离子通道研究方法精要（导读版） 下载 pdf 百度网盘 epub 免费 2025 电子书 mobi 在线

离子通道领域研究热点的快速增长，证明其在维持生命状态过程中起到的基础性作用。分子生物学和物理学是离子通道研究中极为重要的两种方法，本书基于这一角度选取了部分有代表性的研究方法。章节作者在阐述某种研究方法时，使用了大量的图表并与其他方法进行比较，提供一些窍门和捷径使其可以适用于其他研究体系。本书语言简明易懂，适合初涉离子通道领域的研究者，对有经验的研究人员也极具参考价值。

撰稿人

前言

部分 组装

    1.离子通道的组装

        Ⅰ.引言

        Ⅱ.策略和方法

        参考文献

第二部分 遗传学

    2.通过酵母双杂交系统鉴定离子通道关联的蛋白质

        Ⅰ.引言

        Ⅱ.酵母双杂交系统的原理

        Ⅲ.材料和方法

        Ⅳ.酵母双杂交系统的局限、前景和展望

        附录:溶液

        参考文献

第三部分 电生理学

    3.囊性纤维化跨膜转运调节因子氯离子通道的膜片钳研究

        Ⅰ.引言

        Ⅱ.表达体系的选择

        Ⅲ.膜片钳技术

        Ⅳ.CFTR通道的通透性

        Ⅴ.CFTR氯离子通道的调控

Ⅵ.DRSCAN:一种用于长时间记录分析的兼容性程序

        附录

        参考文献

    4.秀丽隐杆线虫(C. elegans)神经元的紧密封接全细胞膜片钳

        Ⅰ.引言

        Ⅱ.概述

        Ⅲ.用于原位电生理的C.

