MOLECULAR BIOLOGY OF GENES INVOLVED IN INSULIN SECRETION

该项目的总体目标是克隆，测序和表征 参与胰岛素分泌的关键β细胞基因。 而 调节细胞内浓度的基因的功能重要性 Ca2+和K+在产生胰岛素分泌反应中的作用已被证实。 清楚地表明，很少有人知道的分子结构， 在β细胞中表达的这些蛋白质的同种型。 我们将 因此，克隆和表征似乎发挥作用的b细胞K+通道， 在胰岛素分泌的产生和调节中起重要作用 信号了 这些包括ATP敏感的K+通道和几种类型的 电压依赖性K+通道。 我们最近克隆了 其特征在于在人胰岛素瘤中表达的电压依赖性K+通道 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 涉及许多策略，包括表达克隆、基于PCR的 cDNA文库的扩增和低严格性交叉杂交。 我们 还将克隆和表征β细胞Ca2+通道，包括电压 依赖性Ca 2+通道（VDCC）和三磷酸肌醇受体。 我们 初步研究已经确定了VDCC的同种型， 在β细胞和大脑中表达。 克隆将完成，我们 将尝试克隆所建议的β细胞VDCC的其他形式 电生理学研究。 一种补充机制， 细胞内Ca 2+的调节是通过其从 内质网中的隐蔽位点。 这一过程是中介的 通过IP3与特定受体的结合。 我们利用最近的 克隆大鼠脑IP3受体以鉴定相关的同种型， 在大鼠胰岛中表达。 克隆完成后， 其结构与类似蛋白质如神经元IP3的比较 和ryanodine受体。 的生物物理和药理学特性 克隆的通道基因及其在胰岛素分泌中的作用 异源系统将与项目2合作研究， 3. 我们预计，这些研究将提供一个更好的理解， 参与调节的蛋白质的结构和功能 在分子水平上促进胰岛素分泌，并促进旨在 以阐明糖尿病中存在的改变。 此外我们 我希望定义一个最小的系统，使β细胞耦合 分泌刺激到电兴奋性。

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.