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

我们的长期目标是获得血栓素A2- 前列腺素受体（TPR）和大电导Ca ~（2+）激活的K ~+ 通道（MaxiK，BK）相互作用 来调节血管功能TPR和MaxiK在血管生成中起重要作用， 健康此外，已知这两种蛋白质都参与肿瘤发生和心肌细胞凋亡的调节。 梗塞在冠状动脉中，TPR被前列腺素类血栓烷A2（TXA 2）激活，导致 强有力的血管收缩;而MaxiK通道在其β1亚基的帮助下可以微调动脉张力， 渠道的活跃程度。我们的前期工作表明TXA 2模拟物U46119抑制MaxiK通道， 冠状动脉平滑肌膜在脂质双层中重建的活性，这表明 TPR和MaxiK之间的功能关联经受分离重建程序。 最近的初步实验还表明：1）TPR通过以下途径调节MaxiK成孔α亚基（Slo 1） 一种不依赖于G蛋白的新机制，其中MaxiK通道活性可以通过 特异性TPR激动剂U46619，2）TPR和MaxiK通道亚基在细胞内形成异多聚体复合物， 天然动脉和表达系统中，3）TPR和MaxiK通道功能偶联发生在天然动脉中， 人冠状动脉心肌细胞，并可在TPR和Slo 1异位表达后复制， 和4）激动剂刺激增强TPR和Slo 1缔合。在这里，我们将测试假设， 激动剂刺激改变TPR-Slo 1-β1缔合，导致Slo 1通道抑制。具体目标 1.揭示激动剂（U46619）激活的Slo 1通道抑制的分子机制 TPR和2.明确β1亚基在激动剂-TPR-Slo 1通道抑制中的作用。实验将 使用现代方法如生物化学、分子生物学和光生物物理方法进行 包括衍射极限的荧光显微术。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研究哺乳动物生理学提供新的机会。 不仅预防心血管系统的疾病，也预防其他系统的疾病。

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.