CIC-3 chloride ion channels in vascular smooth muscle

氯（Cl）电流有助于激动剂诱导的血管平滑肌（VSM）细胞的去极化。 作为原始拨款提案的一部分，本申请是一个延续，我们创造了一个缺乏特定氯离子通道的小鼠，CIC-3（由CIcn 3基因编码）。 本实验室的五项发现证实了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，但响应于A11而插入质膜，3）存在由可能改变膜运输的可变剪接导致的CIC-3蛋白结构的未被认识到的多样性，4）Clcn 3-/-细胞具有增加的细胞内活性氧物质（ROS）水平，5）超氧阴离子可通过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。 这些不一致性可能反映了缺乏深入的知识，在细胞内的通道定位和机制，移动细胞区室之间的通道。 我们将首先仔细定义CIC-3的本地化和贩运。 然后，我们将测试的假设，即在Clcn 3-/-小鼠中观察到的微血管功能的改变是有关的电导，通常提供了一种机制，超氧阴离子移动跨生物膜的情况下。 在此更新申请中，我们将：1）确定天然CIC-3蛋白在鼠VSM中的亚细胞定位，并鉴定响应于血管紧张素II调节CIC-3向质膜运输的因子，2）使用重组CIC-3的FIV驱动的表达，确定CIC-3的六种不同剪接变体在VSM细胞中的亚细胞定位。3）确定细胞内ROS水平在Clcn 3-/-细胞和组织中升高的原因，并辨别这种升高是否是生理学相关的。 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Clcn 3-/-小鼠的生理缺陷将产生对细胞ROS代谢以及ROS与高血压之间的联系的重要见解。

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.