Molecular Characterisation of the Voltage-Gated Sodium Channel from Aphids

Abstract

We describe the gene structure and primary sequence of the voltage-gated sodium channel (VGSC) of Acyrthosiphon pisum (Harris) and the characterisation of the corresponding VGSC from the closely-related aphid Myzus persicae (Sulzer), an economically important crop pest. The A. pisum sodium channel sequence spans more than 46 Kb on a single scaffold of the recently-released second assembly of the pea aphid genome. In-silico analysis of the VGSC suggests that channel domains I and II are encoded separately from domains III and IV on two different genes. Northern and Southern blots of A. pisum genomic material are currently being employed to confirm our findings. The aphid genes show a high degree of sequence homology to VGSCs from other species, although closer analysis of the pore region reveals an unusual selectivity filter within the P-loops of the aphid channels consisting of DENS (domains I-IV) rather than the standard sodium selective DEKA filter. Site-directed mutagenesis of the Drosophila melanogaster ‘para’ VGSC was undertaken to recreate part or all of the aphid selectivity filter and subsequent expression of these modified channels in Xenopus oocytes yielded voltage-dependent currents with unusual properties. Homology modelling and ligand docking studies suggested that the aphid VGSCs may be insensitive to TTX. We have confirmed this prediction experimentally using both bioassay studies and voltage-clamp recordings of oocytes expressing ‘para’ channels modified within the TTX binding site. This is the first report of a VGSC encoded by two separate genes and containing an alternative selectivity filter.