Novel interactions of Slo1 channel and Thromboxane A2 receptor in blood vessels

我们的长期目标是对血栓素A2- 前列腺素受体(TPR)与大电导钙激活K+ 渠道(Maxik、BK)互动 来调节血管功能。TPR和MAXIK在血管检测中的重要作用 健康。此外，已知这两种蛋白都参与了肿瘤发生和心肌梗死的调节。 脑梗塞。在冠状动脉中，TPR被前列腺素A2(TXA2)激活，导致 强大的血管收缩；而MAXIK通道在其β1亚单位的辅助下可以微调所确定的动脉张力 由通道活跃度决定。我们早期的工作表明，TXA2模拟物U46119抑制Maxik通道 在脂质双层中重组的冠脉平滑肌膜的活性，这表明 TPR和Maxik之间的功能关联经历了解离重建过程。 最新的初步实验还表明：1)Tpr通过以下途径调控Maxik成孔α亚基(Slo1) 一种不依赖G蛋白的新机制，其中Maxik通道的活性可以通过 特异性TPR激动剂U46619，2)TPR与Maxik通道亚基形成异多聚体复合体 在天然动脉和表达系统中，3)TPR和Maxik通道功能偶联在天然动脉中发生 人冠状动脉肌细胞，在异位表达TPR和sO11后可复制， (4)激动剂刺激可增强TPR和SL1的关联性。在这里，我们将检验假设 激动剂刺激改变Tpr-slo1-β1关联，导致slo1通道抑制。具体目标 1.揭示激动剂(U46619)激活抑制sL1通道的分子机制(S) 2.明确β1亚基在激动剂-TpR-slo1通道抑制中的作用。实验将是 使用现代方法，如生物化学、分子生物学和光生物物理方法 包括衍射极限处的荧光显微镜。由于Maxik和TPR在 哺乳动物生理学，从这些研究中获得的知识可能为 不仅在心血管系统而且在其他系统中预防疾病。

Our long term goal is to obtain an integral view of the mechanisms by which Thromboxane A2- prostanoid receptor (TPR) and the large conductance Ca2+-activated K+ channel (MaxiK, BK) interact with each other to regulate vascular function. TPR and MaxiK play significant roles in determining vascular health. In addition, both proteins are known to be involved in the modulation of tumorigenesis and myocardial infarction. In coronary arteries, TPR are activated by the prostanoid thromboxane A2 (TXA2) leading to powerful vasoconstrictions; while MaxiK channel aided by its β1 subunit can fine tune arterial tone determined by the degree of channel activity. Our early work showed that TXA2 mimetic U46119 inhibits MaxiK channel activity in membranes from coronary smooth muscle reconstituted in lipid bilayers, which suggested a strong functional association between both TPR and MaxiK that endured dissociative reconstitution procedures. Recent preliminary experiments also show that: 1) TPR modulates MaxiK pore-forming α subunit (Slo1) via a novel mechanism that is G-protein independent, where MaxiK channel activity can be reduced by the specific TPR agonist U46619, 2) TPR and MaxiK channel subunits form heteromultimeric complexes in native arteries and in expression systems, 3) TPR and MaxiK channel functional coupling occurs in native human coronary arterial myocytes and can be reproduced after ectopic expression of TPR and Slo1, and 4) agonist-stimulation enhances TPR and Slo1 association. Here, we will test the hypothesis that agonist stimulation changes TPR-Slo1-β1 associations resulting in Slo1 channel inhibition. The specific aims are to: 1. Unravel the molecular mechanism(s) of Slo1 channel inhibition by agonist (U46619)- activated TPR, and 2. Define the role of β1 subunit in agonist-TPR-Slo1 channel inhibition. Experiments will be performed using modern approaches such as biochemistry, molecular biology, and opto-biophysical methods including fluorescence microscopy at the diffraction limit. Because of the broad impact of MaxiK and TPR in mammalian physiology, the knowledge derived from these studies may provide new opportunities for the prevention of disease not only in the cardiovascular system but in other systems as well.