Development of novel means to stimulate Ca2+-dependent exocytotic secretion

该项目的长期目标是发现新的方法来刺激Ca 2+依赖的胞吐， 通过调节细胞兴奋性从而调节细胞内Ca 2+信号来调节神经递质或激素。这些 新的方法将被用来开发有用的治疗策略，以对抗人类疾病。我们 目前的目标是开发特异性试剂以增加膜兴奋性，从而刺激Ca 2 +- 胰腺β细胞的胰岛素依赖性胞吐作用。葡萄糖浓度升高会增加新陈代谢 和β细胞中的胞浆ATP，其反过来抑制ATP敏感性K+（KATP）通道。KATP降低 通道活性使细胞膜去极化，激活电压门控Ca 2+（CaV）通道。CaV介导的Ca 2+内流升高细胞内Ca 2+浓度（[Ca 2 +]in），触发胰岛素释放。β细胞的KATP通道由内向整流K+通道Kir6.2（KCNJ 11）和调节亚基磺酰脲受体SUR 1（ABCC 8）组成。磺酰脲类药物是主要的一类抗糖尿病药物， 据信通过作用于SUR 1从而间接抑制Kir6.2活性来刺激胰岛素分泌。 从概念上讲，Kir6.2本身应该代表一个更直接，潜在有效的抗糖尿病药物靶点。 值得注意的是，几项研究表明，SUR 1除了抑制Kir6.2外，还发挥其他作用，如 与突触融合蛋白相互作用，突触融合蛋白是参与胞吐作用的关键蛋白质。此外，磺酰脲格列本脲 似乎也通过KATP非依赖性机制促进胰岛素的胞吐作用。此外，在过去 十年来，发现Kir6.2或SUR 1的许多功能获得性突变（降低KATP的ATP敏感性）， 是大多数永久性新生儿糖尿病（PNDM）的基础。这些患者中的大多数已经 成功地用磺脲类药物治疗，而不是传统的胰岛素治疗。然而，一些PNDM 具有某些突变的患者对磺脲类药物无反应。原则上，这些患者可能 可以用合适的Kir6.2抑制剂治疗。不幸的是，到目前为止，还没有Kir6.2抑制剂可用。到 获得实验证据，以科学的理由判断是否应该投入大量资源， 开发临床上有用的Kir6.2抑制剂以治疗糖尿病，我们提出：i.开发一个特定的原型 Kir6.2抑制剂，从而证明这种奋进的可行性，ii.调查Kir6.2是否 抑制剂可以刺激足够胰岛素分泌，和iii.应用这种抑制剂来测试假设， 磺酰脲也通过KATP非依赖性机制刺激胰岛素分泌。如果这个假设是 正确的，Kir6.2抑制剂可能没有磺脲类药物的所有副作用。我们将进行研究 结合电生理学、Ca 2+成像、蛋白质生物化学、分子生物学、细胞生物学 和组织学。我们的建议构成了一个创新的，概念验证的研究，一种新的抗糖尿病的战略; 全球有3.5亿人患有糖尿病， 这一流行病需要开发新的有效的治疗策略。

The long-term goal of this project is to discover new methods to stimulate Ca2+-dependent exocytosis of neurotransmitters or hormones by tuning cellular excitability and thereby the intracellular Ca2+ signal. These new methods will then be used to develop useful therapeutic strategies to combat human diseases. Our immediate goal here is to develop specific reagents to increase membrane excitability to stimulate Ca2+- dependent exocytosis of insulin from pancreatic β cells. Elevated glucose concentrations increase metabolism and cytosolic ATP in β cells, which in turn inhibits the ATP-sensitive K+ (KATP) channel. Decreased KATP channel activity depolarizes the cell membrane, activating voltage-gated Ca2+ (CaV) channels. The CaV-mediated Ca2+ influx elevates intracellular Ca2+ concentration ([Ca2+]in), triggering insulin release. A KATP channel of the β cell consists of the inward-rectifier K+ channel Kir6.2 (KCNJ11) and the modulatory subunit sulphonylurea receptor, SUR1 (ABCC8). Sulphonylureas, a major class of antidiabetic drugs, are generally believed to stimulate insulin secretion by acting on SUR1 and thereby indirectly suppressing Kir6.2 activity. Conceptually, Kir6.2 itself should then represent a more direct, and potentially effective antidiabetic drug target. It is noteworthy that several studies suggest that SUR1 also plays other roles besides inhibiting Kir6.2, such as interacting with syntaxin, a key protein involved in exocytosis. Furthermore, the sulphonylurea glibenclamide appears to promote exocytosis of insulin also by KATP-independent mechanisms. Additionally, in the past decade, many gain-of-function mutations of Kir6.2 or SUR1 (lowering KATP's ATP sensitivity) were found to underlie most cases of permanent neonatal diabetes mellitus (PNDM). Most of these patients have since been successfully treated with sulphonylureas, instead of the traditional insulin therapy. However, some PNDM patients with certain mutations are unresponsive to sulphonylureas. In principle, these patients might be treatable with a suitable Kir6.2 inhibitor. Unfortunately, to date, there are no Kir6.2 inhibitors available. To obtain experimental evidence for judging, on scientific grounds, if serious resources should be invested in developing a clinically useful Kir6.2 inhibitor to treat diabetes, we propose to: i. develop a prototype specific Kir6.2 inhibitor and thereby demonstrate the feasibility of such an endeavor, ii. investigate whether a Kir6.2 inhibitor can stimulate adequate insulin secretion, and iii. apply this inhibitor to test the hypothesis that a sulphonylurea stimulates insulin secretion also via KATP-independent mechanisms. If this hypothesis is correct, a Kir6.2 inhibitor may then not have all the side effects of sulphonylureas. We will perform our studies with a combination of electrophysiology, Ca2+ imaging, protein biochemistry, molecular biology, cell biology, and histology. Our proposal constitutes an innovative, proof-of-concept study of a novel antidiabetic strategy; its significance is underscored by the fact that 350 million people worldwide have diabetes, a growing pandemic that calls for the development of new, effective therapeutic strategies.