Caractérisation des sous-unités principales et auxiliaires des canaux sodium dépendant du potentiel exprimées dans le système nerveux central de l'insecte Periplaneta americana (Thèse de Doctorat d'Université)

Abstract

In insects, only one gene encodes the α pore-forming subunit of voltage-gated sodium channel (Nav). In addition, four to five genes encode the β auxiliary subunits. Although the American cockroach, Periplaneta americana, is a model in insect neurophysiology, few data describe the molecular structures at the base of the Nav. The aim of our study was to characterize the subunits expressed in the nerve cord (NC), the terminal abdominal ganglion (TAG) and DUM neurons of this particular species. First, we showed that the molecular diversity of the α subunit is generated by alternative splicing in NC and TAG. The main alternative exons identified in the cloned cDNA are A (loop L1), EFGHG1129 (loop L2) and G1 (IIIS3-S4). Despite of the molecular diversity, only one 6153-bp cDNA encoding PaNav1 has been identified. This isoform is characterized by the alternative exons (J, A; E; F; G1129, H and G1) highly expressed in the NC and TAG. On the opposite, only one type of cDNA has been identified in DUM neurons. Altogether, these results show that molecular diversity is lower at the cellular level. Moreover, a bioinformatic analysis showed the optional exons I and F are the most divergent currently known exons between insect orders. In addition, we discovered two full-length cDNAs encoding atypical α subunits named PaFPC1 and PaFPC2. They contain 1553 amino acid residues and differ from each other by eight punctual mutations. They share 59% protein identity with PaNav1. The absence of the inactivation particle MFMT suggests that these novel subunits mediate original electrophysiological properties. However, a detailed phylogenetic analysis puts forward that PaFPC is a novel member of the insect Nav channels. Hence; this discovery shows for the first time the existence of an ancestral duplication event of the Nav gene in insects. Finally, we cloned two cDNA populations encoding auxiliary subunits named PaTEH1.1 and PaTEH1.2. A phylogenetic analysis clarifies the relationships between the different families of insect auxiliary subunit. Moreover, a preliminary electrophysiological study showed that PaTEH1.1 increased significatively the expression of the pore-forming subunit of insect Nav in Xenopus oocytes. In conclusion, our results constitute new insights in insect ion channels field. In addition, they open new research perspectives for understanding the molecular basis of the membrane excitability in insects