The functional dyad of scorpion toxin Pi1 is not itself a prerequisite for toxin binding to the voltage-gated Kv1.2 potassium channels

摘要

pPi1 is a 35-residue scorpion toxin cross-linked by four disulphide bridges that acts potently on both small-conductance Casup2+/sup-activated (SK) and voltage-gated (Kv) Ksup+/sup channel subtypes. Two approaches were used to investigate the relative contribution of the Pi1 functional dyad (Tyr-33 and Lys-24) to the toxin action: (i) the chemical synthesis of a [Asub24/sub,Asub33/sub]-Pi1 analogue, lacking the functional dyad, and (ii) the production of a Pi1 analogue that is phosphorylated on Tyr-33 (P-Pi1). According to molecular modelling, this phosphorylation is expected to selectively impact the two amino acid residues belonging to the functional dyad without altering the nature and three-dimensional positioning of other residues. P-Pi1 was directly produced by peptide synthesis to rule out any possibility of trace contamination by the unphosphorylated product. Both Pi1 analogues were compared with synthetic Pi1 for bioactivity. iIn vivo/i, [Asub24/sub,Asub33/sub]-Pi1 and P-Pi1 are lethal by intracerebroventricular injection in mice (LDsub50/sub values of 100 and 40 μg/mouse, respectively). iIn vitro/i, [Asub24/sub,Asub33/sub]-Pi1 and P-Pi1 compete with sup125/supI-apamin for binding to SK channels of rat brain synaptosomes (ICsub50/sub values of 30 and 10 nM, respectively) and block rat voltage-gated Kv1.2 channels expressed in iXenopus laevis/i oocytes (ICsub50/sub values of 22 μM and 75 nM, respectively), whereas they are inactive on Kv1.1 or Kv1.3 channels at micromolar concentrations. Therefore, although both analogues are less active than Pi1 both iin vivo/i and iin vitro/i, the integrity of the Pi1 functional dyad does not appear to be a prerequisite for the recognition and binding of the toxin to the Kv1.2 channels, thereby highlighting the crucial role of other toxin residues with regard to Pi1 action on these channels. The computed simulations detailing the docking of Pi1 peptides on to the Kv1.2 channels support an unexpected key role of specific basic amino acid residues, which form a basic ring (Arg-5, Arg-12, Arg-28 and Lys-31 residues), in toxin binding./p