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

The large-conductance calcium-activated potassium (BK_Ca) 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 BK_Ca 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 BK_Ca 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 BK_Ca-related pathophysiological conditions.