K+ Channel Expression in Pancreatic Beta-Cells

描述(由申请人提供)：这项修订提案的总体目标是阐明正常和糖尿病状态下细胞的电兴奋性与葡萄糖诱导的胰岛素分泌(GSIS)耦合的分子机制。我们试图验证有关电压依赖性K+(Kv)通道在调节电活动中的作用以及触发GSIS的细胞内游离钙离子浓度([Ca~(2+)]i)变化的假设。一旦代谢导致产生动作电位(AP)的KATP通道关闭，Kv通道在重新极化细胞膜方面起着明显的作用，导致胰岛素分泌所需的钙瞬变。用于治疗糖尿病的胰岛素激动剂通过依赖PKA的机制减少Kv电流，但所涉及的Kv通道的身份仍不明确。Kv通道Kv2.1是细胞内主要的Kv通道，被认为是细胞膜复极化的关键通道。我们发现，Kv2.1-/-小鼠，一种新的基因敲除模型，表现出异常的葡萄糖稳态，伴随着显著的静息性低血糖和胰岛素分泌增加，以响应葡萄糖浓度的生理步骤。胰岛具有宽的异常动作电位(AP)。令人惊讶的是，Kv2.1-/-胰岛仍然对Kv通道和钙激活K+通道(Kca)的阻断剂四乙基铵敏感。这些结果表明，其他K+通道参与了膜复极化和AP的产生，可能是调控的靶点。我们建议在野生型小鼠模型和Kv2.1-/-小鼠模型中研究这些以前未被研究过的Kv电流的特性，目的如下：1.明确Kv通道调节胰岛素分泌和钙信号的机制。目的2.明确在细胞内表达的复极化K+通道的分子同一性，并了解它们在细胞兴奋-分泌偶联中的作用。这些研究的结果将加深我们对Kv通道在胰岛素分泌中的重要性以及它们在糖尿病发病机制和潜在治疗中的作用的理解。公共卫生相关性：糖尿病是一个重要的健康问题，由胰岛素分泌异常引起的胰岛素抵抗程度导致众多并发症。这个项目涉及胰岛素分泌调节的重要生物物理方面，重点是钾离子进出细胞如何控制胰岛素分泌。

DESCRIPTION (provided by applicant): The overall objective of this revised proposal is to elucidate the molecular mechanisms coupling electrical excitability of ¿-cells to glucose-induced insulin secretion (GSIS) in normal and diabetic states. We seek to test hypotheses concerning the role of voltage- dependent K+ (Kv) channels in regulating electrical activity and changes in intracellular free Ca2+ concentration ([Ca2+]i) that triggers GSIS. Once metabolism leads to closure of KATP channels generating action potentials (APs), Kv channels serve a distinct role in repolarizing the ¿-cell membrane, resulting in Ca2+ transients necessary for insulin secretion. Incretin agonists used to treat diabetes reduce Kv currents by a PKA- dependent mechanism, but the identity of the Kv channels involved remains undefined. The Kv channel Kv2.1 is the predominant Kv channel in ¿-cells, thought to be a critical channel for ¿-cell membrane repolarization. We found that Kv2.1-/- mice, a new knockout model, exhibit abnormal glucose homeostasis with a significant resting hypoglycemia and increased insulin secretion in response to physiological steps in glucose concentration. The islets have wide and aberrant action potentials (APs). Surprisingly the Kv2.1-/- islets remain sensitive to tetraethylammonium, a blocker of Kv channels and Ca2+-activated K+ channels (KCa). These results reveal that other K+ channels participate in membrane repolarization and generation of APs, and could be targets for regulation. We propose to study the properties of these Kv currents not previously studied in normal mouse models in wild type and Kv2.1-/- mice with the following two specific aims: Aim 1. To define mechanisms underlying regulation of insulin secretion and calcium signaling by Kv channels. Aim 2. To define the molecular identity of repolarizing K+ channels expressed in ¿-cells and understand the role they play in ¿-cell excitation-secretion coupling. The results of these studies will enhance our understanding of the importance of Kv channels in insulin secretion and their role in the pathogenesis and potential treatment of diabetes. PUBLIC HEALTH RELEVANCE: Diabetes Mellitus is an important health problem, caused by abnormal insulin secretion relative to the degree of insulin resistance leading to numerous complications. This project addresses important biophysical aspects of the regulation of insulin secretion focusing on how potassium ion movement in and out of the cell controls insulin secretion.