CIC-3 chloride ion channels in vascular smooth muscle

氯(CI)电流参与激动剂诱导的血管平滑肌细胞去极化。作为最初拨款方案的一部分，我们创造了一种缺乏特定氯离子通道的小鼠，CIC-3(由CIcn3基因编码)。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、左室肥厚和舒张期功能障碍，以及阻力血管内皮依赖的松弛受损；2)CIC-3通道位于静止的VSM细胞内，但作为对ALL的响应被插入质膜；3)通过选择性剪接可能改变膜的运输，导致CIC-3蛋白结构的未知多样性，4)Clcn3-/-细胞内活性氧(ROS)水平增加，以及5)超氧阴离子可能通过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了对细胞内通道定位和在细胞间移动通道的机制缺乏深入了解。我们将首先仔细界定CIC-3的本地化和贩运。然后，我们将测试这一假设，即在Clcn3-/-小鼠中观察到的微血管功能的改变与缺乏电导有关，而电导通常提供超氧阴离子通过生物膜移动的机制。在这一新的应用中，我们将：1)确定天然CIC-3蛋白在小鼠VSM中的亚细胞定位，并确定响应血管紧张素II而调节CIC-3向质膜转运的因子；2)通过FIV驱动的重组CIC-3蛋白的表达，定义CIC-3在VSM细胞中的六种不同剪接变体的亚细胞定位，并确定调控CIC-3膜转运的基序和生理因素；以及3)确定为什么在Clcn3-/-细胞和组织中细胞内ROS水平升高，并区分这种增加是否与生理相关。CIC-3基因敲除小鼠代表了一种新的高血压单基因缺陷模型。仔细分析Clcn3-/-小鼠的生理缺陷将对细胞ROS代谢以及ROS与高血压之间的联系产生重要的见解。

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.