Modulation of afterpotentials by calcium-dependent cationic conductances in pyramidal neurons

Afterpotentials are known to occur in pyramidal neurons of the hippocampus, where they help to shape the firing pattern. A novel afterdepolarizing current (IADP) has been previously characterised by the laboratory of Professor Paola Pedarzani. This current underlies a medium duration afterdepolarization. The aim of this PhD study was to determine whether TRPM2 (transient receptor potential, subfamily melastatin, member 2) ion channels mediate this IADP current. Due to the lack of channel-specific pharmacological tools, the approach of choice was downregulation of TRPM2 by generating mutant constructs. Firstly, the rat TRPM2 channel was cloned and its function and expression characterised in a heterologous expression system (HEK293 cells) by electrophysiological recordings, Western blotting and immunocytochemistry. Subsequently, dominant-negative rat TRPM2 mutant subunits were generated by introducing point mutations in the pore region or deleting parts of the amino and carboxyl terminal regions. All constructs were shown to reduce TRPM2 currents in HEK293 cells with varying efficiencies, when co-expressed with non-mutated TRPM2 subunits. The expression of a deletion mutant with an enhanced Green Fluorescent Protein (EGFP) tag was further quantified in HEK293 cells by Western blotting and immunocytochemistry, and was shown to interact with non-mutated TRPM2 subunits by co-immunoprecipitation. Transfection of this deletion mutant devoid of the EGFP-tag into rat primary hippocampal culture elicited a reduction in the frequency of action potential firing, suggesting changes in neuronal excitability. A contribution of TRPM2 in mediating the IADP is therefore possible, but further research is required to determine the extent