Synthesis of a Biotin Derivative of Iberiotoxin:  Binding Interactions with Streptavidin and the BK Ca²⁺-Activated K⁺ Channel Expressed in a Human Cell Line

Iberiotoxin (IbTx) is a scorpion venom peptide that inhibits BK Ca²⁺-activated K⁺ channels with high affinity and specificity. Automated solid-phase synthesis was used to prepare a biotin-labeled derivative (IbTx−LC-biotin) of IbTx by substitution of Asp19 of the native 37-residue peptide with N-ε-(d-biotin-6-amidocaproate)-l-lysine. Both IbTx−LC-biotin and its complex with streptavidin (StrAv) block single BK channels from rat skeletal muscle with nanomolar affinity, indicating that the biotin-labeled residue, either alone or in complex with StrAv, does not obstruct the toxin binding interaction with the BK channel. IbTx−LC-biotin exhibits high affinity (K_D = 26 nM) and a slow dissociation rate (k_off = 5.4 × 10^-4 s^-1) in a macroscopic blocking assay of whole-cell current of the cloned human BK channel. Titration of IbTx−LC-biotin with StrAv monitored by high performance size exclusion chromatography is consistent with a stoichiometry of two binding sites for IbTx−LC-biotin per StrAv tetramer, indicating that steric interference hinders simultaneous binding of two toxin molecules on each of the two biotin-binding faces of StrAv. In combination with fluorescent conjugates of StrAv or anti-biotin antibody, IbTx−LC-biotin was used to image the surface distribution of BK channels on a transfected cell line. Fluorescence microscopy revealed a patch-like surface distribution of BK channel protein. The results support the feasibility of using IbTx−LC-biotin and similar biotin-tagged K⁺ channel toxins for diverse applications in cellular neurobiology.