心肌细胞核Ca2+库特点及其调节的离体研究

研究核外Ca~(2+)浓度对核Ca~(2+)的影响,及细胞核Ca~(2+)摄取和释放的关系,以探讨核Ca~(2+)转运的调节机制。采用差速离心和密度梯度离心法分离纯化心肌细胞核,以Fluo-4/AM荧光指示剂负载心肌细胞核,应用激光共聚焦扫描显微镜和荧光分光光度计进行观察和测定。结果显示,分离纯化的成年大鼠心肌细胞核内自由[Ca~(2+)]随着核外[Ca~(2+)]的增加而逐渐增加,孵育液[Ca~(2+)]为1000 nmol/L达高峰,但二者增加的程度并不一致,之后随核外[Ca~(2+)]浓度的增加而呈降低趋势。ATP和100—600nmol/L的核外游离Ca~(2+),使心肌细胞核显示核被膜腔Ca~(2+)荧光,ATP和1000nmol/L的核外游离Ca~(2+)则进一步引起核浆内的Ca~(2+)荧光强度升高。荧光染色观察可见IP_3受体染色主要位于核内膜,而钙泵和ryanodine受体染色主要位于核外膜。IP_3和Ryancodine使核Ca~(2+)短暂升高1.68倍和1.93倍(P<0.001),而钙泵抑制剂Thapsigargin和IP_3受体抑制剂Heparin则分别使核Ca~(2+)降低64%和35.6%(p<0.05)。ryanodine使IP_3升高的核Ca~(2+)显著回落至正常水平以下(p<0.001)。Thapsigargin不能阻断IP_3和Ryanodine所致的核Ca~(2+)释放增加(p<0.05),但事先采用钙泵抑制剂Thapsigargin预处理心肌细胞核,则能显著的阻断IP_3和Ryanodine所致的核Ca~(2+)升高作用(Ca~(2+)释放作用)(p<0.05)。结果提示大鼠心肌细胞核可能也是细胞内的钙库之一,心肌细胞核上存在Ca~(2+)-ATPase、ryanodine受体和IP_3受体等Ca~(2+)转运系统,可能参与核Ca~(2+)摄取和释放的调节。     

Expression and reconstitution of the bioluminescent Ca2+ reporter aequorin in human embryonic stem cells,and exploration of the presence of functional IP3 and ryanodine receptors during the early stages of their differentiation into cardiomyocytes

In order to develop a novel method of visualizing possible Ca~(2+) signaling during the early differentiation of h ESCs into cardiomyocytes and avoid some of the inherent problems associated with using fluorescent reporters, we expressed the bioluminescent Ca~(2+) reporter, apo-aequorin, in HES2 cells and then reconstituted active holo-aequorin by incubation with f-coelenterazine. The temporal nature of the Ca~(2+) signals generated by the holo-f-aequorin-expressing HES2 cells during the earliest stages of differentiation into cardiomyocytes was then investigated. Our data show that no endogenous Ca~(2+) transients(generated by release from intracellular stores) were detected in 1–12-day-old cardiospheres but transients were generated in cardiospheres following stimulation with KCl or Ca Cl_2, indicating that holo-f-aequorin was functional in these cells. Furthermore, following the addition of exogenous ATP, an inositol trisphosphate receptor(IP_3R) agonist, small Ca~(2+) transients were generated from day 1 onward. That ATP was inducing Ca~(2+) release from functional IP_3 Rs was demonstrated by treatment with 2-APB, a known IP_3 R antagonist. In contrast, following treatment with caffeine, a ryanodine receptor(Ry R) agonist, a minimal Ca~(2+) response was observed at day 8 of differentiation only. Thus, our data indicate that unlike Ry Rs, IP_3 Rs are present and continually functional at these early stages of cardiomyocyte differentiation.     

