Probing the pH‐dependent structural features of α‐KTx12.1, a potassium channel blocker from the scorpion Tityus serrulatus

摘要: Animal venoms are constantly being studied in search of new pharmacologically active molecules that can be used for therapeutic purposes. In this respect, scorpion venom is an incredible resource molecules, most which peptides and small proteins characterized by a variety biological activities. A good number them able to bind specifically ion channels, producing mainly neurotoxic effects. On the other hand, it worth remembering from point view scorpions, spiders, snakes, these toxins essential tools their survival. As result, they must quite robust order reach targets exert functions. fact, have resist overcome defensive systems hosts such as, instance, proteases and/or significant local pH variations may result inflammation states (Steen et al. 1996; Waldmann Lasdunski 1998) induced animal bite itself. Potassium channels widely spread living kingdom part large processes. addition, now well recognized involved increasing human pathologies (Ashcroft 2000; Shieh 2000). Thus, perception potassium important explains growing efforts comprehend three-dimensional structure mechanism action. Scorpion selective K+ (KTx) short-chain (23–43 amino acids) cross-linked three or four disulfide bridges. They been classified into α-, β-, γ- (Tytgat 1999; Corona 2002). The α-KTx family best date. At first, was divided 12 subfamilies comprising total 49 members 1999). More recently, six added (Batista 2002; Goudet Figure 1A ▶ shows sequence alignment selected toxins. Their global fold displays same α/β scaffold formed α-helix two- three-stranded β-sheet linked two bridges (Tenenholz This fold, however, shared polypeptides with diverse functions, including on Na+ (Cohen 2004), some antimicrobial as defensins (Bauer 2001; Sawai 2001), γ-thionins (Bloch 1998), antifungal (Fant thus suggesting does not correlate directly specific target. Figure 1. (A) Sequence carried out using CLUSTAL W (Thompson 1994) followed manual adjustment. Identical highly conserved residues indicated stars colons, respectively. (B) ... Overall, despite similar architecture, exhibit distinct selectivity affinity (Rodriguez de la Vega 2003), posing interesting questions concerning molecular factors influence diversified channel blocker activity. α-KTx12.1 disulfide-bridged neurotoxin (Fig. 1B ▶), first isolated Brazilian Tityus serrulatus. Due lack homology any toxin belonging family, has considered member subfamily Originally, named TsTX-IV (Tityus IV) (Arantes 1989) described 41-residue protein (Novello However, according results reported here agreement data recently published another group (Pimenta turns polypeptide lacks residue Asn41. Based findings, follows α-KTx12.1 40-residue-long butantoxin (BuTX) originally serrulatus subsequently T. bahiensis stigmurus (Holaday 2000) molecule. Finally, peptide purified Argentinean trivittatus, presenting primary α-KTx12.1, α-KTx12.2 because different geographic origin (Coronas 2003). As its activity, at nM concentration, shown block high-conductance Ca2+-activated Leydig cells Furthermore, while compete 125I-apamin, very ligand low-conductance activated inhibit binding 125I-kaliotoxin receptor rat brain synaptosomal preparations, IC50 46 nM, also Kv1.3 2003). block, reversibly, Shaker B K+-channels, Kd ~660 summary, appears interact exhibiting variable each them. Such behavior suggests that, bridges, conformational flexibility allows fit pore region channels. The current models describing functionality assume type position acids exposed surface critical determining functions properties particular toxin. suggest inhibition conductance ability occlude extracellular opening. process, reversible, bimolecular reaction governed electrostatic interactions between negatively charged positively Consequently, expected affected both ionic strength external media (Ellis Garcia 2001). In work, aiming disclose how succeeds actively various types knowing influenced structural features possesses just before interacting channel, we investigated step. Recognizing experience environments where varies significantly, ionization state affect stability (Fersht 1998; Kipping Hu combined NMR spectroscopy dynamics (MD) simulations probe dynamic acidic neutral pH.