Ca2+-dependent Cl− efflux in tonoplast-free cells ofNitellopsis obtusa

Summary In order to demonstrate the presence of a Ca²⁺-activated Cl^–-channel in theNitellopsis plasmalemma, tonoplast-free cells were prepared and their intracellular Ca²⁺ concentration was modified by internal perfusion. An increase in the Ca²⁺ concentration caused a large Cl^– efflux with a concomitant depolarization of the membrane potential. These changes were for the most part reversible. The critical Ca²⁺ concentration was about 4.0 m. Neither the Cl^– efflux nor the membrane depolarization showed a time-dependent inactivation. A Cl^–-channel blocker, A-9-C (9-anthracenecarboxylic acid) reduced both the Cl^– efflux and the magnitude of the membrane potential depolarization. A small increase in the intracellular Ca²⁺ concentration, which is caused by membrane excitation of tonoplast-free cells is not sufficient to activate this Ca²⁺-dependent Cl^–-channel.     

Intracellular Ca2+-dependent formation of N-acyl-phosphatidylethanolamines by human cytosolic phospholipase A2ε

N-Acyl-phosphatidylethanolamines (NAPEs) are known to be precursors of bioactive N-acylethanolamines (NAEs), including the endocannabinoid arachidonoylethanolamide (anandamide) and anti-inflammatory palmitoylethanolamide. In mammals, NAPEs are produced by N-acyltransferases, which transfer an acyl chain from the sn-1 position of glycerophospholipid to the amino group of phosphatidylethanolamine (PE). Recently, the ɛ isoform of cytosolic phospholipase A₂ (cPLA₂ɛ) was found to be Ca²⁺-dependent N-acyltransferase. However, it was poorly understood which types of phospholipids serve as substrates in living cells. In the present study, we established a human embryonic kidney 293 cell line, in which doxycycline potently induces human cPLA₂ɛ, and used these cells to analyze endogenous substrates and products of cPLA₂ɛ with liquid chromatography-tandem mass spectrometry. When treated with doxycycline and Ca²⁺ ionophore, the cells produced various species of diacyl- and alkenylacyl-types of NAPEs as well as NAEs in large quantities. Moreover, the levels of diacyl- and alkenylacyl-types of PEs and diacyl-phosphatidylcholines (PCs) decreased, while those of lysophosphatidylethanolamines and lysophosphatidylcholines increased. These results suggested that cPLA₂ɛ Ca²⁺-dependently produces NAPEs by utilizing endogenous diacyl- and alkenylacyl-types of PEs as acyl acceptors and diacyl-type PCs and diacyl-type PEs as acyl donors.     

Ca2+-dependent Inactivation of CaV1.2 Channels Prevents Gd3+ Block: Does Ca2+ Block the Pore of Inactivated Channels?

Lanthanide gadolinium (Gd(3+)) blocks Ca(V)1.2 channels at the selectivity filter. Here we investigated whether Gd(3+) block interferes with Ca(2+)-dependent inactivation, which requires Ca(2+) entry through the same site. Using brief pulses to 200 mV that relieve Gd(3+) block but not inactivation, we monitored how the proportions of open and open-blocked channels change during inactivation. We found that blocked channels inactivate much less. This is expected for Gd(3+) block of the Ca(2+) influx that enhances inactivation. However, we also found that the extent of Gd(3+) block did not change when inactivation was reduced by abolition of Ca(2+)/calmodulin interaction, showing that Gd(3+) does not block the inactivated channel. Thus, Gd(3+) block and inactivation are mutually exclusive, suggesting action at a common site. These observations suggest that inactivation causes a change at the selectivity filter that either hides the Gd(3+) site or reduces its affinity, or that Ca(2+) occupies the binding site at the selectivity filter in inactivated channels. The latter possibility is supported by previous findings that the EEQE mutation of the selectivity EEEE locus is void of Ca(2+)-dependent inactivation (Zong Z.Q., J.Y. Zhou, and T. Tanabe. 1994. Biochem. Biophys. Res. Commun. 201:1117-11123), and that Ca(2+)-inactivated channels conduct Na(+) when Ca(2+) is removed from the extracellular medium (Babich O., D. Isaev, and R. Shirokov. 2005. J. Physiol. 565:709-717). Based on these results, we propose that inactivation increases affinity of the selectivity filter for Ca(2+) so that Ca(2+) ion blocks the pore. A minimal model, in which the inactivation "gate" is an increase in affinity of the selectivity filter for permeating ions, successfully simulates the characteristic U-shaped voltage dependence of inactivation in Ca(2+).     