Con A刺激致T淋巴细胞胞浆游离Ca~(2+)浓度升高

本文分别应用荧光Ca~(2+)指示剂Quin2和Indo-1研究了Con A刺激的T淋巴细胞[Ca~(2+)]i升高过程及其发生机制.结果表明Con A与T淋巴细胞作用可导致细胞[Ca~(2+)]i的迅速升高.这种增加的胞内游离Ca~(2+)不仅来自胞外Ca~(2+)的内流,也来源于胞内钙库的释放.其中Ca~(2+)内流与T细胞钙通道的开放有关.可被钙通道抑制剂戊脉胺抑制,细胞的去极化及钾通道阻断剂四乙胺均不能阻断Ca~(2+)的内流,提示Ca~(2+)内流不是通过电位操纵的钙通道实现的,也与拥通道的开闭无关.Ca~(2+)内流可能是通过Con A受体活化的受体操纵的钙通道而实现的.     

血管紧张素Ⅱ对血管平滑肌细胞中大电导钙激活钾通道的多重调节作用（英文）

肾素-血管紧张素系统(renin-angiotensin system, RAS)是影响血管平滑肌细胞张力的重要因素。RAS主要活性物质血管紧张素Ⅱ (angiotensin Ⅱ, Ang Ⅱ)可通过激活血管紧张素Ⅱ-1型受体(angiotensin Ⅱ type 1 receptor, AT1R)升高胞内Ca~(2+)浓度,收缩平滑肌细胞。大电导钙激活钾(large-conductance Ca~(2+)-and voltage-activated potassium, BK)通道是血管平滑肌细胞中分布最广、表达最多的钾离子通道,在维持细胞膜电位和胞内钾钙平衡中发挥重要作用。血管平滑肌细胞上的BK通道主要包含α与β1两种亚基。其中功能亚基BKα上分布有膜电位及Ca~(2+)感受器。因此当膜电位或细胞内Ca~(2+)浓度升高时会反馈性引起BK通道开放。然而,越来越多的研究显示,尽管Ang Ⅱ可升高胞内Ca~(2+)浓度,但却通过激活PKC通路、促进AT1R与BKα通道形成的异源二聚体内吞、加快α与β1亚基解离等途径抑制BK通道的表达和功能。在一些情况下,Ang Ⅱ对BK通道也可表现出激活作用,但机制尚不完全明确。该综述总结了Ang Ⅱ对BK通道抑制或激活两方面效应的可能原因,为改善细胞内离子失衡提供理论依据。     

芍药苷对大鼠背根神经元细胞内Ca~(2+)的影响

目的建立一种评价芍药苷对大鼠背根神经节神经元细胞内游离Ca~(2+)浓度影响的方法。方法显微解剖获取大鼠背根神经节(DRG),通过胰蛋白酶消化,过筛,用DF-12和抗有丝分裂培养液交替培养纯化,获得原代大鼠DRG神经元细胞,并采用细胞免疫荧光技术测定DRG神经元细胞纯度;采用激光共聚焦显微成像技术,观察细胞内Ca~(2+)荧光强度的变化,并对Ca~(2+)荧光强度变化率进行分析,探讨芍药苷对DRG细胞内游离钙离子浓度及辣椒素受体的影响。结果采用上述方法分离得到的DRG细胞纯度可高达95%以上,辣椒平可通过阻断辣椒素激活的瞬时受体电位通道的作用而抑制细胞内Ca~(2+)的增加。芍药苷表现出与辣椒平类似的作用,可以阻断细胞外Ca~(2+)内流。结论芍药苷可能是通过作用于TRPV1通道,而抑制DRG细胞内Ca~(2+)大量增加,本方法可以用于评价药物对大鼠DRG细胞内Ca~(2+)浓度的影响。     

