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Published on: June 22, 2017
O1-conotoxin Tx6.7 cloned from the genomic DNA of
Maojun Zhou1, Manyi Yang2, Huiling Wen3
1Department of Oncology, NHC Key Laboratory of Cancer Proteomics, State Local Joint Engineering Laboratory for Anticancer Drugs, Xiangya Hospital, Central South University, Changsha, Hunan, China.
Background:
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 of Conus textile using 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.
Insights
Researchers discovered a new conotoxin, Tx6.7, using genomic DNA cloning. This conotoxin shows potential for ion channel research and developing novel therapeutics for pain management.
Area of Science:
- Marine biotechnology
- Neuropharmacology
- Molecular biology
Background:
- Conotoxins are valuable tools in neuropharmacology due to their specific interactions with ion channels and receptors.
- Traditional conotoxin discovery relies on venom purification or gene amplification, which can be labor-intensive.
Purpose of the Study:
- To explore a novel method for conotoxin discovery by directly cloning from genomic DNA.
- To characterize the functional properties and potential applications of the newly identified conotoxin Tx6.7.
Main Methods:
- Direct cloning of O1 superfamily conotoxin Tx6.7 from Conus textile genomic DNA using conserved intronic and 3' UTR sequences.
- Solid-phase chemical synthesis of Tx6.7 and confirmation via mass spectrometry.
- Electrophysiological assessment using patch clamp on rat DRG neurons and specific ion channel subtypes (hCaV1.2, hCaV2.2, hNaV1.8).
Main Results:
- Tx6.7 significantly inhibited peak calcium currents (59.29%) and potassium currents (22.33%) in rat DRG neurons.
- Tx6.7 demonstrated specific inhibition of calcium channel subtypes hCaV1.2 (56.61%) and hCaV2.2 (24.67%), with minimal effect on hNaV1.8 (7.30%).
- Tx6.7 exhibited no significant cellular toxicity and increased pain threshold in a mouse hot plate assay.
Conclusions:
- Direct genomic DNA cloning offers an alternative strategy for discovering novel conotoxins.
- Tx6.7 is a promising conotoxin for use as a research probe for ion channels and as a potential therapeutic candidate for drug development.
