Ion Channels as Effectors for Cell Growth Regulatory Signaling

利用模型细胞系统进行细胞生长控制，主要研究人员和其他人提出了离子通道是中心生长因子/原癌蛋白/癌蛋白生长调控信号网络不可或缺的组成部分的概念。具体地说，首席研究员的实验室已经确定了一种成纤维细胞的中间电导，钙激活的钾通道，FIK，它在生长因子或癌蛋白刺激的有丝分裂信号刺激下选择性上调。此外，用肽基毒素特异性阻断FIK功能已被证明可可逆地抑制细胞增殖。FIK还被证明在10T1/2-MRF4成纤维细胞中调节MRF4肌肉调节转录因子的转录活性，10T1/2-MRF4成纤维细胞是一个模型细胞系统，用于确定细胞膜信号事件和生长调节转录事件之间的联系。FIK如何调节细胞生长的一个关键假设是，它确保细胞膜的超极化一致地达到或接近钾平衡电位。该模型预测，这种超极化作用反过来将通过生长因子激活的电压非依赖性钙通道促进促有丝分裂的钙离子进入。为了支持这一观点，初步研究已经在10T1/2-MRF4成纤维细胞中发现了一种钙通道，该通道可被碱性成纤维细胞生长因子激活，碱性成纤维细胞生长因子是这些细胞的重要生长调节肽。因此，大量已发表的工作和初步数据表明，FIK和多肽生长因子激活的钙通道是细胞生长的关键生理调节因子。该项目的长期目标是确定这些通道如何调节细胞生长，从而为细胞生理学的这一方面提供独特的见解。具体的项目目标是：1.为FIK在细胞生长调节信号中的作用建立一个机制基础。生理学和药理学手段将被用来研究成纤维细胞中FIK通道调控和生长控制之间的联系，并利用10T1/2-MRF4模型系统来了解FIK如何调控生长相关转录。2.同源克隆成纤维细胞IK通道(FIK)根据最近克隆的人钙激活钾通道的序列，将实施同源克隆策略。异源表达的克隆通道的生理学与本实验室广泛表征的成纤维细胞IK非常相似。克隆FIK最终将允许使用许多方法(诱导表达、反义抑制原生表达、点突变扰乱离子选择性等)。以确定该通道如何调节细胞生长。

Using model cell systems for cell growth control, the principal investigator and others have advanced the concept that ion channels are integral components of the central growth factor/proto-oncoprotein/ oncoprotein growth regulatory signaling networks. Specifically, the laboratory of the principal investigator has identified a fibroblast intermediate conductance, calcium-activated, potassium channel, FIK, that is selectively up-regulated in response to growth factor or oncoprotein stimulated mitogenic signaling. In addition, specific block of FIK function with peptidyl toxins has been shown to reversibly inhibit cell proliferation. FIK has also been shown to regulate transcriptional activity of the MRF4 muscle regulatory transcription factor in 10T1/2-MRF4 fibroblasts, a model cell system for defining the linkage between signaling events at the cell membrane and growth regulatory transcription events. A key hypothesis for how FIK regulates cell growth is that it insures consistent hyperpolarization of the cell membrane to or near the potassium equilibrium potential. The model predicts that this hyperpolarizing action will, in turn, promote mitogenic calcium entry via growth factor activated, voltage-independent calcium channels. In support of this idea, preliminary studies have identified a calcium channel in 10T1/2-MRF4 fibroblasts which is activated by bFGF, an important growth regulatory peptide for these cells. Thus, a wealth of published work and preliminary data have identified FIK and peptide growth factor activated calcium channels as key physiologic regulators of cell growth. The long term goal of this project is to define how these channels regulate cell growth, thus providing a unique insight into this aspect of cell physiology. Specific project goals are: 1. Establish a mechanistic basis for the role of FIK in cell growth regulatory signaling. Physiological and pharmacological means will be used to examine the link between FIK channel regulation and growth control in fibroblast cells, with the 10T1/2-MRF4 model system being exploited to understand how FIK modulates growth-associated transcription. 2. Homology clone the fibroblast IK channel (FIK). An homology cloning strategy will be implemented based on the sequence of recently cloned human calcium-activated potassium channels. Physiology of the heterologously expressed cloned channels is remarkably similar to the fibroblast IK which this lab has extensively characterized. Cloning FIK will ultimately allow utilization of a number of approaches (inducible expression, antisense inhibition of native expression, point mutations to perturb ionic selectivity, etc.) to determine how the channel regulates cell growth.