AbstractBackground:Conotoxins exhibit great potential as neuropharmacology tools and therapeutic
candidates due to their high affinity and specificity for ion channels,
neurotransmitter receptors or transporters. The traditional methods to
discover new conotoxins are peptide purification from the crude venom or
gene amplification from the venom duct.Methods:In this study, a novel O1 superfamily conotoxin Tx6.7 was directly cloned
from the genomic DNA ofConus textileusing primers
corresponding to the conserved intronic sequence and 3’ UTR elements. The
mature peptide of Tx6.7 (DCHERWDWCPASLLGVIYCCEGLICFIAFCI) was synthesized by
solid-phase chemical synthesis and confirmed by mass spectrometry.Results:Patch clamp experiments on rat DRG neurons showed that Tx6.7 inhibited peak
calcium currents by 59.29 ± 2.34% and peak potassium currents by 22.33 ±
7.81%. In addition, patch clamp on the ion channel subtypes showed that 10
μM Tx6.7 inhibited 56.61 ± 3.20% of the hCaV1.2 currents, 24.67 ±
0.91% of the hCaV2.2 currents and 7.30 ± 3.38% of the
hNaV1.8 currents. Tx6.7 had no significant toxicity to ND7/23
cells and increased the pain threshold from 0.5 to 4 hours in the mouse hot
plate assay.Conclusion:Our results suggested that direct cloning of conotoxin sequences from the
genomic DNA of cone snails would be an alternative approach to obtaining
novel conotoxins. Tx6.7 could be used as a probe tool for ion channel
research or a therapeutic candidate for novel drug development.