A radiolabeled peptide ligand of the hERG channel, [125I]-BeKm-1.
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.
Angelo
Kamilla
K
The Wolfson Institute for Biomedical Research, Cruciform Building, University College London, Gower Street, London, WC1E 6BT, UK. k.angelo@ucl.ac.uk
Korolkova
Yuliya V
YV
Grunnet
Morten
M
Grishin
Eugene V
EV
Pluzhnikov
Kirill A
KA
Klaerke
Dan A
DA
Knaus
Hans-Günther
HG
Møller
Morten
M
Olesen
Søren-Peter
SP
D000818:Animals; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; D006801:Humans; D007457:Iodine Radioisotopes / Q000378:metabolism; D008024:Ligands; D008297:Male; D010455:Peptides / Q000378:metabolism*; D026902:Potassium Channel Blockers / Q000378:metabolism*; D015221:Potassium Channels / Q000378:metabolism*; D024642:Potassium Channels, Voltage-Gated; D011485:Protein Binding / Q000502:physiology; D051381:Rats; D012604:Scorpion Venoms / Q000378:metabolism*
C425305:BeKm-1 toxin; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; D007457:Iodine Radioisotopes; C000606913:KCNH2 protein, human; D008024:Ligands; D010455:Peptides; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D012604:Scorpion Venoms