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--(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 (KD = 26 nM) and a slow dissociation rate (koff = 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. .
Bingham
Jon-Paul
JP
Department of Biology, Clarkson University, Potsdam, New York 13699, USA.
Bian
Shumin
S
Tan
Zhi-Yong
ZY
Takacs
Zoltan
Z
Moczydlowski
Edward
E
D000595:Amino Acid Sequence; D001710:Biotin / Q000737:chemistry*; D002460:Cell Line; D004594:Electrophysiology; D006801:Humans; D051036:Large-Conductance Calcium-Activated Potassium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000737:chemistry* / Q000494:pharmacology*; D026902:Potassium Channel Blockers / Q000737:chemistry* / Q000494:pharmacology; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D019809:Streptavidin / Q000737:chemistry*
D051036:Large-Conductance Calcium-Activated Potassium Channels; D010455:Peptides; D026902:Potassium Channel Blockers; D001710:Biotin; C064719:iberiotoxin; D019809:Streptavidin