Secretagogue induced mechanisms regulating islet electrical activity

描述（由申请人提供）： 这个建议的总体目标是阐明调节促分泌素诱导的β-细胞电活动变化的离子机制及其对胰岛素分泌的影响。胰高血糖素样肽1（GLP-1）刺激胰岛cAMP的产生并增加餐后胰岛素分泌。然而，缺乏GLP-1在调节胰岛电活动和钙波动中的明确作用。我已经确定，GLP-1刺激胰岛导致葡萄糖和/或甲苯磺丁脲（阻断KATP）诱导的β-细胞动作电位（AP）放电频率显著降低，同时增加AP持续时间，这与快速的细胞内钙浓度变化精确相关。与此相反，甲苯磺丁脲诱导的胰岛AP频率响应于葡萄糖而显著增加，这也引起β细胞的瞬时超极化。葡萄糖和GLP-1均增加胰岛cAMP，这是一种通过磷酸化调节肌肉中电压门控钙通道（CaV）的分子。因此，促分泌素可能通过CaV通道的磷酸化来调节胰岛钙波动。葡萄糖还激活来自胰岛β细胞的麻醉敏感性漏钾传导，引起瞬时超极化，其非常类似于任务-1和任务-3钾通道。基于这些初步研究，本计划将探讨两种机制，包括A。L-型钙通道磷酸化和B对胰岛电活动的调节。任务1和任务3通道的葡萄糖依赖性激活及其在调节β-细胞胰岛素分泌中的作用。这将被调查使用：1。- 细胞附着的整个胰岛电流钳记录结合高速钙成像以测量AP的变化和由葡萄糖、GLP-1和磷酸酶调节引起的钙波动。2.针对CaV1.2和CaV1.3的磷酸化序列的磷酸化特异性抗体将与啮齿动物和人β细胞的电压钳实验结合使用，并结合葡萄糖和GLP-1刺激，以解决L型钙通道振幅的变化和从磷酸化调节的失活中的恢复。特异性激酶抑制剂、磷酸酶抑制剂和siRNA也将用于阐明调节CaV1.2和CaV1.3的调节途径。3.一种shRNA靶向方法，用于确定Task-1和Task-3联合收割机是否在α-细胞中组合形成葡萄糖激活的钾通道，以及它们在葡萄糖刺激的胰岛素分泌（GSIS）期间共同和/或单独的作用。特定的基因敲除和糖尿病小鼠模型也将用于所有三个目标。 公共卫生相关性：KATP是正常GSIS的必需离子通道，然而，缺乏KATP的啮齿动物和用KATP阻断剂治疗的糖尿病患者仍然表现出葡萄糖调节的胰岛素分泌。因此，本项目试图定义离子通道，其独立于KATP，也可以帮助调节葡萄糖诱导的钙内流和胰岛素分泌。了解促分泌素如何调节胰岛电活动，而不是KATP，可能有助于开发针对兴奋-分泌途径特定元件的新疗法。

DESCRIPTION (provided by applicant): The overall objective of this proposal is to elucidate ionic mechanisms regulating secretagogue induced changes in (-cell electrical activity and their effect on insulin secretion. Glucagon-like peptide 1 (GLP-1) stimulates islet cAMP production and augments postprandial insulin secretion. However, a clear role for GLP-1 in modulating islet electrical activity and calcium fluctuations is lacking. I have determined that GLP-1 stimulation of islets causes a significant reduction in glucose and/or tolbutamide (to block KATP) induced (-cell action potential (AP) firing frequency while increasing AP duration, which correlates precisely with fast intracellular calcium concentration changes. In contrast, tolbutamide induced islet AP frequency is significantly increased in response to glucose, which also causes a transient hyperpolarization of (-cells. Both glucose and GLP-1 increase islet cAMP, a molecule that regulates voltage-gated calcium channels (CaVs) in muscle through phosphorylation. Therefore, the modulation of islet calcium fluctuations by secretagogues could occur through phosphorylation of CaV channels. Glucose also activates an anesthetic sensitive leak-potassium conductance from islet (-cells, causing a transient hyperpolarization, which closely resembles Task-1 and Task-3 potassium channels. Based on these preliminary studies this proposal will investigate two mechanisms including A. Modulation of islet electrical activity by L-type calcium channel phosphorylation and B. Glucose dependent activation of Task-1 and Task-3 channels and both of their roles in regulating (-cell insulin secretion. This will be investigated using: 1. (-cell-attached whole islet current clamp recordings in combination with high speed calcium imaging to measure changes in APs and calcium fluctuations caused by glucose, GLP-1, and phosphatase regulation. 2. Phospho-specific antibodies directed to phosphorylation sequences of CaV1.2 and CaV1.3 will be employed together with voltage clamp experiments on rodent and human (-cells in combination with glucose and GLP-1 stimulation to address changes in L-type calcium channel amplitude and recovery from inactivation regulated by phosphorylation. Specific kinase inhibitors, phosphatase inhibitors, and siRNA will also be utilized to clarify the regulatory pathways shown to regulate CaV1.2 and CaV1.3. 3. An shRNA targeted approach to determine if Task-1 and Task-3 combine to form a glucose activated potassium channel in (-cells and their role together and/or individually during glucose stimulated insulin secretion (GSIS). Specific knockout and diabetic mouse models will also be employed in all three aims. PUBLIC HEALTH RELEVANCE: KATP is an essential ion channel for normal GSIS, however, rodents lacking KATP and diabetic patients treated with blockers to KATP still exhibit glucose regulated insulin secretion. Therefore this project seeks to define the ion channels, which are independent of KATP, which can also help regulate glucose induced calcium influx and insulin secretion. Understanding how secretagogues regulate islet electrical activity distinctly from KATP may help to develop new therapies that target specific elements of the excitation-secretion pathway.