SINGLE ION CHANNELS IN PANCREATIC ISLET CELLS

内分泌胰腺生理学中一个有趣的问题是， 环境中葡萄糖或氨基酸浓度的升高，看似简单和 所有细胞都暴露在无处不在的刺激下，导致 B细胞控制的胰岛素搏动性分泌及其抑制作用 A细胞分泌的胰升糖素。这项工作的目标是研究 单离子通道在代谢物刺激偶联中的作用 胰岛素和胰升糖素的分泌。 主要地，我们希望检查一种代谢物的刺激是否会导致 在B细胞去极化(并因此电压依赖的钙内流)中， 导致ATP敏感的K通道关闭。为了做到这一点，我们将 检查该通道的显著特征，包括离子 它的选择性、门控、单通道动力学和药理学 频道。这些实验将涉及标准细胞的附着和切除 贴片记录、全细胞记录、细胞连接贴片记录 部分渗透细胞和一种新的“慢速”全细胞技术。 第二，我们希望检测B细胞内钙的释放， 它也可能受到新陈代谢的刺激，通过钙通道发生在 内质网(ER)，通过测量钙通道活性 胰岛素瘤ER融合成平面双层膜。我们也希望 确定这种细胞内钙的来源是否有助于 参与触发胰岛素胞吐的钙离子池，使用 部分心肌细胞贴壁斑块中钙激活的钾通道 通透性细胞作为局部钙浓度的测定。第三，我们 希望开始探索基础电活动的单通道 免疫细胞化学鉴定A细胞。 这些实验的结果应该有助于我们了解 刺激-分泌偶联在内分泌胰腺和铺平道路 糖尿病中膜缺陷的未来研究。

An intriguing question in the physiology of the endocrine pancreas is how a rise in ambient glucose or amino acid concentration, a seemingly simple and ubiquitous stimulus which all cells are exposed to, results in the controlled pulsatile secretion of insulin by the B cell and inhibition of glucagon secretion by the A cell. The goal of this work is to study the role of single ion channels in the coupling of metabolite stimuli to insulin and glucagon secretion. Principally, we wish to examine whether stimulation by a metabolite results in B cell depolarization (and hence voltage-dependent Ca2+ influx) by inducing closure of an ATP sensitive K+ channel. To do this we shall examine the salient characteristics of this channel including ion selectivity, gating, single channel kinetics and pharmacology of this channel. These experiments will involve standard cell attached and excised patch recording, whole cell recording, cell attached patch recording with partially permeabilized cells and a novel "slow" whole cell technique. Second, we wish to examine whether intracellular release of Ca2+ in B cell, which may also be stimulated by metabolism, occurs through Ca2+ channels in endoplasmic reticulum (ER), by measuring Ca2+ channel activity of insulinoma ER fused into planar bilayer membranes. We also wish to determine whether this source of intracellular calcium contributes to a pool of Ca2+ which participates in triggering insulin exocytosis, using Ca2+-activated K+ channels in cell attached patches of partially permeabilized cells as an assay for local Ca2+ concentration. Third, we wish to begin exploring single channels underlying electrical activity in immunocytochemically identified A cells. Results of these experiments should contribute to our knowledge of stimulus-secretion coupling in the endocrine pancreas and pave the way for future investigations of membrane defects in diabetes mellitus.