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

Kamilla Angelo; Yuliya V Korolkova; Morten Grunnet; Eugene V Grishin; Kirill A Pluzhnikov; Dan A Klaerke; Hans-Günther Knaus; Morten Møller; Søren-Peter Olesen

doi:10.1007/s00424-003-1125-9

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

Kamilla Angelo, Yuliya V Korolkova, Morten Grunnet, Eugene V Grishin, Kirill A Pluzhnikov, Dan A Klaerke, Hans-Günther Knaus, Morten Møller, Søren-Peter Olesen

15 Citations (Scopus)

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 [(125)I]-BeKm-1 were fast (association rate constant, k(on)=3.6 x 10(7) M(-1)s(-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 (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.

Original language	English
Journal	Pflügers Archiv: European Journal of Physiology
Volume	447
Issue number	1
Pages (from-to)	55-63
Number of pages	8
ISSN	0031-6768
DOIs	https://doi.org/10.1007/s00424-003-1125-9
Publication status	Published - 2003

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10.1007/s00424-003-1125-9

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@article{752d0f00ab5511ddb5e9000ea68e967b,

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

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 [(125)I]-BeKm-1 were fast (association rate constant, k(on)=3.6 x 10(7) M(-1)s(-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 (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.",

author = "Kamilla Angelo and Korolkova, {Yuliya V} and Morten Grunnet and Grishin, {Eugene V} and Pluzhnikov, {Kirill A} and Klaerke, {Dan A} and Hans-G{\"u}nther Knaus and Morten M{\o}ller and S{\o}ren-Peter Olesen",

note = "Keywords: Animals; Cell Line; Dose-Response Relationship, Drug; Ether-A-Go-Go Potassium Channels; Humans; Iodine Radioisotopes; Ligands; Male; Peptides; Potassium Channel Blockers; Potassium Channels; Potassium Channels, Voltage-Gated; Protein Binding; Rats; Scorpion Venoms",

year = "2003",

doi = "10.1007/s00424-003-1125-9",

language = "English",

volume = "447",

pages = "55--63",

journal = "Pfl{\"u}gers Archiv: European Journal of Physiology",

issn = "0031-6768",

publisher = "Springer",

number = "1",

}

TY - JOUR

T1 - A radiolabeled peptide ligand of the hERG channel, [125I]-BeKm-1.

AU - Angelo, Kamilla

AU - Korolkova, Yuliya V

AU - Grunnet, Morten

AU - Grishin, Eugene V

AU - Pluzhnikov, Kirill A

AU - Klaerke, Dan A

AU - Knaus, Hans-Günther

AU - Møller, Morten

AU - Olesen, Søren-Peter

N1 - Keywords: Animals; Cell Line; Dose-Response Relationship, Drug; Ether-A-Go-Go Potassium Channels; Humans; Iodine Radioisotopes; Ligands; Male; Peptides; Potassium Channel Blockers; Potassium Channels; Potassium Channels, Voltage-Gated; Protein Binding; Rats; Scorpion Venoms

PY - 2003

Y1 - 2003

N2 - 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 [(125)I]-BeKm-1 were fast (association rate constant, k(on)=3.6 x 10(7) M(-1)s(-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 (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.

AB - 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 [(125)I]-BeKm-1 were fast (association rate constant, k(on)=3.6 x 10(7) M(-1)s(-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 (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.

U2 - 10.1007/s00424-003-1125-9

DO - 10.1007/s00424-003-1125-9

M3 - Journal article

C2 - 12905030

SN - 0031-6768

VL - 447

SP - 55

EP - 63

JO - Pflügers Archiv: European Journal of Physiology

JF - Pflügers Archiv: European Journal of Physiology

IS - 1

ER -

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

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