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

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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.