Previous data from our laboratory demonstrated that acute exposure to cholane derivatives (bile acids and synthetic analogs) increases the activity of large conductance, Ca2+-sensitive K+ (BK) channels in arterial smooth muscle. Activation of smooth muscle BK channels is thought to be a major mechanism underlying vasodilation caused by not only bile acid derivatives, but also other physiologically relevant steroids, such as estradiol, progesterone, and androgens. In contrast, others and we found that increased cholesterol levels lead to smooth muscle BK channel inhibition. The long-term goal of this proposal is to identify both the structural determinants in the steroid molecule and the channel protein regions that are responsible for steroid activation of BK channels. Combining methodologies from Electrophysiology (patch-clamp), Molecular Biology, and Pharmacology, we will specifically address: 1) which chemical determinants in the cholane derivative molecule are critical for activating smooth muscle BK channels; 2) which channel subunit (alpha vs. beta) is necessary/sufficient for cholane derivative action; 3) which protein regions in the channel subunits are responsible for conferring cholane sensitivity to the ion channel complex; 4) whether modulation of BK channel activity by cholane derivatives underlies smooth muscle relaxation caused by these steroids. We will answer these questions going from studies in isolated arteries to evaluate drug action on tissue physiology to studies that used engineered ion channel subunits expressed in Xenopus oocytes to address the more mechanistic aspects of the application. We will initially use the rat cerebral artery to freshly isolate both a resistive artery segment to measure changes in tone caused by steroids, and dissociated myocytes to probe steroid modulation of native BK channels. The same overall strategy can be applied to other relevant vessels in the future. We chose the rat cerebral artery/myocyte model because: a) the key role of BK channel activity as regulator of arterial tone was clearly demonstrated; b) we have experience studying the effect of modulators on the active/passive tone of isolated rat cerebral arteries; c) we have isolated, cloned and successfully expressed in Xenopus oocytes the pore forming subunit of the rat cerebral artery BK channel; d) the therapeutic need for new cerebral vasodilators that act on the smooth muscle itself, i.e., that can be effective in the absence of intact endothelial function. Pinpointing the molecular mechanisms involved in the steroid-cerebrovascular BK channel interaction will bring fundamental insight for the rationale design of steroidal vasodilators of potential therapeutic use, devoid of hormonal side effects.
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