神经轴突的钠离子通道模型

镶嵌在神经轴突膜中的一种蛋白质分子行使钠通道的功能。沿着跨膜方向,蛋白质分子中与类脂的脂肪酸链相邻部分形成数条α-螺旋。氨基酸极性侧链向螺旋之间的空间伸展,侧链的偶极子或离子基团可以成对地形成氢键,并构成通道的活化或失活闸门。每一极性侧链可以有两种稳定构象,一条侧链处于构象Ⅰ或Ⅱ的几率服从波尔茨曼定律。在膜电场作用下,这些极性侧链在两种构象间发生跃迁,形成门控电流并导致通道呈现关闭或开放态。钠离子以跳跃扩散方式越过势垒。根据电场作用下电介质的极化特性,推导了门控电流和钠电导的稳态和动力学公式,它们是膜电位和时间的函数。用这些公式进行定量估算的结果表踞,与实验事实基本相符。本文并对钠通道和门控过程的一些特性进行了讨论。     

电压门控性钾通道的结构和调控机制

电压门控性K 通道是由4个相同亚单位构成的四聚体通道,其中每个亚单位都含有1个电压感受器,并且4个亚单位合起来组成1个中央孔.电压门控性通道蛋白具有3种主要功能,一是离子通透功能,二是门控蛋白构象改变,三是门控与感知机制的偶联.通道具有高通透速率和高选择性,通过构象改变的门控机制有3种,一是S6束交叉门控,二是球链门控,三是选择性滤器的门控.     

甲氧普胺阻滞心肌钠通道的计算机模拟研究

根据闸门相关受体模型,应用计算机模拟研究了甲氧普胺阻滞心肌钠通道的动力学特点及其作用的状态依赖性。本文首次描述和分析了同时受两种门控机制调控的钠通道阻滞剂的作用特点,甲氧普胺即具此特点,其阻滞作用依赖于通道的激活态及失活态,其在刺激频率2.0Hz时的表观阻滞起效速率为0.49/AP,在膜电位—85mV时的阻滞恢复时间常数为2.82s.     

生物膜电压门控离子通道的结构和功能性构象

生物膜离子通道具有多种重要的生理功能.近年,已分离、纯化了电压门控的Na~+、Ca~(2+)和K~+通道的蛋白质组分.Na~+和Ca~(2+)通道分别由一个构成离子孔洞的主要亚单位和数目不同的其他亚单位组成,K~+通道是单一的多肽.对Na~+、Ca~(2+)通道主要亚单位和K~+通道的氨基酸序列的测定表明,它们之间有许多相似性.已分别给出了三种通道跨膜排列的二级结构图象.考虑了Na~+通道的功能特性,包括电压敏感性、通道开放动力学、门控电流、神经毒素的作用等,已提出几种Na~+通道功能性构象模型.     

生物膜电压门控离子通道的结构和功能性构象

生物膜离子通道具有多种重要的生理功能.近年,已分离、纯化了电压门控的Na~+、Ca~(2+)和K~+通道的蛋白质组分.Na~+和Ca~(2+)通道分别由一个构成离子孔洞的主要亚单位和数目不同的其他亚单位组成,K~+通道是单一的多肽.对Na~+、Ca~(2+)通道主要亚单位和K~+通道的氨基酸序列的测定表明,它们之间有许多相似性.已分别给出了三种通道跨膜排列的二级结构图象.考虑了Na~+通道的功能特性,包括电压敏感性、通道开放动力学、门控电流、神经毒素的作用等,已提出几种Na~+通道功能性构象模型.     

大鼠大脑皮层神经元单离子钾通道门控动力学

采用膜片钳制技术,对新生大鼠大脑皮层神经元作了细胞贴附和内膜向外两种模式的单通道电流记录。通道开放和关闭事件的转换过程为一随机过程,开关时间服从指数分布。细胞膜单离子通道的时间常数和对离子通透性接近的构象组成构象集合态。由残差法获得模型参数初值,由非线性最小二乘法获得修正值。新生大鼠大脑皮层神经元在-40mV钳制电位和电极内外对称高钾溶液下,细胞贴附和内膜向外的两种膜片钳制模式的单通道电流的动力学特征有明显差异。通道开放时间分布接近一状态分布。细胞贴附时的通道平均开放时间为2.53ms。内膜向外时的通道平均开放时间为2.04ms。关闭时间分布接近三状态分布,细胞贴附时通道平均关闭时间为3.36ms,内膜向外时通道的平均关闭时间为7.58ms。细胞贴附下,通道关闭时主要处于第一和第二关闭态;内膜向外下,通道关闭时主要处于第一关闭态。经初值估计和参数修正,得到各状态间的转移概率密度常数。     

