[125I]-Omega-Conotoxin GVIA, a blocker of neuronal (N-type) calcium channels labelled 295 +/- 121 fmol per mg protein of high affinity sites (apparent half-saturation at 1.5 to 2.5 pmol/l) in guinea-pig cerebral cortex membranes. Divalent cations (Cd2+ greater than Ni2+ greater than Co2+ greater than Ca2+ greater than Sr2+ = Ba2+ greater than Mg2+) and La3+ were potent inhibitors of Omega-Conotoxin GVIA binding, whereas monovalent cations (Na+, K+, Li+) were ineffective up to 50 mmol/l. Aminoglycosides (neomycin greater than gentamycin = tobramycin greater than streptomycin greater than amikacin greater than kanamycin) and polymyxin B also inhibited [125I]-Omega-Conotoxin GVIA binding with IC50 values in the mumolar range. All other antibiotics tested were ineffective up to 1 mmol/l. With the exception of polymyxin B, which partially inhibited the binding of the 1,4-dihydropyridine (+)-[3H]PN 200-110 and of (-)-[3H]desmethoxyverapamil, the aminoglycosides and the other antibiotics had no effect on the L-type calcium channel labelling. It is suggested, that inhibition of neurotransmitter release by aminoglycosides is mediated via blockade of the N-type calcium channel to which [125I]-Omega-Conotoxin GVIA binds selectively in a quasi irreversible manner.

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.

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