BASOLATERAL MEMBRANE POTASSIUM CHANNELS

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.克隆编码这些蛋白质的cDNA并确定其活性 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.

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.