The neurotoxic cone snail peptide mu-GIIIA specifically blocks skeletal muscle voltage-gated sodium (Na(V)1.4) channels. The related conopeptides mu-PIIIA and mu-SIIIA, however, exhibit a wider activity spectrum by also inhibiting the neuronal Na-V channels Na-V 1.2 and Na-V 1.7. Here we demonstrate that those mu-conopeptides with a broader target range also antagonize select subtypes of voltage-gated potassium channels of the K(v)1 family: mu-PIIIA and mu-SIIIA inhibited K(V)1.1 and K(V)1.6 channels in the nanomolar range, while being inactive on subtypes K(V)1.2-1.5 and K(V)2.1. Construction and electro-physiological evaluation of chimeras between K(V)1.5 and K(V)1.6 revealed that these toxins block K-V channels involving their pore regions; the subtype specificity is determined in part by the sequence close to the selectivity filter but predominantly by the so-called turret domain, i.e. the extracellular loop connecting the pore with transmembrane segment S5. Conopeptides mu-SIIIA and mu-PIIIA thus, are not specific for Na-V channels, and the known structure of some K-V channel subtypes may provide access to structural insight into the molecular interaction between-conopeptides and their target channels. (C) 2016 Elsevier Inc. All rights reserved.