MOLECULAR STUDIES OF VOLTAGE DEPENDENT POTASSIUM CHANNELS

结肠肌肉表现为不同种类的K+电导 它们通过兴奋-收缩耦合来促进运动。 而分散的平滑肌细胞的电生理学研究 产生了大量关于房产的信息 在这些电流中，结肠运动的分子基础还没有 被调查。具体地说，K+通道基因产物负责 胃肠道平滑肌上的K+电流尚未阐明 它们在这些不同组织中的表达模式也没有被研究过。 本申请中提出的实验将检验这些假设 这是：(1)结肠的不同电行为由以下因素决定 几种不同的K+通道基因在结肠平滑肌中的表达(2) 编码这些通道的基因的差异遗传表达 对所展示的电活动的区域模式有贡献 在胃肠道。此外，通过鉴定和分离分子 与结肠中存在的K+电流相对应，它将是 可能表征电生理学和药理学 这些通道在没有污染电流的情况下的特性。 为了解决假设(1)，编码K+通道蛋白的全长cDNA 将从结肠平滑肌cDNA文库中分离得到，包括 纵肌和培养的间质特异性文库 细胞。通过注射由这些cDNA克隆制成的体外转录的RNA 进入非洲爪哇卵母细胞，这些细胞的电生理特性 离子通道将被确定，并与Project收集的数据相关 2，从分离的结肠肌细胞中获得。 将进行生物物理研究，以确定其机制和 4-AP区块的结构关系。假设(2)将被解决 通过测定K+通道表达的分子分布 结肠和胃肠道的其他区域。结肠K+的可能性 将通过实验来探索通道调节，这些实验将确定 酶活性、温度和二价阳离子对酶活性的影响 克隆了结肠K+通道功能。

Colonic muscles display a heterogeneous collection of K+ conductances which contribute to motility through excitation-contraction coupling. While electrophysiological studies in dispersed smooth muscle cells have generated a considerable body of information concerning the properties of these currents, the molecular basis for colonic motility has yet to be investigated. Specifically, K+ channel gene products responsible for the K+ currents demonstrated in GI smooth muscle have not been elucidated nor have their expression patterns in these diverse tissues been studied. The experiments proposed in this application will test the hypotheses that: (1) the diverse electrical behavior of the colon is determined by several distinct K+ channel genes expressed in colonic smooth muscle, (2) the differential genetic expression of the genes encoding these channels contributes to the regional pattern of electrical activity demonstrated in the GI tract. In addition, by identifying and isolating the molecular counterparts for the K+ currents present in the colon, it will be possible to characterize the electrophysiological and pharmacological properties of these channels in the absence of contaminating currents. To address hypothesis (1), full-length cDNAs encoding K+ channel proteins will be isolated from colonic smooth muscle cDNA libraries, including libraries specific for longitudinal muscle and cultured interstitial cells. By injecting in vitro transcribed RNA made from these cDNA clones into Xenopus oocytes, the electrophysiological characteristics of these ion channels will be determined and related to data collected by Project 2, which has been obtained from dissociated colonic myocytes. Biophysical studies will be conducted to determine the mechanism and structural relationships of 4-AP block. Hypothesis (2) will be addressed by determining the molecular distribution of K+ channel expression in the colon and other regions of the GI tract. The possibility of colonic K+ channel regulation will be explored with experiments which will determine the effects of kinase activity, temperature and divalent cations on cloned colonic K+ channel functions.