血管平滑肌细胞的钙动力学

血管平滑肌细胞外的Ca~(2+)通过多种通道进入细胞内。Ca~(2+)通道的本质是镶嵌在膜脂质双分子层中的糖蛋白,神经介质和药物可影响Ca~(2+)通道的功能。靠近胞膜的肌质网和胞膜内侧面的高亲和性Ca~(2+)结合位点是血管平滑肌细胞内储存和释放Ca~(2+)的主要部位。胞浆[Ca~(2+)]增高后在钙调蛋白的介导下引起血管收缩。高血压等血管性疾病的发生与其平滑肌细胞的钙动力学异常有关。     

电压门控通道家族的新成员——Ca~(2+)通道克隆成功

最近日本京都大学医学院 Numa 实验室丛家兔骨骼肌纯化了钙通道阻断剂二氢嘧啶(dihydropyridine,DHP)的受体,并利用重组 DNA 技术推出其一级结构。比较 DHP 受体、Na~+通道和 K~+通道及由此预测的二级结构,发现三者具有十分相似的共同特征,提示 DHP 受体就是骨骼肌细胞膜上起兴奋收缩偶联作用的电压感受器和 Ca~(2+)通道。因此,Na~+、K~+、Ca~(2+)这三种通道构成由一个祖先基因进化而来的电压门控通道家族。     

强声暴露对巴马小型猪海马细胞内Ca~(2+)变化及其信号通道的影响

声音对神经系统有重要影响,本研究旨在探讨噪音或高强度声音刺激对神经系统的影响及其机制。将听力正常的巴马小型猪随机分为正常对照组与强声暴露组。强声暴露组的巴马小型猪暴露于中低频强声(900 Hz-142 dB SPL)环境中15min,暴露结束后即刻分离出海马组织。用Fluo-4探针观察海马组织细胞内Ca~(2+)浓度([Ca~(2+)]_i)的变化,用real-time PCR和Western blot分别检测Ca~(2+)受体、L-型Ca~(2+)通道α2/δ1亚基、PKC和PI3K的mRNA和蛋白表达,用DAPI染色法观察细胞核形态变化。结果显示,相对对照组,强声暴露组小型猪海马组织细胞[Ca~(2+)]_i明显增加,L-型Ca~(2+)通道α2/δ1亚基、PKC和PI3K mRNA表达上调,Ca~(2+)受体和PKC蛋白表达显著上调。此外,强声暴露引起海马组织细胞核出现肿胀变形等损伤样改变。以上结果提示,强声暴露可以通过激活海马组织PKC信号通路,引起[Ca~(2+)]_i上调,最终导致海马组织内细胞的损伤。本研究结果不仅揭示了强声引起神经损伤的可能机制,同时为防护强声对神经系统造成的损伤提供了新的思路。     

气相色谱法检测大鼠脑及颌下腺磷脂酰肌醇代谢环路中肌醇的含量

磷脂酰肌醇(PI)约占细胞总磷脂的5～10％,其代谢十分活跃。近几年来,人们发现PI代谢是许多膜受体信号跨膜转导的重要途径。乙酰胆碱等多种神经递质激动其受体后,经三磷酸鸟苷结合蛋白转导激活细胞膜磷脂酶C,催化PI水解生成两种信使物质:1,4,5一三磷酸肌醇(IP_3)及二酰基甘油,其中IP_3经特异性     

TRP通道与自噬相关性的探讨

TRP通道(Transient Receptor Potential,瞬时受体电位通道)是细胞内重要的Ca~(2+)调控通道,其在细胞内有着独特的分布及其能与其它钙离子传感元件特定地相互作用。TRP通道蛋白通过对Ca~(2+)的调控发挥调节细胞的多种生理活动,如自噬和凋亡等。其中细胞质膜或是细胞器膜上的这些钙离子通道开放后可提升胞质[Ca~(2+)]i,为自噬的启动提供Ca~(2+)。自噬启动的初衷是为了使细胞能够在缺氧、营养匮乏及病理因素等应激状态下降解细胞内某些细胞成分满足某些重要生理活动的物质需求,但有大量的研究显示过度的自噬可导致细胞发生与凋亡相关的细胞程序性死亡,因而TRP通道对自噬的调控作用与疾病的发生、发展紧密相连,如TRPML1(Transient Receptor Potential mucolipin-1)与IV型粘膜脂质沉积症相关。根据目前对TRP通道与自噬的研究结果来看,不同的TRP通道可通过不同的机制升高胞质[Ca~(2+)]i,这都与不同TRP通道蛋白在细胞内的特定分布有关,如TRPV(Transient receptor potential vanilloid)通道主要在细胞质膜或内质网膜上调控Ca~(2+),而TRPML则主要在溶酶体上发挥作用,但具体分子通路激活机制尚需进一步研究。     

