How does alternate splicing of a sodium channel gene generate diversity in neuronal signalling?

The human brain contains a staggeringly large number of nerve cells (neurons) that form networks of connections (synapses) with one another. These neuronal circuits control almost everything we do, including behaviours such as breathing, walking and learning. Diversity of behaviour is mirrored by the need for a similar degree of diversity of function of individual nerve cells. Thus, neurons differ in the target that they contact, the signalling chemicals (neurotransmitters) they release, and the way in which they are able to fire electrical action potentials that trigger release of their neurotransmitter(s). Indeed, the degree of diversity of these characteristics is likely orders of magnitude greater than the number of genes present in the genome and, as such, additional mechanisms are required. Recent research suggests that additional diversity is due, at least in part, to different protein products being produced from the same genes. Alternate splicing, as this is called, results in multiple related proteins being encoded within one gene. Each protein variant produced has the capability of conferring different properties to the neuron in which expression occurs. Although now established, it remains largely to be shown how splicing induces changes to neuron function. We use the fruitfly because it is very amenable to genetic analysis, the complete genome has been sequenced, and because it provides a simple model of the human nervous system. Our study will inform us how our own nerve cells are able to develop to have different properties from one another.

To function correctly, developing neurons must acquire a characteristic mix of voltage-gated ion channels to allow appropriate integration of synaptic excitation and action potential firing. We now know that the extensive diversity that exists in neuronal signalling exceeds that observed in the genome. Thus, additional mechanisms are required. One such mechanism is splicing. Through such mechanisms, single genes can produce up to thousands of related transcripts that encode channels with subtle differences in function; differences that translate to diversity in action potential firing. Although now well understood, the ways in which splicing contributes to modification of channel activity remains poor. The Na+ current present in Drosophila neurons is encoded by paralytic and we have identified multiple splice variants of this gene that results in channels with differing functional properties. The question remains as to how each variant contributes to neuronal signalling. This study will identify the spatial distribution of differing splice variants of this important gene and will correlate this with the signalling properties of those neurons shown to contain different spliced variants. Moreover, we will determine how splicing affects channel function. Primarily we will concentrate on two more probable mechanisms; first the possibility that splicing changes protein-protein interactions of the paralytic channel with other cofactors. Second, we will model the consequence to overall structure of the paralytic channel protein due to inclusion of alternately spliced exons. The information provided will represent one of the most detailed investigations of how regulated splicing underlies the emergence of appropriate behaviour in a developing organism.