PT  - JOURNAL ARTICLE
AU  - Celeste M. Hackney
AU  - Paula Flórez Salcedo
AU  - Emilie Mueller
AU  - Lau D. Kjelgaard
AU  - Maren Watkins
AU  - Linda Grønborg Zachariassen
AU  - Jeffrey R. McArthur
AU  - David J. Adams
AU  - Anders S. Kristensen
AU  - Baldomero Olivera
AU  - Rocio K. Finol-Urdaneta
AU  - Helena Safavi-Hemami
AU  - Jens Preben Morth
AU  - Lars Ellgaard
TI  - Identification of a sensory neuron Cav2.3 inhibitor within a new superfamily of macro-conotoxins
AID  - 10.1101/2022.07.04.498665
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.07.04.498665
4099  - http://biorxiv.org/content/early/2022/07/04/2022.07.04.498665.short
4100  - http://biorxiv.org/content/early/2022/07/04/2022.07.04.498665.full
AB  - 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 StatementThe authors have declared no competing interest.AEXanion exchangeAITCallyl isothiocyanate derived from mustard oilCNGBChina National GenebankAPCautomated patch clampCDcircular dichroism spectroscopyCGRPcalcitonin gene-related peptideC-LTMRsC-low threshold mechanoreceptorsCNScentral nervous systemcsPDIconotoxin-specific PDICVcolumn volumeDDBJDNA Databank of JapanDMEMDulbecco’s Modified Eagle’s MediumDRGdorsal root gangliaDTTdithiothreitolERendoplasmic reticulumFDAU.S. Food and Drug AdministrationFOMfigure of meritGFPgreen fluorescent proteinGPCRG protein-coupled receptorHEK293Thuman embryonic kidney cellshPDIhuman PDIIACUCInstitutional Animal Care and Use CommitteeIB4Alexa-Fluor 647 Azolectin B4IMACimmobilized metal affinity chromatographyIPTGisopropyl ß-D-1-thiogalactopyranosideLBlysogeny brothMALDI-TOFmatrix-assisted laser desorption-ionization-time of flightMPCmanual patch clampNCBINational Center for Biotechnology InformationNSSnormal saline solutionPDIprotein-disulfide isomeraseRMSDroot mean square deviationRP-HPLCreversed-phase high pressure liquid chromatographySAPLIPSaposin like proteinsSAXSsmall angle x-ray scattering SVDSDS-PAGEsodium dodecyl sulfate-polyacrylamide gel electrophoresisSLCsaposin-like conotoxinSRseal resistantTEVtobacco etch virusUbubiquitinUb-His10Ub containing 10 consecutive histidines.