Exact Stochastic Simulation of a Calcium Microdomain Reveals the Impact of Ca2+ Fluctuations on IP3R Gating

In this study, we numerically analyzed the nonlinear Ca²⁺-dependent gating dynamics of a single, nonconducting inositol 1,4,5-trisphosphate receptor (IP₃R) channel, using an exact and fully stochastic simulation algorithm that includes channel gating, Ca²⁺ buffering, and Ca²⁺ diffusion. The IP₃R is a ubiquitous intracellular Ca²⁺ release channel that plays an important role in the formation of complex spatiotemporal Ca²⁺ signals such as waves and oscillations. Dynamic subfemtoliter Ca²⁺ microdomains reveal low copy numbers of Ca²⁺ ions, buffer molecules, and IP₃Rs, and stochastic fluctuations arising from molecular interactions and diffusion do not average out. In contrast to models treating calcium dynamics deterministically, the stochastic approach accounts for this molecular noise. We varied Ca²⁺ diffusion coefficients and buffer reaction rates to tune the autocorrelation properties of Ca²⁺ noise and found a distinct relation between the autocorrelation time τ_ac, the mean channel open and close times, and the resulting IP₃R open probability P_O. We observed an increased P_O for shorter noise autocorrelation times, caused by increasing channel open times and decreasing close times. In a pure diffusion model the effects become apparent at elevated calcium concentrations, e.g., at [Ca²⁺] = 25 μM, τ_ac = 0.082 ms, the IP₃R open probability increased by ≈20% and mean open times increased by ≈4 ms, compared to a zero noise model. We identified the inactivating Ca²⁺ binding site of IP₃R subunits as the primarily noise-susceptible element of the De Young and Keizer model. Short Ca²⁺ noise autocorrelation times decrease the probability of Ca²⁺ association and consequently increase IP₃R activity. These results suggest a functional role of local calcium noise properties on calcium-regulated target molecules such as the ubiquitous IP₃R. This finding may stimulate novel experimental approaches analyzing the role of calcium noise properties on microdomain behavior.     

Inositol 1,4,5-Trisphosphate Receptor Subtype-Specific Regulation of Calcium Oscillations

Oscillatory fluctuations in the cytosolic concentration of free calcium ions (Ca²⁺) are considered a ubiquitous mechanism for controlling multiple cellular processes. Inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃R) are intracellular Ca²⁺ release channels that mediate Ca²⁺ release from endoplasmic reticulum (ER) Ca²⁺ stores. The three IP₃R subtypes described so far exhibit differential structural, biophysical, and biochemical properties. Subtype specific regulation of IP₃R by the endogenous modulators IP₃, Ca²⁺, protein kinases and associated proteins have been thoroughly examined. In this article we will review the contribution of each IP₃R subtype in shaping cytosolic Ca²⁺ oscillations.     