乌拉坦对大鼠海马CA1神经元电压门控钠通道和动作电位的作用

乌拉坦对兴奋性和抑制性配体门控通道具有广泛的可检测的作用.作者运用全细胞膜片钳技术研究乌拉坦对wistar大鼠海马CA1神经元电压门控钠通道和动作电位的作用.结果发现乌拉坦可逆并剂量依赖性地抑制钠电流和动作电位,其中,在10mmol/L浓度时可减小钠电流强度达38%,使激活曲线向去极化方向移动,并延长钠通道失活后的恢复时间,降低动作电位的幅值.这些结果表明乌拉坦对电压门控钠通道的抑制作用可能是乌拉坦全身麻醉作用的机制之一.     

大鼠颈上神经节烟碱电流的整流及失敏

在培养的新生大鼠颈上神经节交感神经元标本上,用全细胞膜片钳技术研究了烟碱型乙酰胆碱受体（ｎＡＣｈＲ）通道的整流及失敏现象。烟碱激动剂引起的全细胞电流在膜电位为负值时随膜电位呈线性改变,而在膜电位达＋６０ｍＶ时仍不出现外向电流,表现出强烈的内向整流。ｎＡＣｈＲ通道电流存在失敏现象,即持续恒压喷射激动剂所引起的全细胞电流随时间呈指数衰减,不能保持在峰值水平,失敏随激剂浓度呈量效关系,膜电位的超极化也加     

电压门控性离子通道的药理

一、引言离子通道是细胞膜上的特殊蛋白质大分子,在脂质双层膜上构成具有高度选择性的亲水性孔道,容许适当大小和适当电荷的离子通过。细胞膜内、外离子浓度差决定离子扩散的方向。大多数离子通道大部分时间是关闭的,只是在特殊刺激下,打开的机率才大大增加,这种现象称为门控(gating)。通道蛋白的构象     

百日咳毒素对棉铃虫神经细胞电压门控钠、钙通道的调节作用

用膜片钳技术研究了百日咳毒素对离体培养的棉铃虫幼虫中枢神经细胞电压门控钠、钙通道的影响。结果表明,对照组细胞钠通道在-50～-40mV激活,在-20mV左右电流达到最大值,在记录的20min内、电流．电压关系曲线(I-V)和电流幅值未有明显变化;细胞与百日咳毒素预孵后,钠通道在-40mV左右激活,电流在0mV左右达到最大值,在记录过程中,激活电压和峰值电压继续向正方向移动约10mV、电流持续下降。对照组钙通道在-40～-30mV激活,在0mV左右电流达峰值;经百日咳毒素处理后,I-V曲线向负电位方向移动约10mV,在记录过程中,I-V曲线继续向负电位方向移动,电流的衰减(rundown)现象比对照组严重．此外,百日咳毒素处理引起钙电流达到峰值的时问显著延长。结果提示,百日咳毒素敏感的G蛋白(Gi)可能通过直接途径或间接途径调节棉铃虫神经细胞钠、钙通道的电压敏感性和开放几率以及钙通道由备用态向激活态转化的速度。同时,经百日咳毒素处理后钠通道的,I-V曲线与抗性棉铃虫I-V曲线非常相似,可能暗示Gi蛋白在棉铃虫抗药性形成中发挥作用。     

Ligand Trapping by Cytochrome c Oxidase: IMPLICATIONS FOR GATING AT THE CATALYTIC CENTER*

Cytochrome c oxidase is a member of the heme-copper family of oxygen reductases in which electron transfer is linked to the pumping of protons across the membrane. Neither the redox center(s) associated with proton pumping nor the pumping mechanism presumably common to all heme-copper oxidases has been established. A possible conformational coupling between the catalytic center (Fe_a3³⁺–Cu_B²⁺) and a protein site has been identified earlier from ligand binding studies, whereas a structural change initiated by azide binding to the protein has been proposed to facilitate the access of cyanide to the catalytic center of the oxidized bovine enzyme. Here we show that cytochrome oxidase pretreated with a low concentration of azide exhibits a significant increase in the apparent rate of cyanide binding relative to that of free enzyme. However, this increase in rate does not reflect a conformational change enhancing the rapid formation of a Fe_a3³⁺–CN–Cu_B²⁺ complex. Instead the cyanide-induced transition of a preformed Fe_a3³⁺–N₃–Cu_B²⁺ to the ternary complex of Fe_a3³⁺–N₃ Cu_B²⁺–CN is the most likely reason for the observed acceleration. Significantly, the slow rate of azide release from the ternary complex indicates that cyanide ligated to Cu_B blocks a channel between the catalytic site and the solvent. The results suggest that there is a pathway that originates at Cu_B and that, during catalysis, ligands present at this copper center control access to the iron of heme a₃ from the bulk medium.     

