Martentoxin as a 37-residue peptide was capable of blocking large-conductance Ca(2+)-activated K(+) (BK) channels in adrenal medulla chromaffin cells. This study investigated the pharmacological discrimination of martentoxin on BK channel subtypes. The results showed that the iberiotoxin-insensitive neuronal BK channels (alpha+beta4) could be potently blocked by martentoxin (IC(50) = approximately 80 nM). In contrast, the iberiotoxin-sensitive BK channel consisting of only alpha-subunit was less sensitive to martentoxin. Distinctively, martentoxin inhibited neuronal BK channels (alpha+beta4) with a novel interaction mode. Two possible interaction sites of neuronal BK channels (alpha+beta4) might be responsible for the binding with martentoxin: one for trapping and the other located at the pore region for blocking. In addition, the inhibition of martentoxin on neuronal BK channels (alpha+beta4) depended on cytoplasmic Ca(2+) concentration. On the other hand, in vivo experiments from EEG recordings suggested that neuronal BK channels (alpha+beta4) were the primary target of martentoxin. Therefore, this research not only sheds light on a unique ligand for neuronal BK channels (alpha+beta4), but also highlights a novel model approach for the interaction between K(+) channels and specific-ligands.