STUDIES ON BASOLATERAL MEMBRANE POTASSIUM CHANNELS
基底外侧膜钾离子通道的研究
基本信息
- 批准号:3246764
- 负责人:
- 金额:$ 18.76万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:1992
- 资助国家:美国
- 起止时间:1992-09-30 至 1997-09-29
- 项目状态:已结题
- 来源:
- 关键词:Urodela Xenopus oocyte basolateral membrane gastrointestinal epithelium hydropathy immunocytochemistry intestines laboratory rabbit lipid bilayer membrane membrane reconstitution /synthesis membrane structure membrane transport proteins molecular cloning potassium channel protein sequence protein structure function site directed mutagenesis
项目摘要
The long-term goals of the proposed research project are logical
extensions of our efforts during the past few years and are designed to
further our understanding of the regulation and structure-function
relations of two K+ channels that are present, in basolateral membranes
of intestinal cells and that have been successfully reconstituted into
planar phospholipid bilayers. The first is an "inwardly rectifying K+
channel that is present in basolateral membrane vesicles isolated from
Necturus small intestinal cells. The second is a Ca-activated, high
("maxi") conductance channel that is present in basolateral membrane
vesicles of rabbit colonic epithelial cells.
The five specific aims of the proposed research program are:
I. To examine the effects of possible intracellular mediators and/or
second messengers on the activities of these channels reconstituted into
planar phospholipid bilayers.
II. To purify these channel proteins in functional forms as determined by
the ability to reconstitute their single channel activities in planar
bilayers. Preliminary studies to be described below have disclosed
feasible approaches towards that goal.
III. To clone the cDNAs encoding these proteins and determine their
primary structures (i.e. amino acid sequence) and putative "membrane
topologies" using Kyte-Doolittle hydropathy analyses.
IV. To express these channels in Xenopus oocytes using the poly(A+)mRNAs
derived from the cloned cDNAs to confirm that we have in fact cloned the
correct cDNAs and open the avenue for the study of structure-function
relations using site-directed mutagenesis.
V. To confirm the putative membranes of origin and explore the tissue
distribution of these channel proteins using immunohistochemical
techniques.
拟议的研究项目的长期目标是合乎逻辑的
这是我们过去几年努力的延伸,旨在
进一步加深了我们对调控和结构-功能的理解
基底外侧膜中存在的两个K+通道的关系
肠细胞,并已成功地重建成
平面磷脂双层。 第一种是“内向整流K+
存在于基底外侧膜囊泡中的通道,分离自
小肠细胞。 第二种是钙激活的,
(“maxi”)存在于基底外侧膜中的传导通道
兔结肠上皮细胞的囊泡。
拟议研究计划的五个具体目标是:
I.检查可能的细胞内介质和/或
第二信使对这些渠道的活动重组成
平面磷脂双层。
二.为了纯化功能形式的这些通道蛋白,
在平面上重建其单通道活动能力
双层。下文所述的初步研究已经披露了
实现这一目标的可行途径。
三.为了克隆编码这些蛋白质的cDNA并确定其表达水平,
一级结构(即氨基酸序列)和推定的“膜
拓扑”使用Kyte-Doolittle亲水性分析。
四.利用poly(A+)mRNA在非洲爪蟾卵母细胞中表达这些通道
从克隆的cDNA中获得,以确认我们实际上克隆了
修正cDNA,为结构-功能研究开辟途径
使用定点诱变的关系。
诉确认假定的原始膜并探索组织
这些通道蛋白的分布使用免疫组织化学
技术.
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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STANLEY G SCHULTZ其他文献
STANLEY G SCHULTZ的其他文献
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{{ truncateString('STANLEY G SCHULTZ', 18)}}的其他基金
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