Does a calmodulin-dependent Ca2+-regulated Mg2+-dependent ATPase contribute to hepatic microsomal calcium uptake?

Solubilization of microsomal proteins followed by calmodulin affinity chromatography resulted in the separation of two distinct Ca2+-Mg2+-ATPases (Ca2+-regulated Mg2+-dependent ATPases), one being insensitive to calmodulin (ATPase-1), the other being stimulated about 5-fold by calmodulin (ATPase-2). ATPase-2 accounts for only 8% of total microsomal Ca2+-Mg2+-ATPase-activity. ATPase-1 and -2 can also be distinguished by different pH optima, different sensitivity towards inhibition by vanadate and LaCl3, and different apparent Mr values of the phosphoenzyme intermediates (115,000 and 150,000 for ATPase-1 and ATPase-2 respectively). ATPase-1 from liver co-migrated with Ca2+-Mg2+-ATPase from rat skeletal-muscle sarcoplasmic reticulum, whereas ATPase-2 from liver co-migrated with calmodulin-dependent Ca2+-Mg2+-ATPase derived from rat skeletal-muscle sarcolemma. After separation of parenchymal and nonparenchymal liver cells, a calmodulin-dependent Ca2+-Mg2+-ATPase of Mr 150,000 was found only in the non-parenchymal cells. The kinetic parameters of ATPase-2 and the similarity of the apparent Mr of its phosphoenzyme intermediate to that of skeletal-muscle sarcolemma Ca2+-Mg2+-ATPase makes it likely that the calmodulin-sensitive Ca2+-Mg2+-ATPase found in rat liver microsomal fractions reflects a contamination with plasma membranes (possibly from non-parenchymal cells) rather than a true location in the endoplasmic reticulum of parenchymal liver cells.     

Calretinin Regulates Ca2+-dependent Inactivation and Facilitation of Cav2.1 Ca2+ Channels through a Direct Interaction with the α12.1 Subunit

Voltage-gated Ca_v2.1 Ca²⁺ channels undergo dual modulation by Ca²⁺, Ca²⁺-dependent inactivation (CDI), and Ca²⁺-dependent facilitation (CDF), which can influence synaptic plasticity in the nervous system. Although the molecular determinants controlling CDI and CDF have been the focus of intense research, little is known about the factors regulating these processes in neurons. Here, we show that calretinin (CR), a Ca²⁺-binding protein highly expressed in subpopulations of neurons in the brain, inhibits CDI and enhances CDF by binding directly to α₁2.1. Screening of a phage display library with CR as bait revealed a highly basic CR-binding domain (CRB) present in multiple copies in the cytoplasmic linker between domains II and III of α₁2.1. In pulldown assays, CR binding to fusion proteins containing these CRBs was largely Ca²⁺-dependent. α₁2.1 coimmunoprecipitated with CR antibodies from transfected cells and mouse cerebellum, which confirmed the existence of CR-Ca_v2.1 complexes in vitro and in vivo. In HEK293T cells, CR significantly decreased Ca_v2.1 CDI and increased CDF. CR binding to α₁2.1 was required for these effects, because they were not observed upon substitution of the II-III linker of α₁2.1 with that from the Ca_v1.2 α₁ subunit (α₁1.2), which lacks the CRBs. In addition, coexpression of a protein containing the CRBs blocked the modulatory action of CR, most likely by competing with CR for interactions with α₁2.1. Our findings highlight an unexpected role for CR in directly modulating effectors such as Ca_v2.1, which may have major consequences for Ca²⁺ signaling and neuronal excitability.     

