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

描述(申请人提供)：在2型糖尿病(T2 DM)患者和该疾病的动物模型中，胰岛素和胰高血糖素的分泌受到干扰，部分原因是胰岛细胞膜电位(�？p)和钙离子进入的扰动。虽然调节兴奋性的离子通道是钙内流的关键调节器，但我们对葡萄糖刺激时动作电位激发处稳定�？p的背景钾电流的理解存在差距。这个缺口的存在代表了一个重要的问题，因为在填补这个缺口之前，我们对胰高血糖素和胰岛素分泌的了解是不完整的，这限制了可用于治疗血糖紊乱的治疗靶点。这项研究的长期目标是在“泄漏”两孔区域(K2P)钾通道活性的背景下了解生理和病理生理上的胰岛激素分泌。这项建议的总体目标是阐明通过K2P通道调节胰岛钙内流和激素分泌的分子机制，这是实现长期目标的下一步。本项目将验证这一中心假设，即K2P通道调节胰岛细胞膜电位(�？P)，从而调节钙内流和激素分泌。这一假说是根据初步数据提出的，发现K2P通道TASK-1和TASK-3调节葡萄糖刺激的�细胞�？P、钙内流和胰岛素分泌。进一步的数据发现，K2P通道TREK-2在胰岛�细胞中表达，在那里它调节电活动和胰高血糖素的分泌。这项研究的理论基础是，了解胰岛细胞K2P通道活动是如何影响血糖的，将揭示治疗糖尿病的新靶点。在强大的初步数据的指导下，这一假说将通过追求两个具体目标来检验：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�细胞兴奋性和胰升糖素分泌方面所起的作用。在第一个目标下，利用已有的TASK-1和TASK-3的可诱导�细胞消融的MIE来测试这些通道对小鼠血糖稳态的影响。人类�细胞任务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，这些方法已经在申请者手中被确定为可行的。最后，在用高脂饮食治疗的动物中，将评估任务通道对应激下�细胞功能的影响。在第二个目标下，利用手头上缺乏Trek-2的�细胞表达荧光蛋白的转基因小鼠，将被用来评估这些通道在�细胞胰升糖素分泌过程中的作用。已经被验证的�细胞特异性D/N方法也将被用于评估人类�细胞TREK-2通道的功能。这项拟议的研究意义重大，因为它是一系列研究的第一步，预计将导致调节胰岛素和胰升糖素分泌的药物策略；这对于发现治疗T2 DM和高胰岛素血症等疾病的血糖紊乱的疗法至关重要。

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.