Synthesis Of A Biotin Derivative Of Iberiotoxin: Binding Interactions With Streptavidin And The Bkca2+-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- epsilon-( (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 x 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.