The Om-toxins are short peptides (23-27 amino acids) purified from the venom of the scorpion Opisthacanthus madagascariensis. Their pharmacological targets are thought to be potassium channels. Like Csalpha/beta (cystine-stabilized alpha/beta) toxins, the Om-toxins alter the electrophysiological properties of these channels; however, they do not share any sequence similarity with other scorpion toxins. We herein demonstrate by electrophysiological experiments that Om-toxins decrease the amplitude of the K+ current of the rat channels Kv1.1 and Kv1.2, as well as human Kv1.3. We also determine the solution structure of three of the toxins by use of two-dimensional proton NMR techniques followed by distance geometry and molecular dynamics. The structures of these three peptides display an uncommon fold for ion-channel blockers, Csalpha/alpha (cystine-stabilized alpha-helix-loop-helix), i.e. two alpha-helices connected by a loop and stabilized by two disulphide bridges. We compare the structures obtained and the dipole moments resulting from the electrostatic anisotropy of these peptides with those of the only other toxin known to share the same fold, namely kappa-hefutoxin1.

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1 To investigate the e ect of the hydrophilic Asn amino acid at position 230 of the human mopioid receptor (hMOR230) on the potency of various agonists, we mutated this residue to Thr and Leu (hMORN230T and hMORN230L respectively). 2 Taking advantage of the functional coupling of the opioid receptor with the heteromultimeric Gprotein-coupled inwardly rectifying K + (GIRK1/GIRK2) channel, either the wild type hMOR or one of the mutated receptors (hMORN230L or hMORN230T) were functionally coexpressed with GIRK1/GIRK2 channels and a regulator of G-protein signalling (RGS4) in Xenopus laevis oocytes. 3 The two-microelectrode voltage clamp technique was used to measure the opioid receptoractivated GIRK1/GIRK2 channel responses. The potency of [D-Ala 2 ,N-MePhe 4 ,Gly 5 -ol]-enkephalin (DAMGO), remained una ected as measured via hMORN230T and hMORN230L, while the potency of fentanyl and morphine signi®cantly increased via these mutated receptors. 4 Our results are indicative for the existence of hydrophobic interactions between a methyl-group of the side chain of Thr or Leu on the one hand and the piperidine-ring of fentanyl and the hexenering of morphine on the other. The mutations also had no in¯uence on the potency of morphine-6-glucuronide (M6G) and morphine-3-glucuronide (M3G). 5 We conclude that the hydrophilic side chain of Asn in position 230 is not involved in the formation of a H-bond with the aliphatic alcohol of morphine and that an enhancement of the potency of morphine and fentanyl can be explained by mutating this residue towards more hydrophobic amino acids.

To investigate the effect of the hydrophilic Ser amino acid in position 329 of the human -opioid receptor (hMORwt) on the potency of various agonists, we mutated this residue to Ala (hMORS329A). Taking advantage of the functional coupling of the opioid receptor with the heteromultimeric G-protein-coupled inwardly rectifying potassium channel (GIRK1/GIRK2), either the wild-type hMOR or the mutated receptor (hMORS329A) was functionally coexpressed with GIRK1 and GIRK2 channels together with a regulator of G-protein signaling (RGS4) in Xenopus laevis oocytes. The two-microelectrode voltageclamp technique was used to measure the opioid receptor activated GIRK1/GIRK2 channel responses. The potency of the peptide agonist [D-Ala 2 ,N-MePhe 4 ,Gly 5 -ol]-enkephalin (DAMGO) decreased as measured via hMORS329A, whereas the potency of nonpeptide agonists like morphine, fentanyl, and ␤-hydroxyfentanyl (R004333) increased via the mutated receptor. Our results are indicative for the existence of hydrophilic interactions between Ser 329 and DAMGO, thereby decreasing the potency of DAMGO via the mutated receptor, whereas hydrophobic interactions between the mutated receptor and the N-phenylethyl of morphine and fentanyl can explain the increased potency. We conclude that the hydroxyl group of Ser 329 is not involved in the formation of a hydrogen bond with the ␤-hydroxy group of fentanyl and that mutation of this residue to alanine caused dual effects depending on the nature of the ligand.

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