The three-finger toxin MTα is a selective α2B-adrenoceptor antagonist

Muscarinic toxins (MTs) are three-finger folded peptides isolated from mamba snake venoms. In this report we describe a selective antagonistic interaction of MT alpha with the human alpha(2B)-adrenoceptor. In a functional assay, measuring the alpha(2B)-adrenoceptor-induced increase in intracellular [Ca2+], we found that both venomous MT alpha and synthetic MT alpha inhibited the response in a concentration-dependent way. MT alpha did not affect the responses of alpha(2A)-, alpha(2c)-, alpha(1A)- or alpha(1B)-adrenoceptors. To further explore the binding of MT alpha to the alpha(2B)-adrenoceptor, we performed ligand binding experiments on Sf9 cell homogenates with [H-3]RX821002 as reporter ligand. MT alpha bound to the receptor rather slowly requiring about 60 min to reach equilibrium. In equilibrium binding experiments, MT alpha displaced the radioligand with an IC50 of 3.2 nM, but was not able to displace all bound radioligand. Using a saturation binding protocol, we found that MT alpha suppressed the maximum binding without any greater impact on the affinity of the radioligand, indicating a non-competitive mode of inhibition. The toxin bound reversibly to alpha(2B)-adrenoceptor, but extensive washing was needed for full recovery of binding sites at high toxin concentrations. Surprisingly, MT alpha did not affect [H-3]-N-methylscopolamine binding to the muscarinic receptor subtypes at concentrations found to fully block alpha(2B)-adrenoceptors, showing that the toxin is a more potent antagonist for the alpha(2B)-adrenoceptor than for muscarinic receptors. These findings should open up new views in terms of selective adrenoceptor drug design as well as in elucidation of alpha(2)-adrenoceptor physiology. (C) 2010 Elsevier Ltd. All rights reserved.