Evidence for β-adrenergic activation of Na+-dependent efflux of Ca2+ from isolated liver mitochondria

The existence of a Na⁺-dependent mechanism for Ca²⁺ efflux from isolated rat liver mitochondria was confirmed. The activity of this system is decreased by 60% in mitochondria isolated from perfused livers. The Na⁺-dependent activity is fully restored by infusion of either 1μm-adrenaline or 1μm-isoprenaline, but the α-adrenergic agonist phenylephrine is ineffective.     

植物细胞Ca2+的微调系统——Ca2+_ATPase

本文对植物体细胞Ca２＋-ATPase的类型、亚细胞定位、生化特性、分子量差异、基因克隆、酶活性调节剂以及生理功能等方面的研究进展进行综述和讨论。     

骨骼肌内质网Ca2+泵转运Ca2+的结构基础

Ca2 泵(Ca2 -ATPase)是调节细胞内Ca2 浓度的重要蛋白质之一.Ca2 泵在转运Ca2 的过程中经历一系列构象变化.其中,E1状态为外向的Ca2 高亲和状态,E2状态则为内向的Ca2 低亲和状态.目前,骨骼肌内质网Ca2 泵转运Ca2 过程中的几个中间状态,包括E1-2Ca2 ,E1-ATP,E1-P-ADP,E2-Pi和E2状态的三维晶体结构已经解析.介绍这几种状态的晶体结构,并分析Ca2 泵在执行功能过程中结构与功能的关系.     

Can Ca2+-dependent competence be repeatedly induced in the same Escherichia coli cells?

Summary With the help of devised multicycle consecutive transformation (MCT) it is shown that Ca²⁺-dependent competence can be repeatedly induced in the same population of Escherichia coli cells. The same fraction of cells is induced to competence and transformed during MCT. In contrast to the results on classical transformation with mixed DNA preparations, no double transformants are observed in MCT. The competent cells and transformants are found to be more fragile than nontransformed cells. The latter are represented presumably by the cells that have not absorbed exogenous plasmid DNA. The results suggest that there is strong interference between plasmid DNAs during MCT, and that the presence of exogenous DNA makes the cells more sensitive to the apparently harmful procedure of repeated competence induction.     

Stargazin Modulates Neuronal Voltage-dependent Ca2+ Channel Cav2.2 by a Gβγ-dependent Mechanism

Loss of neuronal protein stargazin (γ₂) is associated with recurrent epileptic seizures and ataxia in mice. Initially, due to homology to the skeletal muscle calcium channel γ₁ subunit, stargazin and other family members (γ_3–8) were classified as γ subunits of neuronal voltage-gated calcium channels (such as Ca_V2.1-Ca_V2.3). Here, we report that stargazin interferes with G protein modulation of Ca_V2.2 (N-type) channels expressed in Xenopus oocytes. Stargazin counteracted the Gβγ-induced inhibition of Ca_V2.2 channel currents, caused either by coexpression of the Gβγ dimer or by activation of a G protein-coupled receptor. Expression of high doses of Gβγ overcame the effects of stargazin. High affinity Gβγ scavenger proteins m-cβARK and m-phosducin produced effects similar to stargazin. The effects of stargazin and m-cβARK were not additive, suggesting a common mechanism of action, and generally independent of the presence of the Ca_Vβ₃ subunit. However, in some cases, coexpression of Ca_Vβ₃ blunted the modulation by stargazin. Finally, the Gβγ-opposing action of stargazin was not unique to Ca_V2.2, as stargazin also inhibited the Gβγ-mediated activation of the G protein-activated K⁺ channel. Purified cytosolic C-terminal part of stargazin bound Gβγ in vitro. Our results suggest that the regulation by stargazin of biophysical properties of Ca_V2.2 are not exerted by direct modulation of the channel but via a Gβγ-dependent mechanism.     