Comparison of Models for IP3 Receptor Kinetics Using Stochastic Simulations

Inositol 1,4,5-trisphosphate receptor (IP₃R) is a ubiquitous intracellular calcium (Ca²⁺) channel which has a major role in controlling Ca²⁺ levels in neurons. A variety of computational models have been developed to describe the kinetic function of IP₃R under different conditions. In the field of computational neuroscience, it is of great interest to apply the existing models of IP₃R when modeling local Ca²⁺ transients in dendrites or overall Ca²⁺ dynamics in large neuronal models. The goal of this study was to evaluate existing IP₃R models, based on electrophysiological data. This was done in order to be able to suggest suitable models for neuronal modeling. Altogether four models (Othmer and Tang, 1993; Dawson et al., 2003; Fraiman and Dawson, 2004; Doi et al., 2005) were selected for a more detailed comparison. The selection was based on the computational efficiency of the models and the type of experimental data that was used in developing the model. The kinetics of all four models were simulated by stochastic means, using the simulation software STEPS, which implements the Gillespie stochastic simulation algorithm. The results show major differences in the statistical properties of model functionality. Of the four compared models, the one by Fraiman and Dawson (2004) proved most satisfactory in producing the specific features of experimental findings reported in literature. To our knowledge, the present study is the first detailed evaluation of IP₃R models using stochastic simulation methods, thus providing an important setting for constructing a new, realistic model of IP₃R channel kinetics for compartmental modeling of neuronal functions. We conclude that the kinetics of IP₃R with different concentrations of Ca²⁺ and IP₃ should be more carefully addressed when new models for IP₃R are developed.     

Inositol 1,4,5‐trisphosphate receptor 1 degradation in mouse eggs and impact on [Ca2+]i oscillations

The initiation of normal embryo development depends on the completion of all events of egg activation. In all species to date, egg activation requires an increase(s) in the intracellular concentration of calcium ([Ca²⁺]_i), which is almost entirely mediated by inositol 1,4,5‐trisphosphate receptor 1 (IP₃R1). In mammalian eggs, fertilization‐induced [Ca²⁺]_i responses exhibit a periodic pattern that are called [Ca²⁺]_i oscillations. These [Ca²⁺]_i oscillations are robust at the beginning of fertilization, which occurs at the second metaphase of meiosis, but wane as zygotes approach the pronuclear stage, time after which in the mouse oscillations cease altogether. Underlying this change in frequency are cellular and biochemical changes associated with egg activation, including degradation of IP₃R1, progression through the cell cycle, and reorganization of intracellular organelles. In this study, we investigated the system requirements for IP₃R1 degradation and examined the impact of the IP₃R1 levels on the pattern of [Ca²⁺]_i oscillations. Using microinjection of IP₃ and of its analogs and conditions that prevent the development of [Ca²⁺]_i oscillations, we show that IP₃R1 degradation requires uniform and persistently elevated levels of IP₃. We also established that progressive degradation of the IP₃R1 results in [Ca²⁺]_i oscillations with diminished periodicity while a near complete depletion of IP₃R1s precludes the initiation of [Ca²⁺]_i oscillations. These results provide insights into the mechanism involved in the generation of [Ca²⁺]_i oscillations in mouse eggs. J. Cell. Physiol. 222:238–247, 2010. © 2009 Wiley‐Liss, Inc.     

Effects of both glucose and IP<Subscript>3</Subscript> concentrations on action potentials in pancreatic β-cells

Abstarct Considering the ATP-driven (SERCA) pump flux as function of glucose concentration and the calcium flux from the endoplasmic reticulum (ER) through the IP₃R channel, the calcium-based phantom bursting model (PBM) of β-cells (Bertram and Sherman in Bull Math Biol 66:1313, 2004) is theoretically extended to discuss the effects of glucose and inositol 1,4,5-trisphosphate (IP₃) concentration on the membrane potential activities. When IP₃ concentration is fixed, it is found that there is a critical glucose concentration at which electrical bursting oscillations transfer into spiking, and the critical concentration of glucose is increased with the increasing of IP₃ concentration. To get the bursting oscillations in β-cells, our theoretical results show that the stimulatory glucose concentration should be more than 10 mM, which is consistent with the normal physiological IP₃ level. When the stochastic opening and closing of IP₃R channels are considered, it is shown that the membrane potential oscillation transfers from spiking to bursting with the channel number decreasing, and the average cytosolic free Ca²⁺ concentration is increased with the increase of glucose concentration.     

