STUDIES ON BASOLATERAL MEMBRANE POTASSIUM CHANNELS

拟议研究项目的长期目标是合乎逻辑的。 是我们过去几年努力的延伸，旨在 深化对调控和结构功能的认识 基底膜上存在的两个K+通道的关系 它们已经成功地重组成 平面磷脂双层膜。一是“内化整顿K+” 存在于分离的基侧膜小泡中的通道 蜜腺小肠细胞。第二种是钙激活的，高 (“maxi”)存在于基侧膜上的电导通道 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 第二信使对这些活动的渠道进行了重组 平面磷脂双层膜。 II.提纯这些功能形式的通道蛋白 在平面上重建他们的单通道活动的能力 双层的。将在下文描述的初步研究揭示了 实现这一目标的可行办法。 III.克隆编码这些蛋白的cDNA并测定它们的 一级结构(即氨基酸序列)和假定的“膜” 拓扑“，使用Kyte-Doolitel水病分析。 IV.利用Poly(A+)mRNAs在非洲爪哇卵母细胞中表达这些通道 从克隆的DNA中派生出来，以确认我们实际上已经克隆了 纠正cDNA，为结构功能研究开辟道路 使用定点突变的亲缘关系。 V.确认可能的起源膜并探索组织 用免疫组织化学方法研究这些通道蛋白的分布 技巧。

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.