Effects of toxins Pi2 and Pi3 on human T lymphocyte Kv1.3 channels: The role of Glu7 and Lys24

Pandinus imperator scorpion toxins Pi2 and Pi3 differ only by a single amin o acid residue (neutral Pro7 in Pi2 vs. acidic Glu7 in Pi3). The binding ki netics of these toxins to human Kv1.3 showed that the decreased on rate (k( ON) = 2.18 x 10(8) M(-1)sec(-1) for Pi2 and 1.28 x 10(7) M(-1)sec(-1) for P i3) was almost entirely responsible for the increased dissociation constant (K-d) of Pi3 (K-d = 795 pM) as compared to Pi2 (K-d = 44 pM). The ionic st rength dependence of the association rates was exactly the same for the two toxins indicating that through-space electrostatic interactions can not ac count for the different on rates. Results were further analyzed on the basi s of the three-dimensional structural models of the toxins. A 3D structure of Pi3 was generated from the NMR spectroscopy coordinates of Pi2 by comput er modeling. The Pi3 model resulted in a salt bridge between Glu7 and Lys24 in Pi3. Based on this finding our interpretation of the reduced on rate of Pi3 is that the intramolecular salt bridge reduces the local positive elec trostatic potential around Lys24 resulting in decreased short-range electro static interactions during the binding step. To support our finding, we con structed a 3D model of the Ser-10-Asp Charybdotoxin mutant displaying disti nctly reduced affinity for Shaker channels. The mutant Charybdotoxin struct ure also displayed a salt bridge between residues Asp10 and Lys27 equivalen t to the one between Glu7 and Lys24 in Pi3.