Abstract
Animal venom peptides represent valuable compounds for biomedical exploration. The venoms of marine cone snails constitute a particularly rich source of peptide toxins, known as conotoxins. Here, we identify the sequence of an unusually large conotoxin, Mu8.1, that defines a new gene superfamily. The crystal structure of recombinant Mu8.1 shows structural similarity with conotoxins of the con-ikot-ikot superfamily, with both toxins displaying a saposin-like fold. Functional studies demonstrate that Mu8.1 curtails calcium influx in defined classes of murine somatosensory dorsal root ganglion (DRG) neurons. When tested on a variety of voltage-gated ion channels, Mu8.1 preferentially inhibited the R-type (Cav2.3) calcium channel. Ca2+ signals from Mu8.1-sensitive DRG neurons were also inhibited by SNX-482, a known spider peptide modulator of Cav2.3 and voltage-gated K+ (Kv4) channels. Our findings highlight the potential of Mu8.1 as a molecular tool to identify and study neuronal subclasses expressing Cav2.3. Importantly, this multidisciplinary study demonstrates the feasibility of large, disulfide-rich venom-component investigation, an endeavor that will lead to the discovery of novel structures and functions in the previously underexplored group of macro-conotoxins.
Competing Interest Statement
The authors have declared no competing interest.
Abbreviations
- AEX
- anion exchange
- AITC
- allyl isothiocyanate derived from mustard oil
- CNGB
- China National Genebank
- APC
- automated patch clamp
- CD
- circular dichroism spectroscopy
- CGRP
- calcitonin gene-related peptide
- C-LTMRs
- C-low threshold mechanoreceptors
- CNS
- central nervous system
- csPDI
- conotoxin-specific PDI
- CV
- column volume
- DDBJ
- DNA Databank of Japan
- DMEM
- Dulbecco’s Modified Eagle’s Medium
- DRG
- dorsal root ganglia
- DTT
- dithiothreitol
- ER
- endoplasmic reticulum
- FDA
- U.S. Food and Drug Administration
- FOM
- figure of merit
- GFP
- green fluorescent protein
- GPCR
- G protein-coupled receptor
- HEK293T
- human embryonic kidney cells
- hPDI
- human PDI
- IACUC
- Institutional Animal Care and Use Committee
- IB4
- Alexa-Fluor 647 Azolectin B4
- IMAC
- immobilized metal affinity chromatography
- IPTG
- isopropyl ß-D-1-thiogalactopyranoside
- LB
- lysogeny broth
- MALDI-TOF
- matrix-assisted laser desorption-ionization-time of flight
- MPC
- manual patch clamp
- NCBI
- National Center for Biotechnology Information
- NSS
- normal saline solution
- PDI
- protein-disulfide isomerase
- RMSD
- root mean square deviation
- RP-HPLC
- reversed-phase high pressure liquid chromatography
- SAPLIP
- Saposin like proteins
- SAXS
- small angle x-ray scattering SVD
- SDS-PAGE
- sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- SLC
- saposin-like conotoxin
- SR
- seal resistant
- TEV
- tobacco etch virus
- Ub
- ubiquitin
- Ub-His10
- Ub containing 10 consecutive histidines.