Cerebrovascular Dilation via Selective Targeting of the Cholane Steroid-Recognition Site in the BK Channel β1-Subunit by a Novel Nonsteroidal Agent

Abstract

The Ca 21 /voltage-gated K 1 large conductance (BK) channel b1 subunit is particularly abundant in vascular smooth muscle. By determining their phenotype, BK b1 allows the BK channels to reduce myogenic tone, facilitating vasodilation. The endogenous steroid lithocholic acid (LCA) dilates cerebral arteries via BK channel activation, which requires recognition by a BK b1 site that includes Thr169. Whether exogenous nonsteroidal agents can access this site to selectively activate b1-containing BK channels and evoke vasodilation remain unknown. We performed a chemical structure database similarity search using LCA as a template, along with a two-step reaction to generate sodium 3-hydroxyolean-12-en-30-oate (HENA). HENA activated the BK (cbv1 1 b1) channels cloned from rat cerebral artery myocytes with a potency (EC 50 5 53 mM) similar to and an efficacy (Â2.5 potentiation) significantly greater than that of LCA.This HENA action was replicated on native channels in rat cerebral artery myocytes. HENA failed to activate the channels made of cbv1 1 b2, b3, b4, or b1T169A, indicating that this drug selectively targets b1-containing BK channels via the BK b1 steroid-sensing site. HENA (3-45 mM) dilated the rat and C57BL/ 6 mouse pressurized cerebral arteries. Consistent with the electrophysiologic results, this effect was larger than that of LCA. HENA failed to dilate the arteries from the KCNMB1 knockout mouse, underscoring BK b1's role in HENA action. Finally, carotid artery-infusion of HENA (45 mM) dilated the pial cerebral arterioles via selective BK-channel targeting. In conclusion, we have identified for the first time a nonsteroidal agent that selectively activates b1-containing BK channels by targeting the steroid-sensing site in BK b1, rendering vasodilation.

Endothelial Nitric Oxide Mediates Caffeine Antagonism of Alcohol-Induced Cerebral Artery Constriction

“…Rats and mice were decapitated using a guillotine and sharp scissors, respectively. Middle cerebral arteries (,260 mm and ,150 mm external diameter in rat and mouse species, respectively) were isolated and cannulated as described in our earlier work (Bukiya et al, 2013). When required, endothelium was removed by passing an air bubble into the vessel lumen for 90 seconds before vessel cannulation.…”

“…In all experiments where ethanol in solution was applied to tissues, solutions with urea isosmotically replacing for ethanol were used as control solution (Liu et al, 2004). Pial arteriolar diameter was monitored with a video micrometer coupled to a television camera that depicted the brain surface through the cranial window (Bukiya et al, 2013).…”

Multi-generational pharmacophore modeling for ligands to the cholane steroid-recognition site in the β1 modulatory subunit of the BKCa channel

“…[13, 14] Cholane steroids such as the bile acid, lithocholic acid (LCA), increase BK Ca channel activity through interaction with β 1 subunits. [14] This action is β 1 subunit-specific and not observed with β 2-4 subunit-containing BK Ca complexes or with homomeric α channels.…”

“…[16] Previous structure-activity relationship (SAR) studies indicated that the overall shape of the bile acid, as well as the number of hydroxyls, and placement of hydroxyls and carboxylic acids, were important in bile acid interaction with β 1. [14] Among non-steroidal compounds, only one triterpenoid, 3-hydroxyolean-12-en-30-oic acid (3-HENA)[13] has been reported to specifically target β 1 -containing BK Ca complexes. Moreover, although there are several ligands with β 1 -dependent effects on BK Ca channel opening, only LCA and 3-HENA have been reported to interact with a defined recognition site [13] making the other ligands less useful in determination of a pharmacophore.…”

Activation of Calcium- and Voltage-gated Potassium Channels of Large Conductance by Leukotriene B4

“…T169A has been proven to serve as a screening tool to identify possible ligands for the BK ␤1 TM2 domain steroidsensing site (12,30). Successful expression of ␤1T169A and its presence within the channel complexes were validated by detecting the ␤1 subunit-characteristic phenotype of cbv1-␤1T169A macroscopic current (12).…”