Synthesis, Characterization, and Application of Cy-Dye- and Alexa-Dye-Labeled Hongotoxin1 Analogues. The First High Affinity Fluorescence Probes for Voltage-Gated K+ Channels
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- 作者：Bernt Pragl ; Alexandra Koschak ; Maria Trieb ; Gerald Obermair ; Walter A. Kaufmann ; Uli Gerster ; Eric Blanc ; Christoph Hahn ; Heino Prinz ; Gerhard Schü ; tz ; Herve Darbon ; Hermann J. Gruber ; and Hans-G
- 刊名：Bioconjugate Chemistry
- 出版年：2002
- 出版时间：May 2002
- 年：2002
- 卷：13
- 期：3
- 页码：416 - 425
- 全文大小：457K
- 年卷期：v.13,no.3(May 2002)
- ISSN：1520-4812
文摘
Hongotoxin1 (HgTX1), a 39-residue peptide recently isolated from the venom of Centruroides limbatus,blocks the voltage-gated K+ channels Kv1.1, Kv1.2, and Kv1.3 at picomolar toxin concentrations(Koschak, A., Bugianesi, R. M., Mitterdorfer, J., Kaczorowski, G. J., Garcia, M. L., and Knaus, H. G.(1998) J. Biol. Chem. 273, 2639-2644). In this report, we determine the three-dimensional structureof HgTX1 using NMR spectroscopy (PDB-code: 1HLY). HgTX1 was found to possess a structure similarto previously characterized K+ channel toxins (e.g. margatoxin) consisting of a three-strandedantiparallel 
-sheet (residues 2-4, 26-30, and 33-37) and a helical conformation (part 310 helix andpart 
helix; residues 10-20). Due to the importance of residue Lys-28 for high-affinity interactionwith the respective channels, lysine-reactive fluorescence dyes cannot be used to label wild-type HgTX1.On the basis of previous studies (see above) and our NMR data, a HgTX1 mutant (HgTX1-A19C) wasengineered, expressed, and purified. HgTX1-A19C-SH was labeled using sulfhydryl-reactive Cy3-, Cy5-,and Alexa-dyes. Pharmacological characterization of fluorescently labeled HgTX1-A19C in radioligandbinding studies indicated that these hongotoxin1 analogues retain high-affinity for voltage-gated K+channels and a respective pharmacological profile. Cy3- and Alexa-dye-labeled hongotoxin1 analogueswere used to investigate the localization of K+ channels in brain sections. The distribution of toxinbinding closely follows the distribution of Kv1.2 immunoreactivity with the highest expression levelsin the cerebellar Purkinje cell layer. Taken together, these results demonstrate that fluorescentlylabeled HgTX1 analogues comprise novel probes to characterize a subset of voltage-gated K+ channels.
-sheet (residues 2-4, 26-30, and 33-37) and a helical conformation (part 310 helix andpart helix; residues 10-20). Due to the importance of residue Lys-28 for high-affinity interactionwith the respective channels, lysine-reactive fluorescence dyes cannot be used to label wild-type HgTX1.On the basis of previous studies (see above) and our NMR data, a HgTX1 mutant (HgTX1-A19C) wasengineered, expressed, and purified. HgTX1-A19C-SH was labeled using sulfhydryl-reactive Cy3-, Cy5-,and Alexa-dyes. Pharmacological characterization of fluorescently labeled HgTX1-A19C in radioligandbinding studies indicated that these hongotoxin1 analogues retain high-affinity for voltage-gated K+channels and a respective pharmacological profile. Cy3- and Alexa-dye-labeled hongotoxin1 analogueswere used to investigate the localization of K+ channels in brain sections. The distribution of toxinbinding closely follows the distribution of Kv1.2 immunoreactivity with the highest expression levelsin the cerebellar Purkinje cell layer. Taken together, these results demonstrate that fluorescentlylabeled HgTX1 analogues comprise novel probes to characterize a subset of voltage-gated K+ channels.