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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.

Lithocholate (LC) (10-300 mM) in physiological solution is sensed by vascular myocyte large conductance, calcium-and voltage-gated potassium (BK) channel b 1 accessory subunits, leading to channel activation and arterial dilation. However, the structural features in steroid and target that determine LC action are unknown. We tested LC and close analogs on BK channel (pore-forming cbv11b 1 subunits) activity using the product of the number of functional ion channels in the membrane patch (N) and the open channel probability (Po). LC (5b-cholanic acid-3a-ol), 5a-cholanic acid-3a-ol, and 5b-cholanic acid-3b-ol increased NPo (EC 50 ?45 mM). At maximal increase in NPo, LC increased NPo by 180%, whereas 5a-cholanic acid-3a-ol and 5b-cholanic acid-3b-ol raised NPo by 40%. Thus, the a-hydroxyl and the cis A-B ring junction are both required for robust channel potentiation. Lacking both features, 5a-cholanic acid-3b-ol and 5-cholenic acid-3b-ol were inactive. Three-dimensional structures show that only LC displays a bean shape with clear-cut convex and concave hemispheres; 5a-cholanic acid-3a-ol and 5b-cholanic acid-3b-ol partially matched LC shape, and 5a-cholanic acid-3b-ol and 5-cholenic acid-3b-ol did not. Increasing polarity in steroid rings (5b-cholanic acid-3a-sulfate) or reducing polarity in lateral chain (5b-cholanic acid 3a-ol methyl ester) rendered poorly active compounds, consistent with steroid insertion between b 1 and bilayer lipids, with the steroid-charged tail near the aqueous phase. Molecular dynamics identified two regions in b 1 transmembrane domain 2 that meet unique requirements for bonding with the LC concave hemisphere, where the steroid functional groups are located.

Background: BK channels regulate smooth muscle contractility and provide docking site for steroids, such as bile acids. Results: Non-steroid leukotriene B4 activates the BK channel via a steroid-sensing site. Conclusion: Physiological lipids from different chemical families share their recognition site in BK proteins. Significance: Our data underscore a likely cross-talk between different physiological lipids in BK channel-driven pathophysiology.

Large conductance, voltage- and Ca2+-gated K+ (BKCa) channels play a critical role in smooth muscle contractility and thus represent an emerging therapeutic target for drug development to treat vascular disease, gastrointestinal, bladder and uterine disorders. Several compounds are known to target the ubiquitously expressed BKCa channel-forming α subunit. In contrast, just a few are known to target the BKCa modulatory β1 subunit, which is highly expressed in smooth muscle and scarce in most other tissues. Lack of available high-resolution structural data makes structure-based pharmacophore modeling of β1 subunit-dependent BKCa channel activators a major challenge. Following recent discoveries of novel BKCa channel activators that act via β1 subunit recognition, we performed ligand-based pharmacophore modeling that led to the successful creation and fine-tuning of a pharmacophore over several generations. Initial models were developed using physiologically active cholane steroids (bile acids) as template. However, as more compounds that act on BKCa β1 have been discovered, our model has been refined to improve accuracy. Database searching with our best-performing model has uncovered several novel compounds as candidate BKCa β1 subunit ligands. Eight of the identified compounds were experimentally screened and two proved to be activators of recombinant BKCa β1 complexes. One of these activators, sobetirome, differs substantially in structure from any previously reported activator.

Reactive oxygen species (ROS) are important modulators of excitability and may play a critical role in the etiology of many neurodegenerative disorders. Previous studies have shown that the fast N-type inactivation of Kv1.4 channels is suppressed by ROS-mediated cysteine oxidation whereas Kv4 channel N-type inactivation is not. However, in native neurons Kv4 channel subunits form large macromolecular complexes with KChIP and DPL proteins, and fast N-type inactivation is conferred to Kv4 channel complexes by a specific isoform of DPL proteins, either DPP10a or DPP6a. To investigate whether the DPP6a-mediated fast inactivation is regulated by ROS, tert-butyl hydroperoxide (tBHP) was applied to oocytes expressing Kv4.2þKChIP3aþDPP6a channels. tBHP (1 mM) application dramatically increases the peak current amplitude by~44% and slowed inactivation kinetics. The effects of tBHP are reversed by the reducing agent dithiothreitol (DTT, 10 mM). In contrast, ternary complex channels containing another DPP6 isoform (DPP6K) are not affected by tBHP, indicating the importance of the DPP6a variable N-terminal domain for the tBHP effect. Alignment of N-terminal sequences from DPP6a and DPP10a with Kv1.4 reveals a common cysteine residue in position 13 (Cys-13), which in Kv1.4 is critical for the redox-regulation of N-type inactivation. Substituting Cys-13 of DPP6a with serine (DPP6a/C13S) results in a loss of regulation by tBHP, consistent with a similar critical role for DPP6a Cys-13. To test if other cysteines in the channel are also required for this regulation, we switched to mutant Kv4.1 (C11xA) and KChIP3a (KChIP3a/del2-59) constructs that remove most intracellular cysteine residues. Channels composed of C11xA, KChIP3a/del2-59, and normal DPP6a show disrupted regulation by tBHP, suggesting that ROS likely induces an intersubunit disulfide linkage to regulate DPP6a-mediated N-type inactivation.

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