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 b1 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 a subunit (Slo1) in a dual way via novel mechanisms: i. constitutive activation (as a regulatory b subunit?), and ii. agonist-induced inhibition in a G-protein independent manner, 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 new hypothesis that the TPR can act as a positive regulatory subunit of Slo1 in a constitutive manner and that agonist stimulation switches the TPR-induced modulatory action on the channel from activatory to inhibitory. Parallel experiments in model and native systems will link molecular mechanisms to functional consequences resulting from the tripartite interaction among TPR, Slo1 and its b1 subunit.
The specific aims are to: 1. Determine the mechanism(s) underlying constitutive TPR positive modulation of Slo1 channel activity, 2. Investigate the role of b1 in and physiological consequences of TPR-Slo1 constitutive activation, 3. Unravel the molecular mechanism(s) of Slo1 channel inhibition by agonist (U46619)- activated TPR, and 4. Define the role and functional consequences of b1 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.
Thromboxane A2-prostanoid receptors (TPR) and the large conductance Ca2+-activated K+ channel (MaxiK, BK) are proteins involved in the regulation of vascular tone and have been implicated in the development of (i.e. TPR) or protection against (i.e. MaxiK) cardiovascular diseases like hypertension, heart attack and stroke. Our discovery that the 1 (Slo1) and b1 subunits of MaxiK interact with TPR and the studies proposed here will set the basis to understand in detail vasoconstricting mechanisms that afflict cardiovascular function, which is crucial to ultimately design new therapeutics targeting cardiovascular disease.
