高粱根质膜K~＋通道蛋白的溶解及重组

用水两相法纯化的高粱根质膜嵌入平面脂双层膜,可以测得离子通道电流。记录了典型的单离子通道。ＴｒｉｔｏｎＸ—１００使质膜蛋白溶解,除去不溶物后,加入ＡｍｂｅｒｌｉｔｅＸＡＤ—２使膜蛋白重组为蛋白脂质体。将蛋白脂质体嵌入脂双层,测定了脂双层上Ｋ＋通道电流逐渐出现的过程,该典型的通道电流记录的通道电导为１１２ｐＳ。通过改变前后槽ＫＣｌ浓度,测定蛋白脂质体嵌入脂双层后的稳态电流一电压关系,近似计算稳态逆转电位,由ＧＨＫ等式得Ｋ＋和Ｃｌ－的选择通透比Ｐｋ：Ｐｃｌ为８．６主要是通透Ｋ＋。关键词：     

Random Bi Bi机制的非稳态酶动力学布尔函数图论研究

本文以非稳态酶动力学的布尔函数图形方法^[1],来研究一类Random Bi Bi机制的非稳态酶动力学问题,推导同此类反应的非稳态酶动力学方程,并对此动力学方程进行了讨论,分析了此类Random Bi Bi机制酶反应体系的非稳态酶动力学方程。     

Ping Pong Bi Bi机制的非稳态酶动力学分布函数图论研究

以非稳态酶动力学的布尔函数图形方法,来研究一类PingPongBiBi机制的非记酶动力学问题,推导出此类反应的非稳态酶动力学方程,并对此动力学方程进行了讨论,分析了此类PingPongBiBi机制酶反应体系的非稳态酶动力学方程。     

神经细胞膜钠通道的门控电流和门控动力学

根据作者已提出的神经轴突钠离子通道蛋白质分子构象模型,组成通道闸门的极性氨基酸侧链可以有两种类型:偶极子和荷电离子基团.它们在两种构象间的跃迁导致通道呈现关闭或开放态,并产生了门控电流相应的两种组分.对两种侧链跃迁动力学参量的计算结果表明,门控电流曲线的时间常数、稳态开启几率及门控电荷随膜电位的变化是不同的.去极化脉冲引起通道状态的变化,可由关闭态经过活化态转变为失活态,也可由关闭态经部分关闭态转变为失活态,对不同膜电位条件下一个通道各种稳定状态几率的计算表明,活化和失活的耦联程度随膜电位而改变.     

马达蛋白定向运动及能量转换效率的研究

通过建立非平衡涨落下的随机跃迁的两态模型,并利用非对称的周期势和Fokker-Planck方程及其本征值法计算两态模型的几率流和稳态几率流密。结果表明马达蛋白的定向运动与有效势的整体倾斜斜率有关,定性讨论了系统能量转换效率与负载有关。     

周期性静滴给药的稳态动力学研究

对线性一定模型的药物,周期静滴给药的动力学模型为一分段连续函数,本文对其动态动力学特征进行了研究,得到了稳态浓度的ｃ－ｔ方程以及一次给药和多次给药动力学参数之间的关系。为预测稳态浓度提供了依据,在此基础上对给药方案的拟定进行了讨论。     

锌离子压抑鲫鱼视网膜水平细胞内向整流钾离子通道的模型和机理探究

内向整流钾离子通道(inwardly rectifying potassium channel,Kir)广泛存在于神经元和肌肉细胞中,在维持细胞膜静息电位和传导神经信号中起到了重要作用。有研究显示,Zn2+在特定的pH值条件下可以抑制Kir电流。本文使用NEURON软件对Kir通道介导的电流建模,模拟了Kir的电流-电压关系,提出一组动力学状态转换方程来描述Zn2+对Kir的压抑作用。模型可以较好地描述不同浓度Zn2+对Kir电流的调节情况,提示Zn2+通过结合在Kir上的特定位点产生压抑作用,并且此位点会被H+竞争性结合。     

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。     

荧光研究表明去辅基天青蛋白突变体M121L至少存在两种不同的构象

以往对绿脓杆菌去辅基天青蛋白变性机制的研究认为它经历了一个复杂的反应过程,相比之下,锌离子替代的天青蛋白的变性符合简单的二态模型。以脲为变性剂对去辅基天青蛋白突变体M121L的变性过程进行了研究。结果表明,虽然稳态条件下突变体的变性／复性符合二态模型,但其动力学过程复杂,并可用溶液中存在着两种可以相互转化的构象的变性／复性来解释。天然态N1去折叠的速度快,其重折叠的速度也快,N1的折叠机制可用存在着折叠途径上的快速折叠中间体模型来描述;天然态N2的去折叠速度慢,其重折叠主要是首先生成天然态N1,然后再缓慢地转化成N2。添加Zn^2 能够把两种构象整合成一种构象,相应地,Zn^2 替代的天青蛋白突变体的变性过程简化为单指数过程。对该突变体的研究加深了对天青蛋白去折叠机制的理解。     

非稳态酶活化动力学的布尔函数图论分析

以非稳态酶动力学的布尔函数图形方法研究非稳态酶活化动力学问题,推导出此类反应的非稳态酶动力学方程,并对此动力学方程进行了讨论,分析了酶活化反应体系的非稳态酶动力学过程．     

Permeation mechanism of a two-state potassium channel

A two-state hopping model was proposed to study the permeation of ion channel.The Nemst equation in equilibrium and the Michaelis-Menten relation in steady state were derived from the two-state kinetic model.The currentvoltage relationship obtained in the symmetrical solutions case was linear when the applied potential was less than 100 mV,which met Ohm's law.The conductance-concentration relationship exhibited the saturation property.Moreover,the characteristic time reaching the steady state of the KcsA channel was also discussed.     

