、Cd2+离子浓度的变化,并利用非损伤微测技术测定植物根尖在不同处理下对Cd2+和Ca2+的动态吸收。结果表明,随着CdCl2处理浓度的增加,2种红树的根、胚轴、茎和叶4器官中的Cd2+含量均明显增加。而泌盐红树桐花树各器官中Cd2+含量均高于非泌盐红树秋茄,分别达到65%(根)、19%(胚轴)、203%(茎)和96%(叶)。利用非损伤微测技术测定Cd2+流,发现Cd2+内流能被Ca2+通道抑制剂LaCl3抑制,表明Cd2+主要通过Ca2+通道实现内流。在NaCl对Cd2+吸收的影响方面,低浓度NaCl(100~200 mmol/L)能促进秋茄对Cd2+的积累,但高浓度NaCl(400 mmol/L)抑制了桐花树和秋茄对Cd2+的吸收。这是由于:1)红树根系对Na+吸收增加,而Na+能与Cd2+竞争膜上转运蛋白上的金属离子结合位点从而减少Cd2+的吸收,2)NaCl促进了植物根尖对Ca2+的吸收,从而竞争性的抑制了Cd2+通过Ca2+通道的内流,最终减少了2种红树根系对Cd2+的吸收和积累。泌盐红树桐花树Cd2+含量高于非泌盐红树秋茄,表明桐花树根细胞质膜上的转运蛋白与Ca2+通道对Cd2+的吸收能力高于秋茄。      

胞外ATP、H2O2、Ca2+与NO调控木榄根系离子平衡机理研究

运用非损伤微测技术(NMT),研究了短期盐胁迫下胞外ATP(eATP)、H2 O2 、Ca2 + 与NO 对非泌盐红树木榄根 系K+/Na+ 平衡的调控作用。NaCl(100 mmol/L,24 h)与等渗甘露醇处理的实验表明,木榄根尖对盐胁迫的响应具 有高度的离子特异性。盐胁迫增强了木榄根尖的Na+ 外流,但Na+ 外流被Na+ /H+ 逆向转运蛋白抑制剂Amiloride 和质膜H+ -ATPase 抑制剂Vanadate 抑制,表明Na+ 外流源于根尖表皮细胞质膜Na+ /H+ 逆向转运系统驱动的Na+ 外排。短期盐胁迫处理能诱导木榄根尖K+ 外流,但被氯化四乙胺(TEA,外向K+ 通道抑制剂)明显抑制,证明K+ 外流是由激活的去极化外向型离子通道KORCs 介导。胞外ATP(300 mol/L)、H2 O2 (10 mmol/L)、Ca2 + (10 mmol/ L)与SNP(NO 供体,100 mol/L)均能增加短期盐胁迫下的Na+ 外流,同时抑制K+ 外流。其中,促进Na+ 外流效果 较强的是H2 O2 和Ca2 + ,而Ca2 + 和NO 抑制K+ 外流的效果突出。这些实验结果表明,胞外ATP、H2 O2 、Ca2 + 与NO 这4 种盐胁迫信使是通过上调木榄根系细胞质膜Na+ /H+ 逆向转运体系(Na+ /H+ 逆向转运体和H+ 泵)活性,在促 进Na+ 和H+ 逆向跨膜转运的同时,抑制去极化激活的K+ 离子通道来减少盐诱导的K+ 外流。      

植物低温信号的感知、转导与转录调控

低温是植物生长的主要环境胁迫因子之一。植物对低温的应激是一个复杂的过程,包括低温信号的感知、信号转导和转录调控等阶段。低温可以通过质膜流动性的改变被质膜感知,也可以通过质膜上的钙离子通透性通道、组氨酸激酶、受体激酶和磷酸酯酶感知。低温信号转导包括钙信号途径和其他信号途径,其中钙信号途径是低温应答过程中重要的信号途径。在此途径中,因低温增加的胞质钙离子能被CDPK、磷酸酶和MAPK识别并传导;其他信号途径主要与ABA有关。低温信号最终将启动CBF和非CBF介导的转录调控,提高植物的低温抗性。     

Bruchpilot promotes active zone assembly, Ca2+ channel clustering, and vesicle release

The molecular organization of presynaptic active zones during calcium influx-triggered neurotransmitter release is the focus of intense investigation. The Drosophila coiled-coil domain protein Bruchpilot (BRP) was observed in donut-shaped structures centered at active zones of neuromuscular synapses by using subdiffraction resolution STED (stimulated emission depletion) fluorescence microscopy. At brp mutant active zones, electron-dense projections (T-bars) were entirely lost, Ca2+ channels were reduced in density, evoked vesicle release was depressed, and short-term plasticity was altered. BRP-like proteins seem to establish proximity between Ca2+ channels and vesicles to allow efficient transmitter release and patterned synaptic plasticity.     

