2-Pore-Domain K+ Channels as Novel Targets for Modulating Islet Hormone Secretion

描述（由申请人提供）：2型糖尿病（T2DM）患者和该疾病的动物模型中胰岛素和胰高血糖素分泌紊乱，部分原因是胰岛细胞膜电位紊乱(？p)和Ca2+进入。虽然调节兴奋性的离子通道是Ca2+内流的关键调节剂，但我们对稳定钙离子的背景钾电流的理解还存在空白。在葡萄糖刺激过程中，动作电位从这里发出。这一差距的存在代表了一个重要的问题，因为在它被填补之前，我们对胰高血糖素和胰岛素分泌的理解是不完整的，这限制了可用于治疗血糖异常的治疗靶点。本研究的长期目标是了解在“泄漏”双孔结构域（K2P）钾通道活性的背景下胰岛激素分泌的生理和病理生理。这一建议的总体目标，这是实现长期目标的下一步，是阐明通过K2P通道调节胰岛Ca2+内流和激素分泌的促分泌剂依赖调节的分子机制。该项目将验证K2P通道调节胰岛细胞膜电位的中心假设。p)，从而调节Ca2+内流和激素分泌。这一假设是根据初步数据制定的，这些数据发现K2P通道TASK-1和TASK-3调节葡萄糖刺激的“细胞”？p， Ca2+内流和胰岛素分泌。进一步的数据发现，K2P通道TREK-2在胰岛细胞中表达，它调节电活动和胰高血糖素分泌。这项研究的基本原理是，了解血糖如何受到胰岛细胞K2P通道活动的影响，将为治疗糖尿病提供新的治疗靶点。在强有力的初步数据的指导下，这一假设将通过追求两个具体目标来验证：1)确定K2P通道对胰岛细胞胰岛素分泌的影响；2)确定K2P通道在限制胰岛细胞兴奋性和胰高血糖素分泌中的作用。在第一个目标下，我们将利用诱导细胞消融已有的TASK-1和task -3来测试这些通道对小鼠葡萄糖稳态的影响。人类细胞TASK通道的功能也将通过特定和有效的药理学和显性阴性（D/N）方法进行评估，这些方法已在申请人手中建立为可行的。最后，将在高脂肪饮食处理的动物中评估应激下TASK通道对细胞功能的影响。在第二个目标下，将利用具有荧光蛋白表达缺乏TREK- 2的-细胞的转基因小鼠来评估这些通道在-细胞胰高血糖素分泌过程中的作用。已经验证的细胞特异性D/N方法也将用于评估人类细胞TREK-2通道的功能。拟议的研究是重要的，因为它是连续研究的第一步，有望导致调节胰岛素和胰高血糖素分泌的药理学策略；这对于发现治疗糖尿病和高胰岛素血症等疾病中血糖异常的疗法至关重要。

DESCRIPTION (provided by applicant): Insulin and glucagon secretion are disrupted in patients with type-2 diabetes mellitus (T2DM) and in animal models of the disease, which is due in part to perturbations in islet-cell membrane potential (�?p) and Ca2+ entry. While ion channels that regulate excitability are key regulators of Ca2+ influx, there is a gap in our understanding of the background potassium currents that stabilize the �?p from where action potentials fire during glucose stimulation. Existence of this gap represents an important problem because, until it is filled, our understanding of glucagon and insulin secretion is incomplete, which limits the therapeutic targets that can be utilized for treating dysglycemia. The long term goal of this research is to understand physiological and pathophysiological islet hormone secretion in the context of "leak" two-pore-domain (K2P) potassium channel activity. The overall objective of this proposal, which is the next step toward attainment of the long term goal, is to elucidate molecular mechanisms regulating secretagogue dependent modulation of islet Ca2+ influx and hormone secretion via K2P channels. This project will test the central hypothesis that K2P channels modulate the islet-cell membrane potential (�?p), thus, regulating Ca2+ influx and hormone secretion. This hypothesis has been formulated from preliminary data that finds that the K2P channels TASK-1 and TASK-3 regulate glucose stimulated �-cell �?p, Ca2+ influx, and insulin secretion. Further data find that the K2P channel TREK-2 is expressed in islet �-cells where it modulates electrical activity and glucagon secretion. The rationale that underlies the proposed research is that understanding how blood glucose is influenced by islet-cell K2P channel activity will expose new therapeutic targets for treating diabetes. Guided by strong preliminary data, this hypothesis will be tested by pursuing two specific aims: 1) Determine the influence of K2P channels on islet �-cell insulin secretion; and 2) Determine the role that K2P channels play in limiting islet �-cell excitability and glucagon secretion. Under the first aim, mie with inducible �-cell ablation of TASK-1 andTASK-3, which are already on hand, will be utilized to test the influences of these channels on mouse glucose homeostasis. The function of human �-cell TASK channels will also be assessed with specific and potent pharmacology and a dominant negative (D/N) approach, which have been established as feasible in the applicants' hands. Finally, the influence of TASK channels on �-cell function under stress will be assessed in animals treated with a high fat diet. Under the second aim, transgenic mice with fluorescent protein expressing �-cells that are deficient for TREK- 2, which are on hand, will be utilized to assess the roles of these channels during �-cell glucagon secretion. An �-cell specific D/N approach, which has been validated, will also be utilized to assess the function of human �- cell TREK-2 channels. The proposed research is significant because it is the first step in a continuum of research that is expected to lead to pharmacological strategies for regulating insulin and glucagon secretion; it is essential for uncovering therapies for treating dysglycemia in diseases such as T2DM and hyperinsulinemia.