Mutational analysis of the analgesic peptide DrTx(1-42) revealing a functional role of the amino-terminal turn.

BACKGROUND

DrTx(1-42) (a carboxyl-terminally truncated version of drosotoxin) is a potent and selective blocker of tetrodotoxin-resistant (TTX-R) Na(+) channels in rat dorsal root ganglion neurons with analgesic activity. This purpose is to identify key amino acids which are responsible for both blocking and analgesic effects of DrTx(1-42).

METHODS

On the basis of previous study, we designed five mutants of DrTx(1-42) (delN, D8A, D8K, G9A, and G9R) in the amino-terminal turn (N-turn) region, a proposed functional region located in the amino-terminus of the molecule. All these mutants were expressed in E.coli and purified by RP-HPLC. Electrophysiological properties of these analogues were examined by whole-cell patch-clamp recordings and their antinociceptive effects were investigated by the formalin test and acetic acid induced writhing test.

RESULTS

All the mutants except for G9A possess a similar secondary structure to that of DrTx(1-42), as identified by circular dichroism analysis. Three mutants (delN, D8A and G9A) were found almost inactive to TTX-R Na(+) channels, whereas D8K retains similar activity and G9R showed decreased potency when compared with the wild-type molecule. Consistent with the electrophysiological observations, D8K and G9R exhibited antinociceptive effects in the second phase (inflammatory pain) of the formalin test and the acetic acid induced writhing test, while delN, D8A and G9A lack such effects.

CONCLUSIONS

Our results show that the N-turn is closely related to function of DrTx(1-42). The mutant (D8A) as a control peptide further reveals that a charged residue at site 8 of the N-terminus is important for channel blockade and analgesic activity. This study indicates that blocking of voltage-gated TTX-R Na(+) channel in DRG neurons contributes to analgesic effect in rat inflammatory pain. Structural and functional data described here offers support for the development of novel analgesic drugs through targeting TTX-R Na(+) channels.