Structural and functional differences of two toxins from the scorpionPandinus imperator

Abstract

The Pandinotoxins, PiTX-K alpha and PiTX-K beta, are members of the Charybdotoxin family of scorpion toxins that can be used to characterize K+ channels. PiTX-K alpha differs from PiTX-K beta, another peptide from Pandinus imperator, by one residue (P10E). When the two toxins are compared in a physiological assay, the affinity of PiTX-K beta for voltage-gated, rapidly inactivating K+ channels in dorsal root ganglia (DRG) neurons is 800-fold lower than that of PiTX-K alpha (K alpha-IC50 = 8.0 nM versus K beta-IC50 = 6,500 nM). To understand this difference, the three-dimensional structure of PiTX-K beta was determined by nuclear magnetic resonance (NMR) spectroscopy and compared to that of PiTX-K alpha. This comparison shows that structural differences between the two toxins occur at a residue that is critical for blocking K+ channels (K27) as well as at the site of the natural mutation (P10E). In PiTX-K beta, the negatively charged carboxylate oxygen of E10 can approach the positive charge of K27 and presumably reduces the net positive charge in this region of the toxin. This is likely the reason why PiTX-K beta binds K+ channels from DRG neurons with a much lower affinity than does PiTX-K alpha.

Interaction of a Toxin from the Scorpion Tityus serrulatus with a Cloned K+ Channel from Squid (sqKv1A)

“…Elements of secondary structure in TsTX-KR were identified using NOE correlations and chemical shift values as previously described (14,15,24) (Figure 2). Strong d RN connectivities from residues 2 and 3, 26-28, and 33-35 together with a positive chemical shift index (38) for the R-protons in these segments indicate -strand conformation.…”

“…Preparation of Recombinant TsTX-K α. Expression and purification of milligram quantities of TsTX-Kα were performed using procedures similar to those described previously ( , ). The expression plasmid was constructed by inserting a synthetic gene encoding TsTX-Kα into the pSR9 expression plasmid and placing it under the control of a T7 promoter.…”

Structural determinants of scorpion toxin affinity: The charybdotoxin (α-KTX) family of K+-channel blocking peptides

“…Comparison of the 3-dimensional structures shows that the two toxins are very similar except that K27 is situated close to El0 in PiTX-KI3. Evidence for a salt bridge between these two residues is supported by the fact that the pK, of El0 is lower by more than 1 pH unit than a free carboxylate in solution (Klenk et al, 2000). Thus, an interaction between K27 and El0 may be responsible for its decreased affinity in DRG.…”

“…In addition, cx-K5.X toxins have an alanine at position 26 (A26; or $26 for PiTX-Ky) rather than a glycine residue (G26) as found for the other subfamilies of ¢x-K toxins. PiTX-Kc~ and PiTX-KJ3 are further distinguished from some of the other ~-K toxin subfamilies since the ~3-turn between the two remaining strands is type II rather than type I (Klenk et al, 2000;Tenenholz et al, 1997). This subtle difference in backbone structure has a large effect on the position of a residue (R31) that is believed to be critical for binding A-type channels (Tenenholz et al, 1997).…”

“…These two commonly used channel preparations likely contain structurally different voltage-gated K + channels (Klenk et al, 2000). In rat brain synaptosomes, the affinity of PiTX-K[3 for voltage-gated, rapidly inactivating (A-type) K + channels is 8-fold lower than that of PiTX-Kcx (K~IC50=6.0 nM versus K~ICs0=45 nM), whereas for rat dorsal root ganglia (DRG) neurons there is a 800-fold difference (K~ICs0=8.0 nM versus I<~ICso=6500 riM).…”