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A radiolabeled peptide ligand of the hERG channel, [125I]-BeKm-1

  • Ion Channels, Transporters
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Abstract

The wild-type scorpion toxin BeKm-1, which selectively blocks human ether-a-go-go related (hERG) channels, was radiolabeled with iodine at tyrosine 11. Both the mono- and di-iodinated derivatives were found to be biologically active. In electrophysiological patch-clamp recordings mono-[127I]-BeKm-1 had a concentration of half-maximal inhibition (IC50 value) of 27 nM, while wild-type BeKm-1 inhibited hERG channels with an IC50 value of 7 nM. Mono-[125I]-BeKm-1 was found to bind in a concentration-dependent manner and with picomolar affinity to hERG channel protein in purified membrane vesicles from transfected human embryonic kidney cells (HEK-293). Under optimized conditions the equilibrium dissociation constant (K d) values from saturation and kinetic binding analysis were 13 and 14 pM, respectively. Both the association and dissociation of [125I]-BeKm-1 were fast (association rate constant, k on=3.6×107 M−1s−1; dissociation rate constant, k off=0.005 s−1). Wild-type BeKm-1 displaced binding of [125I]-BeKm-1 with half-maximal inhibitory concentrations of 44 pM. In contrast, competition experiments with a BeKm-1 mutant BeKm-1-K18A, in which the toxin interaction site is disrupted, resulted in a drop in affinity by more than 300-fold as compared to the wild-type toxin. Iberiotoxin and apamin, peptide inhibitors of Ca2+-activated K+-channels, had no effect on [125I]-BeKm-1 binding. Adding the classical rapid delayed rectifier current (I Kr) blocker E-4031 reduced binding of [125I]-BeKm-1 to the hERG channel to an IC50 of 7 nM. In autoradiographic studies on rat hearts, binding of [125I]-BeKm-1 was dose-dependent and could partially be displaced by the addition of excess amounts of non-radioactive BeKm-1. The density of the radioactive signal was equally distributed in the myocardium of both the ventricle and atria indicating a homogenous expression of hERG channels throughout the heart.

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Acknowledgements

This work was supported by the Danish Heart Foundation and Russian Foundation of Basic Research. Thank you to Bernt Pragl for help with the iodination.

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Authors and Affiliations

  1. The Wolfson Institute for Biomedical Research, Cruciform Building, University College London, Gower Street, London , WC1E 6BT, UK

    Kamilla Angelo

  2. Department of Medical Physiology and Copenhagen Heart Research Center (CHARC), The Panum Institute, University of Copenhagen, Blegdamsvej 3, 2200, Copenhagen N, Denmark

    Kamilla Angelo, Morten Grunnet, Dan A. Klaerke & Søren-Peter Olesen

  3. Department of Anatomy, The Panum Institute, University of Copenhagen, Blegdamsvej 3, 2200, Copenhagen N, Denmark

    Morten Møller

  4. Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10, 117997 , Moscow, Russia

    Yuliya V. Korolkova, Eugene V. Grishin & Kirill A. Pluzhnikov

  5. Institut fur Biochemische Pharmakologie, Universitat Innsbruck, Peter Mayr-Strasse 1, 6020, Austria

    Hans-Günther Knaus

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  1. Kamilla Angelo
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  2. Yuliya V. Korolkova
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Correspondence to Kamilla Angelo.

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Angelo, K., Korolkova, Y.V., Grunnet, M. et al. A radiolabeled peptide ligand of the hERG channel, [125I]-BeKm-1. Pflugers Arch - Eur J Physiol 447, 55–63 (2003). https://doi.org/10.1007/s00424-003-1125-9

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  • DOI: https://doi.org/10.1007/s00424-003-1125-9

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