Ca2+ entry through plasma membrane IP3 receptors

Inositol 1,4,5-trisphosphate receptors (IP3Rs) release calcium ions, Ca2+, from intracellular stores, but their roles in mediating Ca2+ entry are unclear. IP3 stimulated opening of very few (1.9 +/- 0.2 per cell) Ca2+-permeable channels in whole-cell patch-clamp recording of DT40 chicken or mouse B cells. Activation of the B cell receptor (BCR) in perforated-patch recordings evoked the same response. IP3 failed to stimulate intracellular or plasma membrane (PM) channels in cells lacking IP3R. Expression of IP3R restored both responses. Mutations within the pore affected the conductances of IP3-activated PM and intracellular channels similarly. An impermeant pore mutant abolished BCR-evoked Ca2+ signals, and PM IP3Rs were undetectable. After introduction of an alpha-bungarotoxin binding site near the pore, PM IP3Rs were modulated by extracellular alpha-bungarotoxin. IP(3)Rs are unusual among endoplasmic reticulum proteins in being also functionally expressed at the PM, where very few IP3Rs contribute substantially to the Ca2+ entry evoked by the BCR.     

Residual calcium ions depress activation of calcium-dependent current

Calcium ions enter and accumulate during depolarization of some cells, activating a potassium current, IK(Ca), that depends on the cytoplasmic concentration of calcium ions, [Ca]i. However, elevation of [Ca]i can depress IK(Ca) elicited by a subsequent membrane depolarization. The depression of IK(Ca) is ascribed here to a [Ca]i-mediated inactivation of the voltage-gated calcium conductance, which causes a net reduction in calcium ions available for the activation of IK(Ca). This suggests that other processes dependent on gated calcium entry may also be depressed by small background elevations in cytosolic free calcium ions.     

Rapid chemically induced changes of PtdIns(4,5)P2 gate KCNQ ion channels

To resolve the controversy about messengers regulating KCNQ ion channels during phospholipase C-mediated suppression of current, we designed translocatable enzymes that quickly alter the phosphoinositide composition of the plasma membrane after application of a chemical cue. The KCNQ current falls rapidly to zero when phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2 or PI(4,5)P2] is depleted without changing Ca2+, diacylglycerol, or inositol 1,4,5-trisphosphate. Current rises by 30% when PI(4,5)P2 is overproduced and does not change when phosphatidylinositol 3,4,5-trisphosphate is raised. Hence, the depletion of PI(4,5)P2 suffices to suppress current fully, and other second messengers are not needed. Our approach is ideally suited to study biological signaling networks involving membrane phosphoinositides.     

CRACM1 is a plasma membrane protein essential for store-operated Ca2+ entry

Store-operated Ca2+ entry is mediated by Ca2+ release-activated Ca2+ (CRAC) channels following Ca2+ release from intracellular stores. We performed a genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins that inhibit store-operated Ca2+ influx. A secondary patch-clamp screen identified CRACM1 and CRACM2 (CRAC modulators 1 and 2) as modulators of Drosophila CRAC currents. We characterized the human ortholog of CRACM1, a plasma membrane-resident protein encoded by gene FLJ14466. Although overexpression of CRACM1 did not affect CRAC currents, RNAi-mediated knockdown disrupted its activation. CRACM1 could be the CRAC channel itself, a subunit of it, or a component of the CRAC signaling machinery.     

A G protein directly regulates mammalian cardiac calcium channels

A possible direct effect of guanine nucleotide binding (G) proteins on calcium channels was examined in membrane patches excised from guinea pig cardiac myocytes and bovine cardiac sarcolemmal vesicles incorporated into planar lipid bilayers. The guanosine triphosphate analog, GTP gamma S, prolonged the survival of excised calcium channels independently of the presence of adenosine 3',5'-monophosphate (cAMP), adenosine triphosphate, cAMP-activated protein kinase, and the protein kinase C activator tetradecanoyl phorbol acetate. A specific G protein, activated Gs, or its alpha subunit, purified from the plasma membranes of human erythrocytes, prolonged the survival of excised channels and stimulated the activity of incorporated channels. Thus, in addition to regulating calcium channels indirectly through activation of cytoplasmic kinases, G proteins can regulate calcium channels directly. Since they also directly regulate a subset of potassium channels, G proteins are now known to directly gate two classes of membrane ion channels.     

