Identification of a sensory neuron Cav2.3 inhibitor within a new superfamily of macro-conotoxins

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. * 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.