Structural Studies of Inositol 1,4,5-Trisphosphate Receptor: COUPLING LIGAND BINDING TO CHANNEL GATING*

The three isoforms of the inositol 1,4,5-trisphosphate receptor (IP₃R) exhibit distinct IP₃ sensitivities and cooperativities in calcium (Ca²⁺) channel function. The determinants underlying this isoform-specific channel gating mechanism have been localized to the N-terminal suppressor region of IP₃R. We determined the 1.9 Å crystal structure of the suppressor domain from type 3 IP₃R (IP₃R3_SUP, amino acids 1–224) and revealed structural features contributing to isoform-specific functionality of IP₃R by comparing it with our previously determined structure of the type 1 suppressor domain (IP₃R1_SUP). The molecular surface known to associate with the ligand binding domain (amino acids 224–604) showed marked differences between IP₃R3_SUP and IP₃R1_SUP. Our NMR and biochemical studies showed that three spatially clustered residues (Glu-20, Tyr-167, and Ser-217 in IP₃R1 and Glu-19, Trp-168, and Ser-218 in IP₃R3) within the N-terminal suppressor domains of IP₃R1_SUP and IP₃R3_SUP interact directly with their respective C-terminal fragments. Together with the accompanying paper (Yamazaki, H., Chan, J., Ikura, M., Michikawa, T., and Mikoshiba, K. (2010) J. Biol. Chem. 285, 36081–36091), we demonstrate that the single aromatic residue in this region (Tyr-167 in IP₃R1 and Trp-168 in IP₃R3) plays a critical role in the coupling between ligand binding and channel gating.     

DIURNAL CELL DIVISION REGULATED BY GATING THE G1/S TRANSITION IN ENTEROMORPHA COMPRESSA (CHLOROPHYTA)1

The cell‐cycle progression of Enteromorpha compressa (L.) Nees (=Ulva compressa L.) was diurnally regulated by gating the G₁/S transition. When the gate was open, the cells were able to divide if they had attained a sufficient size. However, the cells were not able to divide while the gate was closed, even if the cells had attained sufficient size. The diurnal rhythm of cell division immediately disappeared when the thalli were transferred to continuous light or darkness. When the thalli were transferred to a shifted photoperiod, the rhythm of cell division immediately and accurately synchronized with the shifted photoperiod. These data support a gating‐system model regulated by light:dark (L:D) cycles rather than an endogenous circadian clock. A dark phase of 6 h or longer was essential for gate closing, and a light phase of 14 h was required to renew cell division after a dark phase of >6 h.     

Signaling to Extracellular Signal-regulated Kinase from ErbB1 Kinase and Protein Kinase C: FEEDBACK,HETEROGENEITY, AND GATING*

Many extracellular signals act via the Raf/MEK/ERK cascade in which kinetics, cell-cell variability, and sensitivity of the ERK response can all influence cell fate. Here we used automated microscopy to explore the effects of ERK-mediated negative feedback on these attributes in cells expressing endogenous ERK or ERK2-GFP reporters. We studied acute rather than chronic stimulation with either epidermal growth factor (ErbB1 activation) or phorbol 12,13-dibutyrate (PKC activation). In unstimulated cells, ERK-mediated negative feedback reduced the population-average and cell-cell variability of the level of activated ppERK and increased its robustness to changes in ERK expression. In stimulated cells, negative feedback (evident between 5 min and 4 h) also reduced average levels and variability of phosphorylated ERK (ppERK) without altering the “gradedness” or sensitivity of the response. Binning cells according to total ERK expression revealed, strikingly, that maximal ppERK responses initially occur at submaximal ERK levels and that this non-monotonic relationship changes to an increasing, monotonic one within 15 min. These phenomena occur in HeLa cells and MCF7 breast cancer cells and in the presence and absence of ERK-mediated negative feedback. They were best modeled assuming distributive (rather than processive) activation. Thus, we have uncovered a novel, time-dependent change in the relationship between total ERK and ppERK levels that persists without negative feedback. This change makes acute response kinetics dependent on ERK level and provides a “gating” or control mechanism in which the interplay between stimulus duration and the distribution of ERK expression across cells could modulate the proportion of cells that respond to stimulation.