Differential Regulation of Multiple Steps in Inositol 1,4,5-Trisphosphate Signaling by Protein Kinase C Shapes Hormone-stimulated Ca2+ Oscillations

How Ca²⁺ oscillations are generated and fine-tuned to yield versatile downstream responses remains to be elucidated. In hepatocytes, G protein-coupled receptor-linked Ca²⁺ oscillations report signal strength via frequency, whereas Ca²⁺ spike amplitude and wave velocity remain constant. IP₃ uncaging also triggers oscillatory Ca²⁺ release, but, in contrast to hormones, Ca²⁺ spike amplitude, width, and wave velocity were dependent on [IP₃] and were not perturbed by phospholipase C (PLC) inhibition. These data indicate that oscillations elicited by IP₃ uncaging are driven by the biphasic regulation of the IP₃ receptor by Ca²⁺, and, unlike hormone-dependent responses, do not require PLC. Removal of extracellular Ca²⁺ did not perturb Ca²⁺ oscillations elicited by IP₃ uncaging, indicating that reloading of endoplasmic reticulum stores via plasma membrane Ca²⁺ influx does not entrain the signal. Activation and inhibition of PKC attenuated hormone-induced Ca²⁺ oscillations but had no effect on Ca²⁺ increases induced by uncaging IP₃. Importantly, PKC activation and inhibition differentially affected Ca²⁺ spike frequencies and kinetics. PKC activation amplifies negative feedback loops at the level of G protein-coupled receptor PLC activity and/or IP₃ metabolism to attenuate IP₃ levels and suppress the generation of Ca²⁺ oscillations. Inhibition of PKC relieves negative feedback regulation of IP₃ accumulation and, thereby, shifts Ca²⁺ oscillations toward sustained responses or dramatically prolonged spikes. PKC down-regulation attenuates phenylephrine-induced Ca²⁺ wave velocity, whereas responses to IP₃ uncaging are enhanced. The ability to assess Ca²⁺ responses in the absence of PLC activity indicates that IP₃ receptor modulation by PKC regulates Ca²⁺ release and wave velocity.     

Role of secretory granules in inositol 1,4,5-trisphosphate-dependent Ca(2+) signaling: from phytoplankton to mammals

The majority of secretory cell calcium is stored in secretory granules that serve as the major IP₃-dependent intracellular Ca²⁺ store. Even in unicellular phytoplankton secretory granules are responsible for the IP₃-induced Ca²⁺ release that triggers exocytosis. The number of secretory granules in the cell is directly related not only to the magnitude of IP₃-induced Ca²⁺ release, which accounts for the majority of the IP₃-induced cytoplasmic Ca²⁺ release in neuroendocrine cells, but also to the IP₃ sensitivity of the cytoplasmic IP₃ receptor (IP₃R)/Ca²⁺ channels. Moreover, secretory granules contain the highest IP₃R concentrations and the largest amounts of IP₃Rs in any subcellular organelles in neuroendocrine cells. Secretory granules from phytoplankton to mammals contain large amounts of polyanionic molecules, chromogranins being the major molecules in mammals, in addition to acidic intragranular pH and high Ca²⁺ concentrations. The polyanionic molecules undergo pH- and Ca²⁺-dependent conformational changes that serve as a molecular basis for condensation-decondensation phase transitions of the intragranular matrix. Likewise, chromogranins undergo pH- and Ca²⁺-dependent conformational changes with increased exposure of the structure and increased interactions with Ca²⁺ and other granule components at acidic pH. The unique physico-chemical properties of polyanionic molecules appear to be at the center of biogenesis, and physiological functions of secretory granules in living organisms from primitive to advanced species.     

