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- Title
A radiolabeled peptide ligand of the hERG channel, [<sup>125</sup>I]-BeKm-1.
- Authors
Angelo, Kamilla; Korolkova, Yuliya V.; Grunnet, Morten; Grishin, Eugene V.; Pluzhnikov, Kirill A.; Klaerke, Dan A.; Knaus, Hans-Günther; Møller, Morten; Olesen, Søren-Peter
- 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 (Kd) 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, kon=3.6×107 M-1s-1; dissociation rate constant, koff=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 (IKr) 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.
- Subjects
ION channels; ACTIVE biological transport; PEPTIDES; LIGANDS (Biochemistry); RADIOLABELING; RADIOACTIVE tracers; TOXINS; IODINE isotopes; RADIOIODINATION
- Publication
Pflügers Archiv: European Journal of Physiology, 2003, Vol 447, Issue 1, p55
- ISSN
0031-6768
- Publication type
Article
- DOI
10.1007/s00424-003-1125-9