Synthesis of a Biotin Derivative of Iberiotoxin: Binding Interactions with Streptavidin and the BK Ca2+-Activated K+ Channel Expressed in a Human Cell Line
| Autor: | Shumin Bian, Zhi-Yong Tan, Jon-Paul Bingham, Zoltan Takacs, Edward Moczydlowski |
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| Rok vydání: | 2006 |
| Předmět: |
Models
Molecular Streptavidin BK channel Patch-Clamp Techniques Molecular Sequence Data Biomedical Engineering Biotin Pharmaceutical Science Bioengineering Peptide Article Mass Spectrometry Cell Line chemistry.chemical_compound Tetramer Potassium Channel Blockers Humans Amino Acid Sequence Large-Conductance Calcium-Activated Potassium Channels Patch clamp Binding site Protein Structure Quaternary Pharmacology chemistry.chemical_classification biology Chemistry Organic Chemistry Iberiotoxin Protein Structure Tertiary Electrophysiology Biochemistry biology.protein Biophysics Peptides Biotechnology |
| Zdroj: | Bioconjugate Chemistry. 17:689-699 |
| ISSN: | 1520-4812 1043-1802 |
| DOI: | 10.1021/bc060002u |
| Popis: | 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. . |
| Databáze: | OpenAIRE |
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