elegans的制备

        Ⅳ.亚微米开度的膜片钳的构成

        Ⅴ.溶液

        Ⅵ.膜片钳设置

        Ⅶ.GFP标记的神经元记录

        Ⅷ.紧密封接的全细胞记录

        Ⅸ.小细胞膜片钳记录的阐释

        Ⅹ.电压的空间控制

        Ⅺ.前景

        Ⅻ.总结

        参考文献

    5.门控电流

        Ⅰ.引言

        Ⅱ.门控电流的研究

        Ⅲ.单通道电荷

        Ⅳ.检测中的问题

        Ⅴ.分离中的问题

        Ⅵ.电压钳

        Ⅶ.记录步骤

        Ⅷ.门控电流的记录

        Ⅸ.基本门控事件

        Ⅹ.频域门控电流的记录

        参考文献

    6.离子通道通透性质的确定

        Ⅰ.引言

        Ⅱ.单离子电势

        Ⅲ.离子通道的选择性

        Ⅳ.离子孔道的分类

        Ⅴ.孔道阻断研究

        Ⅵ.孔道占有情况的确定

        Ⅶ.结论

        参考文献

第四部分 表达体系

    7.通过塞姆利基森林病毒(SFV)和杆状病毒表达配体门控的离子通道

        Ⅰ.引言

        Ⅱ.病毒DNA或RNA的生成

        Ⅲ.宿主细胞的选择和培养

        Ⅳ.病毒的扩增和滴定

        Ⅴ.实验参数的优化

Ⅵ.杆状病毒和SFV表达离子通道的应用

        Ⅶ.结论

        参考文献

    8.由重组腺病毒介导的编码离子通道和突触功能相关分子的基因在神经系统中的表达

        Ⅰ.引言

        Ⅱ.重组腺病毒的准备

        Ⅲ.技术

        Ⅳ.钾离子通道的表达

Ⅴ.腺病毒在急性海马脑片生理学中的应用

        Ⅵ.未来的方向

        参考文献

    9.异源离子通道的瞬时表达

        Ⅰ.更新

        Ⅱ.引言

        Ⅲ.方法

        Ⅳ.结果

        Ⅴ.总结

        参考文献

第五部分 模型模拟

    10.离子通道的电脑模拟和建模

        Ⅰ.引言

        Ⅱ.基础统计方法

        Ⅲ.势能

Ⅳ.非线性Poisson-Boltzmann方程

        Ⅴ.模拟步骤总结

        参考文献

第六部分 物理

    11.测定离子通道活性的荧光技术

        Ⅰ.引言

        Ⅱ.实验步骤

        Ⅲ.药理验证试验

        Ⅳ.钙离子高度应答性离子通道

        Ⅴ.钙离子中度应答性离子通道

        Ⅵ.钙离子低度应答性离子通道

Ⅶ.基于荧光染料测定离子通道活性的钙离子浓度试验的局限

        Ⅷ.基于荧光染料钙离子浓度试验的优势

        参考文献

    12.分析离子通道结构和功能的配体结合方法

        Ⅰ.修改文稿介绍

        Ⅱ.引言

        Ⅲ.方法比较

        Ⅳ.方法

        Ⅴ.荧光配体结合试验

        Ⅵ.配体结合分析

        Ⅶ.热动力循环分析

        参考文献

    13.二维结晶、冷冻电镜和成像分析决定的膜蛋白三维结构

        Ⅰ.引言

Ⅱ.电子冷冻晶体学的膜蛋白结构分析的步骤

        Ⅲ.图谱阐释

        Ⅳ.结论

        参考文献

    14.毛细管电泳的电压钳生物感受器

        Ⅰ.引言

        Ⅱ.毛细管电泳

        Ⅲ.断裂电泳毛细管的构成

        Ⅳ.细胞制备

        Ⅴ.毛细管电泳膜片钳记录

        Ⅵ.双电极电压钳记录

        Ⅶ.总结

        参考文献

    15.离子通道作为监控脂双层与膜蛋白相互作用的工具:短杆菌肽作为分子力的传递者

        Ⅰ.近期发展

        Ⅱ.引言

        Ⅲ.蛋白质构象改变和脂双层扰动

        Ⅳ.膜扰动和通道功能

        Ⅴ.膜变形的能量

        Ⅵ.载体与通道报告蛋白的选择

        Ⅶ.分子力传递者

Ⅷ.测定ΔG(I→II)bilayer和现象的弹力常数

        Ⅸ.结论

        参考文献

第七部分 纯化和重组

    16.上皮囊性纤维化跨膜转运调节因子氯离子通道的纯化和重构

        Ⅰ.更新

        Ⅱ.引言

Ⅲ.CFTR在Sf9-杆状病毒系统中的表达

        Ⅳ.CFTR的溶解和纯化

        Ⅴ.CFTR的重组

        Ⅵ.重组CFTR通道功能特征的估测

        参考文献

    17.天然和克隆的通道在平面脂双层的重组

        Ⅰ.更新

        Ⅱ.引言和概述

Ⅲ.大鼠肌肉T-小管细胞膜:一种K+ca通道和Na+v通道的可靠资源

Ⅳ.天然组织中各种类型离子通道的制备和重组

Ⅴ.克隆和异源表达的通道重组到平面脂双层的方法

        参考文献

第八部分 第二信使和生化方法

    18.配体门控离子通道的蛋白质磷酸化

        Ⅰ.引言

Ⅱ.配体门控离子通道磷酸化的生化性质分析

        Ⅲ.配体门控离子通道磷酸化的功能作用

        Ⅳ.结论

        参考文献

    19.离子通道关联蛋白的分析

        Ⅰ.引言

        Ⅱ.总体考虑

        Ⅲ.重组蛋白的体外结合

        Ⅳ.全长蛋白质在异源细胞中的整合

        Ⅴ.离子通道和关联蛋白在体内的共定位

Ⅵ.天然组织中离子通道和关联蛋白的免疫共沉淀

        Ⅶ.结论

        参考文献

    20.离子通道的第二信使调控/植物膜片钳

        Ⅰ.更新

        Ⅱ.引言

        Ⅲ.暴露膜

        Ⅳ.植物细胞与动物细胞膜片钳的对比

        Ⅴ.离子通道的第二信使调控

        Ⅵ.结论性评语

        附录

        参考文献

第九部分 特殊离子通道