Requirement of the inositol trisphosphate receptor for activation of store-operated Ca2+ channels

The coupling mechanism between endoplasmic reticulum (ER) calcium ion (Ca2+) stores and plasma membrane (PM) store-operated channels (SOCs) is crucial to Ca2+ signaling but has eluded detection. SOCs may be functionally related to the TRP family of receptor-operated channels. Direct comparison of endogenous SOCs with stably expressed TRP3 channels in human embryonic kidney (HEK293) cells revealed that TRP3 channels differ in being store independent. However, condensed cortical F-actin prevented activation of both SOC and TRP3 channels, which suggests that ER-PM interactions underlie coupling of both channels. A cell-permeant inhibitor of inositol trisphosphate receptor (InsP3R) function, 2-aminoethoxydiphenyl borate, prevented both receptor-induced TRP3 activation and store-induced SOC activation. It is concluded that InsP3Rs mediate both SOC and TRP channel opening and that the InsP3R is essential for maintaining coupling between store emptying and physiological activation of SOCs.     

alpha-Klotho as a regulator of calcium homeostasis

alpha-klotho was identified as a gene associated with premature aging-like phenotypes characterized by short lifespan. In mice, we found the molecular association of alpha-Klotho (alpha-Kl) and Na+,K+-adenosine triphosphatase (Na+,K+-ATPase) and provide evidence for an increase of abundance of Na+,K+-ATPase at the plasma membrane. Low concentrations of extracellular free calcium ([Ca2+]e) rapidly induce regulated parathyroid hormone (PTH) secretion in an alpha-Kl- and Na+,K+-ATPase-dependent manner. The increased Na+ gradient created by Na+,K+-ATPase activity might drive the transepithelial transport of Ca2+ in cooperation with ion channels and transporters in the choroid plexus and the kidney. Our findings reveal fundamental roles of alpha-Kl in the regulation of calcium metabolism.     

钙离子在植物生理调节中的作用

钙是植物必须的营养元素,同时也是植物体内转导多种生理过程的胞内胞外信号物质之一。胞外Ca2+通过Ca2+通道内流进入胞质,并通过Ca2+-ATPase和Ca2+/H+反向转运蛋白外流,以保持胞质内低Ca2+浓度。同时为了应对植物发育和环境胁迫信号,Ca2+由质膜、液泡膜和内质网膜的Ca2+通道内流进入胞质,导致胞质Ca2+浓度迅速增加,产生钙瞬变和钙振荡,传递到钙信号靶蛋白(如钙调素、钙依赖型蛋白激酶及钙调磷酸酶B类蛋白,引起特异的生理生化反应),这一系列钙信号调节、应答机制构成了植物的钙信号系统。对钙转运系统、钙信号调节和放大及应答方式进行了综述。     

NMDA receptor-mediated K+ efflux and neuronal apoptosis

Neuronal death induced by activating N-methyl-D-aspartate (NMDA) receptors has been linked to Ca2+ and Na+ influx through associated channels. Whole-cell recording from cultured mouse cortical neurons revealed a NMDA-evoked outward current, INMDA-K, carried by K+ efflux at membrane potentials positive to -86 millivolts. Cortical neurons exposed to NMDA in medium containing reduced Na+ and Ca2+ (as found in ischemic brain tissue) lost substantial intracellular K+ and underwent apoptosis. Both K+ loss and apoptosis were attenuated by increasing extracellular K+, even when voltage-gated Ca2+ channels were blocked. Thus NMDA receptor-mediated K+ efflux may contribute to neuronal apoptosis after brain ischemia.     

How synaptotagmin promotes membrane fusion

Synaptic vesicles loaded with neurotransmitters are exocytosed in a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent manner after presynaptic depolarization induces calcium ion (Ca2+) influx. The Ca2+ sensor required for fast fusion is synaptotagmin-1. The activation energy of bilayer-bilayer fusion is very high (approximately 40 k(B)T). We found that, in response to Ca2+ binding, synaptotagmin-1 could promote SNARE-mediated fusion by lowering this activation barrier by inducing high positive curvature in target membranes on C2-domain membrane insertion. Thus, synaptotagmin-1 triggers the fusion of docked vesicles by local Ca2+-dependent buckling of the plasma membrane together with the zippering of SNAREs. This mechanism may be widely used in membrane fusion.     

Regulation of chloride channels by protein kinase C in normal and cystic fibrosis airway epithelia

Apical membrane chloride channels control chloride secretion by airway epithelial cells. Defective regulation of these channels is a prominent characteristic of cystic fibrosis. In normal intact cells, activation of protein kinase C (PKC) by phorbol ester either stimulated or inhibited chloride secretion, depending on the physiological status of the cell. In cell-free membrane patches, PKC also had a dual effect: at a high calcium concentration, PKC inactivated chloride channels; at a low calcium concentration, PKC activated chloride channels. In cystic fibrosis cells, PKC-dependent channel inactivation was normal, but activation was defective. Thus it appears that PKC phosphorylates and regulates two different sites on the channel or on an associated membrane protein, one of which is defective in cystic fibrosis.