Activation mechanism and novel binding sites of the BKCa channel activator CTIBD.

The large-conductance calcium-activated potassium (BKCa) channel, which is crucial for urinary bladder smooth muscle relaxation, is a potential target for overactive bladder treatment. Our prior work unveiled CTIBD as a promising BKCa channel activator, altering V 1/2 and G max This study investigates CTIBD's activation mechanism, revealing its independence from the Ca2+ and membrane voltage sensing of the BKCa channel. Cryo-electron microscopy disclosed that two CTIBD molecules bind to hydrophobic regions on the extracellular side of the lipid bilayer. Key residues (W22, W203, and F266) are important for CTIBD binding, and their replacement with alanine reduces CTIBD-mediated channel activation. The triple-mutant (W22A/W203A/F266A) channel showed the smallest V 1/2 shift with a minimal impact on activation and deactivation kinetics by CTIBD. At the single-channel level, CTIBD treatment was much less effective at increasing P o in the triple mutant, mainly because of a drastically increased dissociation rate compared with the WT. These findings highlight CTIBD's mechanism, offering crucial insights for developing small-molecule treatments for BKCa-related pathophysiological conditions.

Discovery and characterization of a potent activator of the BKCa channel that relives overactive bladder syndrome in rats.

The large-conductance Ca2+-activated K+ channel (BKCa channel) is involved in repolarizing the membrane potential and has a variety of cellular functions. The BKCa channel is highly expressed in bladder smooth muscle and mediates muscle relaxation. Compounds that activate the BKCa channel have therapeutic potential against pathological symptoms associated with the overactivity of bladder smooth muscle. In this regard, we screened a chemical library of 9938 compounds to identify novel BKCa channel activators. A cell-based fluorescence assay identified a structural family of compounds containing a common tricyclic quinazoline ring that activated the BKCa channel. The most potent compound TTQC-1 (7-bromo-N-(3-methylphenyl)-5-oxo-1-thioxo-4,5-dihydro[1,3]thiazolo[3,4-a]quinazoline-3-carboxamide) directly and reversibly activated the macroscopic current of BKCa channels expressed in Xenopus oocytes from both sides of the cellular membrane. TTQC-1 increased the maximum conductance and shifted the half activation voltage to the left. The apparent half-maximal effective concentration and dissociation constant were 2.8 µM and 7.95 µM, respectively. TTQC-1 delayed the kinetics of channel deactivation without affecting channel activation. The activation effects were observed over a wide range of intracellular Ca2+ concentrations and dependent on the co-expression of ß1 and ß4 auxiliary subunits, which are highly expressed in urinary bladder. In the isolated smooth muscle cells of rat urinary bladder, TTQC-1 increased the K+ currents which can be blocked by iberiotoxin. Finally, oral administration of TTQC-1 to hypertensive rats decreased the urination frequency. Therefore, TTQC-1 is a BKCa channel activator with a novel structure that is a potential therapeutic candidate for BKCa channel-related diseases, such as overactive bladder syndrome.

Identification and Characterization of a Novel Large-Conductance Calcium-Activated Potassium Channel Activator, CTIBD, and Its Relaxation Effect on Urinary Bladder Smooth Muscle.

The large-conductance calcium-activated potassium channel (BKCa channel) is expressed on various tissues and is involved in smooth muscle relaxation. The channel is highly expressed on urinary bladder smooth muscle cells and regulates the repolarization phase of the spontaneous action potentials that control muscle contraction. To discover novel chemical activators of the BKCa channel, we screened a chemical library containing 8364 chemical compounds using a cell-based fluorescence assay. A chemical compound containing an isoxazolyl benzene skeleton (compound 1) was identified as a potent activator of the BKCa channel and was structurally optimized through a structure-activity relationship study to obtain 4-(4-(4-chlorophenyl)-3-(trifluoromethyl)isoxazol-5-yl)benzene-1,3-diol (CTIBD). When CTIBD was applied to the treated extracellular side of the channel, the conductance-voltage relationship of the channel shifted toward a negative value, and the maximum conductance increased in a concentration-dependent manner. CTIBD altered the gating kinetics of the channel by dramatically slowing channel closing without effecting channel opening. The effects of CTIBD on bladder muscle relaxation and micturition function were tested in rat tissue and in vivo. CTIBD concentration-dependently reduced acetylcholine-induced contraction of urinary bladder smooth muscle strips. In an acetic acid-induced overactive bladder (OAB) model, intraperitoneal injection of 20 mg/kg CTIBD effectively restored frequent voiding contraction and lowered voiding volume without affecting other bladder function parameters. Thus, our results indicate that CTIBD and its derivatives are novel chemical activators of the bladder BKCa channel and potential candidates for OAB therapeutics. SIGNIFICANCE STATEMENT: The novel BKCa channel activator CTIBD was identified and characterized in this study. CTIBD directly activates the BKCa channel and relaxes urinary bladder smooth muscle of rat, so CTIBD can be a potential candidate for overactive bladder therapeutics.