    21.ATP敏感性钾离子通道

        Ⅰ.引言

        Ⅱ.药物合成

        Ⅲ.组织培养

        Ⅳ.转染操作

        Ⅴ.铷外流检测

        Ⅵ.膜的分离

        Ⅶ.光标操作

        Ⅷ.受体溶解

        Ⅸ.SUR1的部分纯化

        Ⅹ.额外纯化步骤

        Ⅺ.沉降

        Ⅻ.结合检测

        参考文献

    22.研究机械力门控通道的简化快速压力钳技术

        Ⅰ.引言

        Ⅱ.简化压力钳的机械排布

        Ⅲ.压力钳的电子控制

        Ⅳ.构建的一些实践经验

        附录

        参考文献

    23.抑制性甘氨酸受体的异源表达和纯化

        Ⅰ.引言

        Ⅱ.哺乳动物脊索中甘氨酸受体的纯化

        Ⅲ.甘氨酸受体的异源表达

        Ⅳ.HEK293细胞中瞬时表达

        Ⅴ.2×BBS

        Ⅵ.杆状病毒系统

        参考文献

    24.Aquaporin水分子通道蛋白的功能分析

        Ⅰ.引言

        Ⅱ.红血球AQP1蛋白的纯化

        Ⅲ.AQP1在酵母中的表达

        Ⅳ.AQP1重组到蛋白脂质体中

        Ⅴ.AQP1蛋白脂质体的水渗透

Ⅵ.用简并寡核苷酸PCR对Aquaporins的同源克隆

        Ⅶ.表达AQP1蛋白的蛙卵的水渗透

        参考文献

第十部分 毒素和其他膜活性化合物

    25.离子通道的Conus多肽探针

        Ⅰ.引言

        Ⅱ.Conus探针的生化概述

Ⅲ.处理Conus多肽中的一些实践经验

        参考文献

    26.蝎毒作为研究钾离子通道的工具

        Ⅰ.引言

Ⅱ.蝎毒液中钾离子通道多肽抑制剂的纯化

Ⅲ.通过重组技术合成钾离子通道抑制剂多肽

        Ⅳ.钾离子通道抑制性多肽的放射性标记

        Ⅴ.受体结合研究

        Ⅵ.总结

        参考文献

    27.利用平面脂双层快速筛选膜活性化合物

        Ⅰ.更新

        Ⅱ.引言

        Ⅲ.一种新的双层膜系统

        Ⅳ.平面脂膜的设置和区室

        Ⅴ.材料

        Ⅵ.平面脂膜灌流技术的应用

        参考文献

    28.离子通道抗体

        Ⅰ.引言

        Ⅱ.人类疾病中自发产生的抗体

        Ⅲ.配体门控的受体

        Ⅳ.电压门控型离子通道

        Ⅴ.商业产品

        参考文献

索引

Director,Office of Research Advocacy (OHSU)

Senior Scientist,Divisions of Reproductive Sciences and

Neuroscience (ONPRC)

Professor,Departments of Pharmacology and Physiology,Cell and

Developmental Biology,and Obstetrics and Gynecology (OHSU)

Beaverton,Oregon

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PART I

Assembly

CHAPTER 1

Assembly of Ion Channels

ZuFang Sheng and Carol Deutsch

Department of Physiology

University of Pennsylvania

Philadelphia

Pennsylvania

USA

I.Introduction

II.Strategies and Methods

A.Identification of Putative Regions Involved in Intersubunit

Interactions

B.Characterization of Intersubunit Interactions

C.Determination of Subunit Stoichiometry and History During

Assembly

References

I.Introduction

Most ion channels are multisubunit conglomerates.Because synthesis

and assembly

of many different types of pore-forming subunits occur in a single

cell,how

do the right subunits find each other to give the correct

stoichiometry and avoid

scrambling to channel homogeneity?This problem is even more

striking if we

consider the vast number of nonchannel transmembrane proteins made

simultaneously

in a cell.Assembly is a multistep process that requires specific

intersubunit

recognition events.Each of these steps may include intermediate

folded conformations

of subunits and/or intermediate subunit stoichiometries.Such

possibilities

have not been explored for most types of ion channels,including Kt

channels,nor

is it known which regions of the subunits actually interact during

each assembly

step.

