MOLECULAR BIOLOGY OF GENES INVOLVED IN INSULIN SECRETION

这个项目的总体目标是克隆、测序和鉴定 参与胰岛素分泌的关键β细胞基因。而当 调控细胞内浓度的基因在功能上的重要性 钙离子和钾离子在胰岛素分泌反应中的作用 清楚地证明，人们对该分子的分子结构知之甚少 在β细胞中表达的这些蛋白质的异构体。我们会 因此，克隆和鉴定b细胞K+通道似乎扮演着一种 胰岛素分泌的产生和调节中的重要作用 信号。其中包括对ATP敏感的K+通道和几种类型的 电压依赖性K+通道。我们最近克隆并实现了功能 人胰岛素瘤中表达的电压依赖性K+通道的特征 和人类胰岛细胞，并已开始表征其他K+通道 在胰岛素分泌细胞中表达。我们的实验方法将 涉及多种策略，包括基于PCR的表达克隆 文库的扩增和低严格交叉杂交。我们 还将克隆和鉴定包括电压在内的β细胞钙通道 依赖钙通道(VDCC)和三磷酸肌醇受体。我们的 初步研究确定了VDCC的一种异构体，即 在β细胞和大脑中表达。克隆工作将完成，我们将 将尝试克隆建议的其他形式的β细胞VDCC 通过电生理学研究。一种补充机制，用于 细胞内钙离子的调节是通过其动员来实现的 内质网中的隔离部位。这一过程是通过中介 通过IP3与特定受体结合。我们已经利用了最近的 克隆大鼠脑IP3受体以鉴定其相关亚型 在大鼠胰岛中表达。克隆的完成将允许详细的 其结构与神经元IP3等相似蛋白的结构比较 和兰尼定受体。青蒿素的生物物理和药理性质 血管通道基因的克隆及其在胰岛素分泌中的作用 将与项目2和项目2合作研究异质系统 3.我们预计这些研究将使我们更好地了解 参与调控的蛋白质的结构和功能 在分子水平上的胰岛素分泌，并促进旨在 以澄清糖尿病中存在的变化。此外，我们还 希望定义使β细胞能够耦合的最小系统 对电刺激性的分泌性刺激。

The overall goals of this project are to clone, sequence and characterize key beta-cell genes which participate in insulin secretion. While the functional importance of genes which regulate intracellular concentrations of Ca2+ and K+ in the generation of the insulin secretory response has been clearly demonstrated, little is known about the molecular structure of the isoforms of these proteins which are expressed in the beta-cell. We will therefore clone and characterize b-cell K+ channels which appear to play an important role in the generation and regulation of the insulin secretory signal. These include the ATP-sensitive K+ channel and several types of voltage-dependent K+ channels. We have recently cloned and functionally characterized a voltage-dependent K+ channel expressed in human insulinoma and human islet cells, and have begun to characterize other K+ channels expressed in insulin-secreting cells. Our experimental approach will involve a number of strategies including expression cloning, PCR based amplification and low stringency cross-hybridization of cDNA libraries. We will also clone and characterize beta-cell Ca2+ channels including voltage dependent Ca2+ channels (VDCC) and inositol trisphosphate receptors. Our preliminary studies have identified an isoform of the VDCC which is expressed in beta-cells and brain. This cloning will be completed and we will attempt to clone the additional forms of the beta-cell VDCCs suggested by electrophysiologic studies. A complementary mechanism for the regulation of intracellular Ca2+ is through its mobilization from sequestered sites in the endoplasmic reticulum. This process is mediated through binding of IP3 to specific receptors. We have utilized the recent cloning of the rat brain IP3 receptor to identify a related isoform that is expressed in rat islets. Completion of the cloning will allow detailed comparison of its structure with similar proteins such as the neuronal IP3 and ryanodine receptors. The biophysical and pharmacological properties of the cloned channel genes and their role in insulin secretion in heterologous systems will be studied in collaboration with Projects 2 and 3. We anticipate that these studies will provide a better understanding of the structure and function of proteins involved in the regulation of insulin secretion at the molecular level and facilitate studies which aim to clarify the alterations which are present in diabetes. In addition we hope to define the minimal system which enables the beta cell to couple secretory stimuli to electrical excitability.