Permeation mechanism of a two-state potassium channel

A two-state hopping model was proposed to study the permeation of ion channel. The Nernst equation in equilibrium and the Michaelis-Menten relation in steady state were derived from the two-state kinetic model. The current-voltage relationship obtained in the symmetrical solutions case was linear when the applied potential was less than 100 mV, which met Ohm’s law. The conductance-concentration relationship exhibited the saturation property. Moreover, the characteristic time reaching the steady state of the KcsA channel was also discussed. Translated from Acta Biophysica Sinica, 2005, 21(4): 289–294 [译自: 生物物理学报]     

One-way fluxes of alpha-aminoisobutyric acid in Ehrlich ascites tumor cells. Trans effects and effects of sodium and potassium

One-way fluxes in the steady state and one-way influxes at zero intracellular concentrations were measured for alpha-aminoisobutyric acid (AIB) in Ehrlich ascites tumor cells at 32 degrees C. The one-way fulxes show trans effects in the concentration of AIB and are dependent on sodium levels. The one-way fluxes for initial influx and for the steady state were fitted with the equations derived for the frequently used two-state carrier model. Estimates of the parameters of these equations were obtained with use of nonlinear least squares. These gave relatively good fits of the flux data and the data on steady-state distribution ratios. The two-state carrier model predicted a trans inhibition of one-way influx and a trans stimulation of one-way efflux. The former phenomenon has been demonstrated for AIB transport in Ehrlich ascites cells and there is evidence, through less firm, for the latter.     

Access channel model for the voltage dependence of the forward-running Na+/K+ pump

The voltage dependence of steady state current produced by the forward mode of operation of the endogenous electrogenic Na+/K+ pump in Na(+)- loaded Xenopus oocytes has been examined using a two-microelectrode voltage clamp technique. Four experimental cases (in a total of 18 different experimental conditions) were explored: variation of external [Na+] ([Na]o) at saturating (10 mM) external [K+] ([K]o), and activation of pump current by various [K]o at 0, 15, and 120 mM [Na]o (tetramethylammonium replacement). Ionic current through K+ channels was blocked by Ba2+ (5 mM) and tetraethylammonium (20 mM), thereby allowing pump-mediated current to be measured by addition or removal of external K+. Control measurements and corrections were made for pump current run-down and holding current drift. Additional controls were done to estimate the magnitude of the inwardly directed pump-mediated current that was present in K(+)-free solution and the residual K(+)- channel current. A pseudo two-state access channel model is described in the Appendix in which only the pseudo first-order rate coefficients for binding of external Na+ and K+ are assumed to be voltage dependent and all transitions between states in the Na+/K+ pump cycle are assumed to be voltage independent. Any three-state or higher order model with only two oppositely directed voltage-dependent rate coefficients can be reduced to an equivalent pseudo two-state model. The steady state current-voltage (I-V) equations derived from the model for each case were simultaneously fit to the I-V data for all four experimental cases and yielded least-squares estimates of the model parameters. The apparent fractional depth of the external access channel for Na+ is 0.486 +/- 0.010; for K+ it is 0.256 +/- 0.009. The Hill coefficient for Na+ is 2.18 +/- 0.06, and the Hill coefficient for K+ (which is dependent on [Na]o) ranges from 0.581 +/- 0.019 to 1.35 +/- 0.034 for 0 and 120 mM [Na]o, respectively. The model provides a reasonable fit to the data and supports the hypothesis that under conditions of saturating internal [Na+], the principal voltage dependence of the Na+/K+ pump cycle is a consequence of the existence of an external high- field access channel in the pump molecule through which Na+ and K+ ions must pass in order to reach their binding sites.     

A Conserved Pre-Block Interaction Motif Regulates Potassium Channel Activation and N-Type Inactivation

N-type inactivation occurs when the N-terminus of a potassium channel binds into the open pore of the channel. This study examined the relationship between activation and steady state inactivation for mutations affecting the N-type inactivation properties of the Aplysia potassium channel AKv1 expressed in Xenopus oocytes. The results show that the traditional single-step model for N-type inactivation fails to properly account for the observed relationship between steady state channel activation and inactivation curves. We find that the midpoint of the steady state inactivation curve depends in part on a secondary interaction between the channel core and a region of the N-terminus just proximal to the pore blocking peptide that we call the Inactivation Proximal (IP) region. The IP interaction with the channel core produces a negative shift in the activation and inactivation curves, without blocking the pore. A tripeptide motif in the IP region was identified in a large number of different N-type inactivation domains most likely reflecting convergent evolution in addition to direct descent. Point mutating a conserved hydrophobic residue in this motif eliminates the gating voltage shift, accelerates recovery from inactivation and decreases the amount of pore block produced during inactivation. The IP interaction we have identified likely stabilizes the open state and positions the pore blocking region of the N-terminus at the internal opening to the transmembrane pore by forming a Pre-Block (P state) interaction with residues lining the side window vestibule of the channel.     