In some cases,the NH2-terminal domains of ion channels can function

as

specific recognition motifs between subunits(Babila et al.,1994;Li

et al.,1992;

Shen et al.,1993;Verrall and Hall,1992;see also Xu and Li,1998,this

volume),but

it is not clear that such elements contribute to stabilization of

the mature multimeric

protein or whether additional subunit–subunit interactions

between

transmembrane segments provide the energy to shift the equilibrium

in a

lipid bilayer toward multimerization and the final,mature channel

that functions

in the plasma membrane.Most voltage-gated Kt channels are

homotetrameric

membrane proteins,each subunit containing six putative

transmembrane segments,

S1–S6.It is not clear what holds the tetramer together;intersubunit

covalent

linkages do not appear to be responsible(Boland et al.,1994).In

these

channels the cytoplasmic NH2 terminus contains a recognition

domain,T1

(“first tetramerization”),that tetramerizes in vitro and confers

subfamily specificity

(Li et al.,1992;Shen and Pfaffinger,1995;Shen et al.,1993;Xu et

al.,1995).

However,in the native channel there are also intramembrane

association(IMA)

sites in the central core of voltage-gated Kt channels that provide

sufficient

recognition and stabilization interactions for channel assembly,and

disruption

of one or more of these interactions may suppress channel

formation(Sheng et al.,

1997;Tu et al.,1996).The relative contributions of different domain

interactions

(e.g.,T1 and IMA)may vary from channel isoform to isoform.What are

these

T1 and IMA domains in the native full-length Kt channel,and what

are their

relative contributions to channel formation?

Identification of the recognition and stabilization motifs in the

primary sequence

of channel proteins is a good beginning to understanding channel

assembly;

however,it still leaves many questions unanswered.How specific are

these intersubunit

interactions?How strong are they? At which stage in assembly are

subunits

integrated into the membrane?What are the spatial and temporal

events involved

in channel assembly?What is the subunit stoichiometry of the

channel?What is the

history of the subunits during assembly?Is recruitment of subunits

a random

event?What is the nature of the subunit pool?Where is it

located?When are

subunits recruited into multimeric channels,and where?We can

address these

issues both biochemically and biophysically,as described in the

next section,

using a variety of in vitro translation systems and in vivo

expression systems.

The in vitro translation systems include rabbit reticulocyte

lysate(RRL)and

wheat germ agglutinin(WGA)systems,which contain cellular components

necessary

for protein synthesis(tRNA,ribosomes,amino acids,and

initiation,elongation,

and termination factors)and are capable of a variety of

posttranslational

processing activities(acetylation,isoprenylation,proteolysis,and

some phosphorylation

activity).Signal peptide cleavage and core glycosylation can be

reconstituted

and studied by adding canine pancreatic microsomal membranes to

the

translation reaction.These systems permit studies,for example,of

transcriptional

and translational control,association of proteins,and their

membrane integration.

However,the translation efficiency of high molecular weight

proteins(>100,000)is

relatively poor,and it is not clear that all aspects of in vivo

processing have been

reconstituted.Thus,caution must be used in extrapolating findings

with the in vitro

system to in vivo events.

The in vivo expression system most used for study of channel

function and

assembly has been Xenopus oocytes(Rudy and Iverson,1992).Mammalian

cells

are also used frequently and involve DNA transfection

techniques(Rudy and

Iverson,1992).Oocytes typically require injection of channel

mRNA(typically

50 nl/oocyte;<0.1–100 ng mRNA/oocyte).This system is an intact

cell system that

expresses at high levels for both electrophysiological and

biochemical measurements,

which can be done simultaneously in parallel samples.Both the

oocyte and

a mammalian T-cell expression system are described later,as well as

the methods

used to study channel protein synthesis,integration into

membranes,and

oligomerization.

Broadly defined,assembly also involves

trafficking,posttranslational modification,

and localization of channel proteins in specific subcellular

compartments,as

well as the aforementioned processes of recognition and

association(oligomerization).

This chapter,however,focuses only on strategies and methods that

can be

used(1)to identify regions of a protein that are potentially

involved in intersubunit

interactions during assembly of the pore-forming unit of ion

channels,(2)to

determine the strength,kinetics,spatial,and temporal

characteristics of the intersubunit

interactions,and(3)to determine the subunit stoichiometry and

history of

subunits during assembly.For some cases we illustrate the

approaches by describing

experiments in our laboratory involving a voltage-gated Kt

channel,Kvl.3.