Stochastic reaction-diffusion modeling of calcium dynamics in 3D dendritic spines of Purkinje cells

Calcium (Ca²⁺) is a second messenger assumed to control changes in synaptic strength in the form of both long-term depression and long-term potentiation at Purkinje cell dendritic spine synapses via inositol trisphosphate (IP₃)-induced Ca²⁺ release. These Ca²⁺ transients happen in response to stimuli from parallel fibers (PFs) from granule cells and climbing fibers (CFs) from the inferior olivary nucleus. These events occur at low numbers of free Ca²⁺, requiring stochastic single-particle methods when modeling them. We use the stochastic particle simulation program MCell to simulate Ca²⁺ transients within a three-dimensional Purkinje cell dendritic spine. The model spine includes the endoplasmic reticulum, several Ca²⁺ transporters, and endogenous buffer molecules. Our simulations successfully reproduce properties of Ca²⁺ transients in different dynamical situations. We test two different models of the IP₃ receptor (IP₃R). The model with nonlinear concentration response of binding of activating Ca²⁺ reproduces experimental results better than the model with linear response because of the filtering of noise. Our results also suggest that Ca²⁺-dependent inhibition of the IP₃R needs to be slow to reproduce experimental results. Simulations suggest the experimentally observed optimal timing window of CF stimuli arises from the relative timing of CF influx of Ca²⁺ and IP₃ production sensitizing IP₃R for Ca²⁺-induced Ca²⁺ release. We also model ataxia, a loss of fine motor control assumed to be the result of malfunctioning information transmission at the granule to Purkinje cell synapse, resulting in a decrease or loss of Ca²⁺ transients. Finally, we propose possible ways of recovering Ca²⁺ transients under ataxia.     

Analysis of IP3 receptors in and out of cells

Background

Inositol 1,4,5-trisphosphate receptors (IP₃R) are expressed in almost all animal cells. Three mammalian genes encode closely related IP₃R subunits, which assemble into homo- or hetero-tetramers to form intracellular Ca^2 + channels.

Scope of the review

In this brief review, we first consider a variety of complementary methods that allow the links between IP₃ binding and channel gating to be defined. How does IP₃ binding to the IP₃-binding core in each IP₃R subunit cause opening of a cation-selective pore formed by residues towards the C-terminal? We then describe methods that allow IP₃, Ca^2 + signals and IP₃R mobility to be examined in intact cells. A final section briefly considers genetic analyses of IP₃R signalling.

Major conclusions

All IP₃R are regulated by both IP₃ and Ca^2 +. This allows them to initiate and regeneratively propagate intracellular Ca^2 + signals. The elementary Ca^2 + release events evoked by IP₃ in intact cells are mediated by very small numbers of active IP₃R and the Ca^2 +-mediated interactions between them. The spatial organization of these Ca^2 + signals and their stochastic dependence on so few IP₃Rs highlight the need for methods that allow the spatial organization of IP₃R signalling to be addressed with single-molecule resolution.

General significance

A variety of complementary methods provide insight into the structural basis of IP₃R activation and the contributions of IP₃-evoked Ca^2 + signals to cellular physiology. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signaling.     

Frequency and Relative Prevalence of Calcium Blips and Puffs in a Model of Small IP3R Clusters

In this work, we model the local calcium release from clusters with a few inositol 1,4,5-trisphosphate receptor (IP₃R) channels, focusing on the stochastic process in which an open channel either triggers other channels to open (as a puff) or fails to cause any channel to open (as a blip). We show that there are linear relations for the interevent interval (including blips and puffs) and the first event latency against the inverse cluster size. However, nonlinearity is found for the interpuff interval and the first puff latency against the inverse cluster size. Furthermore, the simulations indicate that the blip fraction among all release events and the blip frequency are increasing with larger basal [Ca²⁺], with blips in turn giving a growing contribution to basal [Ca²⁺]. This result suggests that blips are not just lapses to trigger puffs, but they may also possess a biological function to contribute to the initiation of calcium waves by a preceding increase of basal [Ca²⁺] in cells that have small IP₃R clusters.