Ciguatoxins (CTXs) are marine toxins that cause ciguatera fish poisoning, a debilitating disease dominated by sensory and neurological disturbances that include cold allodynia and various painful symptoms as well as long-lasting pruritus. Although CTXs are known as the most potent mammalian sodium channel activator toxins, the etiology of many of its neurosensory symptoms remains unresolved. We recently described that local application of 1 nM Pacific Ciguatoxin-1 (P-CTX-1) into the skin of human subjects induces a long-lasting, painful axon reflex flare and that CTXs are particularly effective in releasing calcitonin-gene related peptide (CGRP) from nerve terminals. In this study, we used mouse and rat skin preparations and enzyme-linked immunosorbent assays (ELISA) to study the molecular mechanism by which P-CTX-1 induces CGRP release. We show that P-CTX-1 induces CGRP release more effectively in mouse as compared to rat skin, exhibiting EC50 concentrations in the low nanomolar range. P-CTX-1-induced CGRP release from skin is dependent on extracellular calcium and sodium, but independent from the activation of various thermosensory transient receptor potential (TRP) ion channels. In contrast, lidocaine and tetrodotoxin (TTX) reduce CGRP release by 53-75%, with the remaining fraction involving L-type and T-type voltage-gated calcium channels (VGCC). Using transgenic mice, we revealed that the TTX-resistant voltage-gated sodium channel (VGSC) NaV1.9, but not NaV1.8 or NaV1.7 alone and the combined activation of the TTX-sensitive VGSC subtypes NaV1.7 and NaV1.1 carry the largest part of the P-CTX-1-caused CGRP release of 42% and 34%, respectively. Given the contribution of CGRP to nociceptive and itch sensing pathways, our findings contribute to a better understanding of sensory symptoms of acute and chronic ciguatera that may help in the identification of potential therapeutics.

Department of Anesthesiology Friedrich Alexander University Erlangen Nuremberg University Hospital Erlangen Krankenhausstraße 12 91054 Erlangen Germany

Department of Anesthesiology Friedrich Alexander University Erlangen Nuremberg University Hospital Erlangen Krankenhausstraße 12 91054 Erlangen Germany Department of Cellular Neurophysiology Institute of Physiology of the Czech Academy of Sciences Videnska 1083 142 20 Prague 4 Czech Republic

Department of Physiology and Pathophysiology Friedrich Alexander University Erlangen Nuremberg Universitätsstraße 17 91054 Erlangen Germany

Institute for Molecular Biosciences IMB Centre for Pain Research University of Queensland 4072 St Lucia Australia

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