CIC-3 chloride ion channels in vascular smooth muscle

血管平滑肌中的CIC-3氯离子通道

基本信息

批准号：
7052847
负责人：
FRED S LAMB
金额：
$ 28.81万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-04-01 至 2008-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7052847
关键词：
angiotensin II calmodulin dependent protein kinase cell membrane chloride channels genetically modified animals laboratory mouse membrane activity muscle cells protein localization protein protein interaction protein structure function protein transport recombinant proteins superoxides tissue /cell culture vascular smooth muscle voltage /patch clamp

项目摘要

Chloride (CI) currents contribute to agonist-induced depolarization of vascular smooth muscle (VSM) cells. As part of the original grant proposal for which this application is a continuation, we created a mouse lacking a specific chloride channel, CIC-3 (encoded by the CIcn3 gene). Five findings from our laboratory demonstrate the importance of CIC-3 to cardiovascular function: 1) Animals lacking CIC-3 CI channels display multiple cardiovascular abnormalities including; hypertension, left ventricular hypertrophy and diastolic dysfunction, and impaired endothelium-dependent relaxation in resistance vessels, 2) CIC-3 channels are located intracellularly in resting VSM cells, but are inserted into the plasma membrane in response to All, 3) there is an unappreciated diversity of CIC-3 protein structure resulting from alternative splicing that may alter membrane trafficking, 4) Clcn3-/- cells have increased levels of intracellular reactive oxygen species (ROS), and 5) superoxide anion may pass through CIC-3 channels. There is conflicting data in the literature as to the biophysical nature of CIC-3. These inconsistencies may reflect a lack of in-depth knowledge of channel localization within the cell and the mechanisms that move the channel between cellular compartments. We will first carefully define the localization and trafficking of CIC-3. We will then test the hypothesis that the altered microvascular function observed in Clcn3-/- mice is related to the absence of a conductance that normally provides a mechanism by which superoxide anion moves across biological membranes. In this renewal application, we will; 1) Define the subcellular localization of native CIC-3 protein in murine VSM and identify factors that regulate the trafficking of CIC-3 to the plasma membrane in response to angiotensin II, 2) Define the subcellular localization of the six distinct splice variants of CIC-3 in VSM cells using FIV-driven expression of recombinant CIC-3 protein and identify motifs and physiologic factors that regulate membrane trafficking of CIC-3, and 3) Determine why intracellular ROS levels are elevated in Clcn3-/- cells and tissues and discern if this increase is physiologically relevant. The CIC-3 knockout mouse represents a novel single-gene defect model of hypertension. Careful analysis of the physiological defects in Clcn3-/- mice will yield important insight into cellular ROS metabolism and the link between ROS and high blood pressure.

氯化物 (CI) 电流有助于激动剂诱导的血管平滑肌 (VSM) 细胞去极化。作为本申请的延续的原始资助提案的一部分，我们创造了一种缺乏特定氯离子通道 CIC-3（由 CIcn3 基因编码）的小鼠。我们实验室的五项发现证明了 CIC-3 对心血管功能的重要性：1) 缺乏 CIC-3 CI 通道的动物表现出多种心血管异常，包括；高血压、左心室肥大和舒张功能障碍，以及阻力血管中内皮依赖性舒张受损，2) CIC-3 通道位于静息 VSM 细胞的细胞内，但插入质膜以响应所有，3)选择性剪接导致的 CIC-3 蛋白质结构多样性未被充分认识，可能会改变膜运输，4) Clcn3-/- 细胞的细胞内活性氧水平增加(ROS)和5)超氧阴离子可以通过CIC-3通道。关于 CIC-3 的生物物理性质，文献中存在相互矛盾的数据。这些不一致可能反映出缺乏对细胞内通道定位以及在细胞区室之间移动通道的机制的深入了解。我们将首先仔细定义 CIC-3 的本地化和贩运。然后，我们将测试以下假设：在 Clcn3-/- 小鼠中观察到的微血管功能改变与电导的缺失有关，而电导通常提供超氧阴离子跨生物膜移动的机制。在此续签申请中，我们将； 1) 定义小鼠 VSM 中天然 CIC-3 蛋白的亚细胞定位，并确定响应血管紧张素 II 调节 CIC-3 运输至质膜的因子，2) 定义 CIC 六种不同剪接变体的亚细胞定位-3 在 VSM 细胞中使用 FIV 驱动的重组 CIC-3 蛋白表达，并识别调节 CIC-3 膜运输的基序和生理因素，以及 3) 确定细胞内 ROS 水平的原因在 Clcn3-/- 细胞和组织中升高，并辨别这种升高是否具有生理相关性。 CIC-3 基因敲除小鼠代表了一种新型的单基因缺陷高血压模型。仔细分析 Clcn3-/- 小鼠的生理缺陷将有助于深入了解细胞 ROS 代谢以及 ROS 与高血压之间的联系。