Inhibition of Ca2+-dependent Cl− channels from secretory epithelial cells by low internal pH

The actions of intracellular pH (pH _i ) on Ca²⁺dependent Cl^? channels were studied in secretory epithelial cells derived from human colon carcinoma (T84) and in isolated rat parotid acinar cells. Channel currents were measured with the whole cell voltage clamp technique with pipette solutions of different pH. Ca²⁺dependent Cl^? channels were activated by superfusing ionomycin to increase the intracellular calcium concentration ([Ca²⁺] _i ) or by using pipette solutions with buffered Ca²⁺ levels. Large currents were activated in T84 and parotid cells by both methods with pH _i levels of 7.3 or 8.3. Little or no Cl^? channel current was activated with pH _i at 6.4. We used on-cell patch clamp methods to investigate the actions of low pH _i on single Cl^? channel current amplitude in T84 cells. Lowering the pH _i had little or no effect on the current amplitude of a 8 pS Cl^? channel, but did reduce channel activity. These results suggest that cytosolic acidification may be able to modulate stimulus-secretion coupling in fluid-secreting epithelia by inhibiting the activation of Ca²⁺-activated Cl^? channels.     

Smoke-derived cues in the regulation of seed germination: are Ca2+-dependent signals involved?

Plant Growth Regulation - Seed dormancy and germination are two distinct physiological processes in the life cycle of plants. Dormancy alleviation and the attendant transition to seed germination...     

苹果果肉质膜微囊主动运输Ca2+的Ca2+-ATP酶特性

应用４５Ｃａ２ ＋ 示踪法研究了苹果果肉质膜微囊依赖于Ｃａ２＋ 的ＡＴＰ 酶（Ｃａ２＋ＡＴＰ酶）活性与Ｃａ２＋ 运输之间的关系及激素对该酶活性的影响。结果表明：Ｃａ２ ＋ＡＴＰ 酶存在于质膜上并受载体Ａ２３１８７ 刺激而活性增加,该酶活性与依赖于ＡＴＰ 的Ｃａ２ ＋ 运输依抑制剂ＥＢ、游离Ｃａ２＋ 和ＡＴＰ浓度的变化并呈极为相似的饱和动力学特征;而其ＥＢ 半抑制浓度,Ｃａ２＋ 和ＡＴＰ 半饱和浓度分别为０ ．１ ,０ ．１ 和５０ μｍｏｌ／Ｌ,从而证实了正是Ｃａ２＋ＡＴＰ酶推动苹果果肉质膜微囊的Ｃａ２＋ 的主动运输。生长素与萘乙酸均可促进苹果果肉质膜微囊Ｃａ２＋ＡＴＰ酶活性和Ｃａ２＋ 吸收,而赤霉素则无此作用。     

Ca2+和突触细胞融合

神经突触传递对于神经系统功能的实现具有十分重要的意义,而神经突触传递涉及到突触囊泡膜和突触前膜的融合,3种膜蛋白SNARE特异性识别并形成复合物,从而介导了神经递质的释放。Ca^2 通过其感受器突触结合蛋白而调节了突触细胞的融合过程,也最终影响了神经元的胞吐作用。     

Ca2+依赖和Ca2+不依赖的囊泡分泌研究进展

Ca2 是促发囊泡胞吐的关键调节因子.最近的研究表明,分泌囊泡和通道之间的空间距离调节囊泡分泌的过程和性质.Ca2 通道开口附近形成的Ca2 微区和Ca2 钠区和囊泡快速递质释放有非常紧密的联系.SNARE蛋白和钙离子传感器synaptotagmins等在触发分泌中起调控作用.同时另有一类不依赖于Ca2 的囊泡分泌存在.Latrotoxin和mastoparan等可以激活这一类不依赖于Ca2 的信号通路,从而触发囊泡释放.本文主要从ca2 对囊泡胞吐的调控作用着手,综述了Ca2 依赖和Ca2 不依赖的囊泡分泌过程和可能的调控机制.     

