Characterisation of Drosophila tipE as a functional orthologue of the voltage gated Na+ channel β1 subunit

Voltage gated Na+ channels (VGSCs) are heteromeric protein complexes responsible for the initiation and propagation of action potentials. They consist of pore forming α subunits (Nav1.1 – Nav1.9) and auxiliary β subunits (β1- β4), which modulate channel activity and are cell adhesion molecules. The β1 subunit regulates cell adhesion, neurite outgrowth and neuronal migration. Drosophila melanogaster possess many orthologous genes associated with human disease. Drosophila Temperature Induced Paralysis Locus E (tipE) is considered an auxiliary VGSC subunit due to its ability to increase functional VGSC expression in Xenopus oocytes, although unlike β1 it is not thought to regulate adhesion. The Drosophila stock tipE1,se carries a point mutation at the tipE locus which results in a reversible, temperature-sensitive paralytic phenotype. The purpose of this study was to interrogate the functional effects of β1 and tipE expression on physiological behaviour in Drosophila, thus ascertaining whether tipE may be a functional orthologue to the mammalian VGSC β1 subunit. Through physiological assays, we found a locomotor deficit in both tipE1,se mutant adults and larvae, which is exacerbated by an increase in temperature. Furthermore, we saw an evident trend towards decreased ERG amplitude at elevated temperatures in tipE1,se mutants. Knocking down tipE using RNAi and RNAi-dicer2 in various tissues suggested an increased climbing ability and hyperactivity at room temperature in adult Drosophila. In contrast, RNAi-dicer2 expressing flies did not show a temperature-sensitive phenotype. Expression of mammalian β1 subunit in the motor neurons of Drosophila larvae mildly inhibited larval locomotor function. The work described in this thesis adds new information on tipE and β1 function. Our results provide evidence that the tipE1,se mutation may disrupt motor function due to a protein truncation. However, further work is still required to address whether tipE can recapitulate β1 function