Noise and Information Transmission in Promoters with Multiple Internal States

Based on the measurements of noise in gene expression performed during the past decade, it has become customary to think of gene regulation in terms of a two-state model, where the promoter of a gene can stochastically switch between an ON and an OFF state. As experiments are becoming increasingly precise and the deviations from the two-state model start to be observable, we ask about the experimental signatures of complex multistate promoters, as well as the functional consequences of this additional complexity. In detail, we i), extend the calculations for noise in gene expression to promoters described by state transition diagrams with multiple states, ii), systematically compute the experimentally accessible noise characteristics for these complex promoters, and iii), use information theory to evaluate the channel capacities of complex promoter architectures and compare them with the baseline provided by the two-state model. We find that adding internal states to the promoter generically decreases channel capacity, except in certain cases, three of which (cooperativity, dual-role regulation, promoter cycling) we analyze in detail.     

Multiple-state model of ionic channel in reproducing the time course of electrophysiological events.

BACKGROUND: The predictions of the Hodgkin-Huxley model do not accurately fit all the measurements of voltage-clamp currents, gating charge and single-channel currents. There are many quantitative differences between the predicted and measured characteristics of the sodium and potassium channels. For example, the two-state gate model has exponential onset kinetics, whereas the sodium and potassium conductances show S-shaped activation and the sodium conductance shows an exponential inactivation. In this paper we shall examine a more general channel model that can more faithfully represent the measured properties of ionic channels in the membrane of the excitable cell. METHODS: The model is based on the generalisation of the notion of a channel with a discrete set of states. Each state has state attributes such as the state conductance, state ionic current and state gating charge. These variables can have quite different waveforms in time, in contrast with a two-state gate channel model, in which all have the same waveforms. RESULTS: The kinetics of all variables are equivalent: gating and ionic currents give equivalent information about channel kinetics; both the equilibrium values of the current and the time constants are functions of membrane potential. The results are in almost perfect concordance with the experimental data regarding the characteristics of nerve impulse. CONCLUSIONS: The expected values of the gating charge and the ionic conductance are weighted sums of the state occupancy probabilities, but the weights differ: for the expected value of the gating charge the weights are the state gating charges and for the expected value of the ionic conductance the weights are the state conductances. Since these weights are different, the expected values of the gating charge and the ionic conductance will differ.     

Two elementary models for the regulation of skeletal muscle contraction by calcium

It is shown by use of an extremely simple explicit two-state model that two basic ideas may be sufficient to understand at least qualitatively the sensitive activation of isometric muscle contraction by Ca2+. (a) Ca2+ binds much more strongly on troponin if myosin is already attached to actin. The steady state analogue of this is that the single rate constant (in the two-state model) for myosin attachment plus Pi release is much larger if Ca2+ is bound to troponin. (b) End-to-end tropomyosin interactions are responsible for positive cooperativity. Although these ideas seem to be sufficient, this of course does not mean that they are necessary. These same ingredients were used in two previous, more elaborate models for the cooperative equilibrium binding of myosin subfragment-1 on actin-tropomyosin-troponin, with and without Ca2+, and for a study of the steady state ATPase activity of the same system. Essentially as an appendix, the above-mentioned simple treatment is extended to a somewhat more realistic and complicated model of isometric contraction.     

Adenine-adenine interaction in adenylyladenosine and polyadenylic acid measured with emission spectroscopy

Dynamical behavior of a melting of stacking interaction in adenylyladenosine (ApA) and polyadenylic acid (polyA) has been investigated with steady state and time resolved emission spectroscopy. Temperature dependence of monomer-like emission and excimer emission shows that the melting behavior of ApA can be analyzed by a simple two-state model whereas that of polyA exhibits the influence of neighboring adenine molecules which can interact in the excited state.     

Biological effects of oscillating electric fields: Role of voltage-sensitive ion channels

An alternating component of potential across the membrane of an excitable cell may change the membrane conductance by interacting with the voltagesensing charged groups of the protein macromolecules that form voltage-sensitive ion channels. Because the probability that a voltage sensor is in a given state is a highly nonlinear function of the applied electric field, the average occupancy of a particular state will change in an oscillating electric field of sufficient magnitude. This “rectification” at the level of the voltage sensors could result in conformational changes (gating) that would modify channel conductance. A simplified two-state model is examined where the relaxation time of the voltage sensor is assumed to be considerably faster than the fastest changes of ionic conductance. Significant changes in the occupancy of voltage sensor states in response to an applied oscillating electric field are predicted by the model.