However,these strategies and methods can be,and have been,used for

other

multimeric channels.

II.Strategies and Methods

The strategies used to address the issues just stated entail either

direct or indirect

determinations of various aspects of subunit association.The former

category

includes primarily biochemical approaches;the latter makes use of

functional

readouts.These strategies are protein based,yet each can have

additional strategies

at the DNA level.For example,strategies that entail constructing

genes that link

multiple channel domains in tandem,swapping channel domains to

create chimeras,

and/or deleting or mutating domains can be combined with the

protein

assays to elucidate mechanisms of channel assembly.

A.Identification of Putative Regions Involved in Intersubunit

Interactions

Intersubunit association can be assessed by direct and indirect

methods as

described in the following subsections.To discover which regions of

the channel

interact across subunit boundaries,physical association between

channel subunits

or between peptide fragments of a channel and the full-length

channel protein must

be demonstrated.This can be done directly by(1)immunoprecipitation

of one

member of a complex by antibody against the other

member,(2)cross-linking

interacting proteins using bifunctional reagents,or(3)binding

assays of interacting

peptides.Such binding assays have been employed to show that Kt

channel

subunits,or parts of these subunits,multimerize both in vitro and

in vivo(Babila

et al.,1994;Li et al.,1992;Shen and Pfaffinger,1995;Shen et

al.,1993).But these

studies have been concerned primarily with cytoplasmic NH2-terminal

interactions.

We describe one of these methods used in our

laboratory,namely,immunoprecipitation.

One important caveat concerning the association of peptide

fragments of a channel with the channel protein is that it is not

clear that such

association faithfully reflects native associations between

full-length subunits

in situ.For instance,constraints imposed on a segment of the

channel in the

context of the full-length folded protein may lead to different

interactions with

another subunit compared with the isolated truncated channel

peptide fragment.

Therefore,for a transmembrane segment,it is ultimately important to

determine

not only whether these interactions occur in the native protein but

also the

topology and orientation of the peptide fragment.

1.Immunoprecipitation

This method requires the use of antibodies(antisera)to a protein or

a peptide

construct.If the antibodies to native epitopes are not sufficiently

good,an epitope

tag may be used;c-myc(MEQKLI-SEEDL)(Evans et al.,1985)is excellent

for this

purpose.Such nonnative epitopes,however,should be inserted into a

primary

sequence at a nonperturbing distance(>15 amino acids)from

putative topogenic

determinants.The first step in this approach involves making the

appropriate

plasmid DNA either for use in transfections for subsequent in vivo

expression,or

for in vitro transcription to produce mRNA for subsequent use in

either in vivo or

in vitro experiments.Standard methods of restriction enzyme

analysis,agarose gel

electrophoresis,and bacterial transformation are used for these

studies.Plasmid

DNA are purified using Qiagen columns(Valencia,CA),and capped mRNA

is

synthesized in vitro from linearized templates using Sp6 or T7 RNA

polymerase

(Promega,Madison,WI).

For in vitro immunoprecipitation experiments,proteins are

translated in vitro

with [35S]methionine(2 ml/25 ml translation mixture; 10 mCi/ml

Dupont/NEN

Research Products,Boston,MA)in RRL(commercial preparations are

available

from Promega,and from MBI Fermentas,Amherst,NY;laboratory

preparations

can be made according to Jackson and Hunt,1983;Walter and

Blobel,1983)in the

presence(1.8 ml membrane suspension/25 ml translation mixture)or

absence of

canine pancreatic microsomal membranes(Promega or MBI

Fermentas),according

to the Promega Protocol and Application Guide.Two proteins that are

proposed

to interact are then cotranslated.Relative mRNA concentrations

should be

determined from the efficiencies of each construct to yield protein

ratios that are

desired.To maximize coimmunoprecipitation,microsomal membranes

should be

used in limiting concentration compared with the total mRNA

concentration.The

translation reaction can be visualized and quantitated using

SDS–PAGE and

phosphor imaging.

To perform immunoprecipitation from an in vitro translation

system(RRL,

microsomal membranes),1–5 ml of cell-free translation products is

mixed in 400 ml

of buffer A [0.1 M NaCl,0.1 M Tris(pH 8.0),10 mM EDTA,and

1%(v/v)

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