Activation mechanism and novel binding sites of the BK_Ca channel activator CTIBD

Area of Science:

• Biophysics
• Molecular Pharmacology
• Ion Channel Physiology

Background:

• Large-conductance calcium-activated potassium (BK_Ca) channels are vital for urinary bladder smooth muscle relaxation and are a therapeutic target for overactive bladder.
• CTIBD has been identified as a potential activator of BK_Ca channels.

Purpose of the Study:

• To elucidate the molecular mechanism by which CTIBD activates BK_Ca channels.
• To investigate CTIBD's interaction with the BK_Ca channel and its functional consequences.

Main Methods:

• Cryo-electron microscopy was used to determine the binding site of CTIBD on the BK_Ca channel.
• Site-directed mutagenesis was employed to identify key residues involved in CTIBD binding and activation.
• Single-channel electrophysiology was performed to assess the functional impact of mutations on CTIBD activity.

Main Results:

• CTIBD binds to hydrophobic regions on the extracellular side of the lipid bilayer, independent of the channel's Ca²⁺ and voltage sensing domains.
• Residues W22, W203, and F266 are critical for CTIBD binding, as their mutation to alanine significantly reduced CTIBD-mediated channel activation.
• The triple mutant (W22A/W203A/F266A) exhibited minimal shifts in voltage dependence and altered activation/deactivation kinetics with CTIBD, alongside a drastically increased dissociation rate, reducing CTIBD's efficacy.

Conclusions:

• CTIBD activates BK_Ca channels through a novel mechanism involving direct binding to the lipid bilayer interface, rather than through the canonical Ca²⁺ or voltage sensors.
• The identified key residues provide crucial insights into the CTIBD binding site and mechanism.
• Understanding CTIBD's activation pathway offers a promising avenue for developing targeted small-molecule therapies for BK_Ca-related conditions, such as overactive bladder.