As previously reported, the activity of the large-conductance calcium (Ca(2+))-activated potassium (K(+)) (BK(Ca)) channel is strongly potentiated from the extracellular side of the cell membrane by certain benzofuroindole derivatives. Here, the mechanism of action of one of the most potent activators, 4-chloro-7-(trifluoromethyl)-10H-benzofuro[3,2-b]indole-1-carboxylic acid (CTBIC), is characterized. This compound, Compound 22 in the previous report (Chembiochem 6:1745-1748, 2005), potentiated the activity of the channel by shifting its conductance-voltage relationship toward the more negative direction. Cotreatment with CTBIC reduced the affinity of charybdotoxin, a peptide pore-blocker, whereas that of tetraethylammonium, a small pore-blocking quaternary ammonium, was not significantly altered. Guided by these results, scanning mutagenesis of the outer vestibule of the BK(Ca) channel was launched to uncover the molecular determinants that affect CTBIC binding. Alanine substitution of several amino acid residues in the turret region and the S6 helix of the channel decreased potentiation by CTBIC. Homology modeling and molecular dynamics simulation showed that some of these residues formed a CTBIC binding pocket between two adjacent α-subunits in the outer vestibule of the channel. Thus, it can be envisioned that benzofuroindole derivatives stabilize the open conformation of the channel by binding to the residues clustered across the extracellular part of the subunit interface. The present results indicate that the interface between different α-subunits of the BK(Ca) channel may play a critical role in the modulation of channel activity. Therefore, this interface represents a potential therapeutic target site for the regulation of K(+) channels.