川楝素对K+、Ca2+通道活动及细胞内Ca2+浓度的调控

川楝素是我国学者从驱蛔中药中分离、鉴定的一个三萜化合物,已证明具选择地影响神经递质释放,有效地对抗肉毒中毒,促进细胞分化、凋亡,抑制肿瘤增殖,抑制昆虫发育和取食,影响K 、Ca2 通道活动等多种生物效应.综述了证明川楝素抑制多种K 通道,选择地易化L型Ca2 通道和进而升高胞内Ca 浓度的研究资料,并对川楝素产生这些生物效应的机制进行了讨论.     

Ca2+ Influx through Store-operated Ca2+ Channels Reduces Alzheimer Disease β-Amyloid Peptide Secretion

Alzheimer disease (AD), the leading cause of dementia, is characterized by the accumulation of β-amyloid peptides (Aβ) in senile plaques in the brains of affected patients. Many cellular mechanisms are thought to play important roles in the development and progression of AD. Several lines of evidence point to the dysregulation of Ca²⁺ homeostasis as underlying aspects of AD pathogenesis. Moreover, direct roles in the regulation of Ca²⁺ homeostasis have been demonstrated for proteins encoded by familial AD-linked genes such as PSEN1, PSEN2, and APP, as well as Aβ peptides. Whereas these studies support the hypothesis that disruption of Ca²⁺ homeostasis contributes to AD, it is difficult to disentangle the effects of familial AD-linked genes on Aβ production from their effects on Ca²⁺ homeostasis. Here, we developed a system in which cellular Ca²⁺ homeostasis could be directly manipulated to study the effects on amyloid precursor protein metabolism and Aβ production. We overexpressed stromal interaction molecule 1 (STIM1) and Orai1, the components of the store-operated Ca²⁺ entry pathway, to generate cells with constitutive and store depletion-induced Ca²⁺ entry. We found striking effects of Ca²⁺ entry induced by overexpression of the constitutively active STIM1_D76A mutant on amyloid precursor protein metabolism. Specifically, constitutive activation of Ca²⁺ entry by expression of STIM1_D76A significantly reduced Aβ secretion. Our results suggest that disruptions in Ca²⁺ homeostasis may influence AD pathogenesis directly through the modulation of Aβ production.     

ATP activates a Ca2+-dependent Cl− current in the rat thyroid cell line,FRTL-5

The effect of external ATP on both the membrane potential and the transmembrane current of the thyroid cell line FRTL-5 has been investigated in the patch-clamp whole-cell recording configuration. In the resting situation the membrane potential is around -70 mV and the membrane acts like a K(+)-sensitive electrode. Application of ATP at concentrations higher than 1 microM elicited an increase in Cl- conductance, responsible for a membrane depolarization which could be blocked by preincubation with the P2-antagonist quinidine. Chelation of intracellular Ca2+ also blocked the ATP induced changes in membrane potential and Cl- current. Intracellular perfusion with inositol trisphosphate (IP3) (50 microM) also stimulated a Cl- current which mimicked the response induced by ATP. ATP is able to initiate a response in the absence of extracellular Ca2+, but also opens a Ca(2+)-influx pathway, as demonstrated by a secondary response upon Ca2+ readmission in the external medium, in the continued presence of ATP. ADP and ATP gamma S were able to mimic the ATP response, whereas AMP and adenosine were unable to elicit a Cl- current. The P2X receptor agonist alpha,beta-methyleneATP was without effect as was the P2Y receptor agonist 2-methylthio ATP. We conclude that ATP is able to elicit a large IP3-mediated Ca(2+)-dependent Cl- current and membrane depolarization via a novel P2-type purinergic receptor.