An unusual fold for potassium channel blockers: NMR structure of three toxins from the scorpion Opisthacanthus madagascariensis.

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.

Assignment of voltage-gated potassium channel blocking activity to kappa-KTx1.3, a non-toxic homologue of kappa-hefutoxin-1, from Heterometrus spinifer venom.

A new family of weak K(+) channel toxins (designated kappa-KTx) with a novel "bi-helical" scaffold has recently been characterized from Heterometrus fulvipes (Scorpionidae) venom. Based on the presence of the minimum functional dyad (Y5 and K19), kappa-hefutoxin-1 (kappa-KTx1.1) was investigated and found to block Kv 1.2 (IC(50) approximately 40 microM) and Kv 1.3 (IC(50) approximately 150 microM) channels. In the present study, kappa-KTx1.3, that shares approximately 60% identity with kappa-hefutoxin 1, has been isolated from Heterometrus spinifer venom. Interestingly, despite the presence of the functional dyad (Y5 and K19), kappa-KTx1.3 failed to reproduce the K(+) channel blocking activity of kappa-hefutoxin-1. Since the dyad lysine in kappa-KTx1.3 was flanked by another lysine (K20), it was hypothesized that this additional positive charge could hinder the critical electrostatic interactions known to occur between the dyad lysine and the Kv 1 channel selectivity filter. Hence, mutants of kappa-KTx1.3, substituting K20 with a neutral (K20A) or a negatively (K20E) or another positively (K20R) charged amino acid were synthesized. kappa-KTx1.3 K20E, in congruence with kappa-hefutoxin 1 with respect to subtype selectivity and affinity, produced blockade of Kv 1.2 (IC(50) = 36.8+/-4.9 microM) and Kv 1.3 (IC(50)=53.7+/-6.7 microM) but not Kv 1.1 channels. kappa-KTx1.3 K20A produced blockade of both Kv 1.2 (IC(50) = 36.9+/-4.9 microM) and Kv 1.3 (IC(50)=115.7+/-7.3 microM) and in addition, acquired affinity for Kv 1.1 channels (IC(50) =1 10.7+/-7.7 microM). kappa-KTx1.3 K20R failed to produce any blockade on the channel subtypes tested. These data suggest that the presence of an additional charged residue in a position adjacent to the dyad lysine impedes the functional block of Kv 1 channels produced by kappa-KTx1.3.

Synthesis and electrophysiological characterization of cyclic morphiceptin analogues.

A challenge in opioid peptide chemistry and pharmacology is the possibility to develop novel peptides with peripheral selectivity. An enzymatically stable opioid peptide could involve an antidiarrheal effect. For this reason, we constrained the highly selective and potent tetrapeptide morphiceptin with a 6-atom bridge, resulting in a cyclic amide and an ester analogue, 2 and 3, respectively. Taking advantage of the functional coupling of the opioid receptor with the heteromultimeric G-protein-coupled inwardly rectifying K+ (GIRK1/GIRK2) channel, either the wild-type mu-, kappa-, delta- or a mutated mu-opioid receptor (hMORS329A) was functionally co-expressed with GIRK1/GIRK2 channels and a regulator of G-protein signaling (RGS4) in Xenopus laevis oocytes. The two-microelectrode voltage clamp technique was used to measure the opioid receptor activated GIRK1/GIRK2 channel responses. Both cyclic analogues were equally potent via the wild-type mu-opioid receptor hMORwt (EC(50) value 976.5 +/- 41.7 for 2 and 1017.7 +/- 60.7 for 3), while the EC(50) value for Tyr-Pro-Phe-D-Pro-NH(2) measured 59.3 +/- 4.8 nM. These three agonists displayed a four to five times decreased potency via hMORS329A as compared to the wild type. Interestingly, no effect on kappa- and delta-opioid receptors was observed. The intramolecular bridge created by cyclization of morphiceptin prevents dipeptidyl peptidase IV from interacting with these analogues. We conclude that constraining morphiceptin with a 6-atom bridge resulted in enzymatically stable peptidomimetics that are exclusively active on mu-opioid receptors. These analogues provide an interesting template in the promising approach for the design of potential antidiarrheal agents.

Serine 329 of the mu-opioid receptor interacts differently with agonists.

To investigate the effect of the hydrophilic Ser amino acid in position 329 of the human mu-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 voltage-clamp 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 beta-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 beta-hydroxy group of fentanyl and that mutation of this residue to alanine caused dual effects depending on the nature of the ligand.