MOLECULAR STUDIES OF VOLTAGE DEPENDENT POTASSIUM CHANNELS

电压依赖性钾通道的分子研究

• 批准号: 6270682
• 负责人: BURTON HOROWITZ
• 金额: $ 16.52万
• 依托单位: UNIVERSITY OF NEVADA RENO
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-01 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6270682
• 关键词: aminopyridines; animal genetic material tag; colon; cyclic AMP; cyclic GMP; divalent cations; dogs; electrophysiology; enzyme activity; gastrointestinal motility /pressure; gene expression; genetic library; inhibitor /antagonist; molecular cloning; potassium channel; protein kinase; protein structure function; second messengers; smooth muscle; temperature; transfection

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.

结肠肌肉显示出K+电导的异质性集合 其通过兴奋-收缩偶联促进运动性。 虽然在分散的平滑肌细胞中的电生理学研究 产生了大量的信息， 在这些电流中，结肠运动的分子基础还没有被证实。 追究 具体而言，K+通道基因产品负责 在GI平滑肌中证实的K+电流尚未阐明 也没有研究过它们在这些不同组织中的表达模式。 本申请中提出的实验将检验这些假设 （1）结肠的各种电行为由以下因素决定： 几个不同的K+通道基因在结肠平滑肌中表达，（2） 编码这些通道的基因的差异遗传表达 有助于显示的区域性电活动模式 在胃肠道里。 此外，通过鉴定和分离分子， 在结肠中存在的K+电流的对应物中，它将是 可以表征电生理学和药理学 在没有污染电流的情况下，这些通道的特性。 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，包括 纵行肌特异性文库和培养的间质 细胞通过注射由这些cDNA克隆制备的体外转录RNA， 非洲爪蟾卵母细胞的电生理特性，这些 离子通道将被确定，并与项目收集的数据相关 2，其已从分离的结肠肌细胞获得。 将进行生物物理研究，以确定机制， 4-AP块的结构关系。 假设（2）将得到解决 通过确定K+通道表达的分子分布， 结肠和胃肠道的其它区域。结肠K+的可能性 将通过实验探索通道调节， 激酶活性、温度和二价阳离子对 克隆结肠钾通道功能。