Regulation of splicing in a model voltage-gated Na+ channel

The mammalian central nervous system is composed of a multitude of nerve cell types. These cell types differ in the targets 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). The ability to fire action potentials is due to the presence of a variety of ion channels in the neuronal membrane. These proteins are encoded by genes, but we now know that the diversity of ion channel types present in the nervous system far exceeds the number of genes that encode for them. This increased diversity is due, in greater part, to a process termed alternative splicing. Essentially genes are composed of coding regions termed exons that are separated by non-coding regions called introns. When genes are expressed the initial transcript produced (called a pre-mRNA) contains both introns and exons. The first step on the road to making protein is to splice out (i.e. remove) the introns to produce a continuous coding sequence. However, we now know that in addition to introns, some exons can also be spliced out. This results in coding sequences that can differ and that in turn produce related, but similarly different proteins that ultimately form ion channels that have different functional properties. Thus, one gene can in fact encode many related proteins due to the existence of alternative splicing. Although the mechanics of splicing are quite well understood, how individual nerve cells 'decide' how to splice specific pre-mRNA transcripts remains unknown.

Diversity in neuronal signalling exceeds the capacity of the genome. We now appreciate that alternative splicing of ion channel transcripts underpins diversity in ion channel proteins observed in all nervous systems. In mammalian systems, each super-class of ion channel (e.g. voltage-gated Na channels) is encoded by multiple genes (9 in this case) which are each subjected to alternative splicing that results in a plethora of channel subtypes, each of which differs in function. These differences in function are critical to support appropriate neural network activity.

By contrast to mammals, insects contain far fewer genes but still utilise alternative splicing to generate diversity in proteins expressed. This fact is advantageous because it allows us to investigate how splicing is regulated and how splice variants differ in function without the complication of multiple related genes. For example, in Drosophila the voltage-gated Na channel is encoded by just a single gene (paralytic) that can be easily manipulated and/or removed using genetics. However, analysis of splicing of paralytic pre-mRNA identifies upwards to 60 individual splice variants that are expressed in a cell-specific manner. The key question we address is how do individual neurons 'choose' which splice variants they will express? Using a combination of molecular biology and electrophysiology we aim to understand the role and regulation of ion channel splicing in the determination of biophysical properties of individual neurons.

The work contained within this grant is basic in nature but has clear strategic relevance in relation to the treatment and possible cure for epilepsy. This work will also validate the use of an invertebrate model organism - Drosophila melanogaster - for the continued investigation of the causes and treatments for this significant disease. Thus it has the potential to reduce the usage of larger animals that are currently used for such research. As such this programme of work falls under the 3R's initiative.

The beneficiaries of this work can be divided into 2 main groupings:

1. Our published demonstration that splicing of voltage-gated Na channels has marked effects on function has significant implications for understanding electrical signaling and the diseases that arise when signaling is aberrant. Of particular relevance here is our demonstration that inclusion of either exon K or L markedly affects the magnitude of the persistent voltage-gated Na current. This current component is already implicated in epilepsy and, indeed, a number of antiepileptic drugs target this specific component of the Na current in neurons. There is considerable controversy in the mammalian neuroscience field as to how the persistent current is produced: our finding that exons K/L affect this current component identify a region of the channel that is important in this regard. This has significant implications to both basic Neuroscientists who are attempting to understand structure:function but also to Neuroscientists in the clinic investigating the underlying causes and treatments for epilepsy.

2. The development of treatments for disease requires the involvement of large pharmaceutical companies. However, nearly all treatments currently available can have their origins traced back to basic research undertaken in Universities. We are very conscious of the roles that pharmaceutical companies play in development of treatments and our research will be of direct benefit to those companies actively pursuing treatments for this disease.

Communications & Engagement

In addition to traditional means (research publications and conferences) we will disseminate our research as follows:

1. Through direct contact with Charities such as Epilepsy Research UK and The International League Against Epilepsy. We will inform such charities of our work and to highlight, in particular, the utility of using non-mammalian animal models (which is usually under-appreciated).
2. Through contact with the Media. For example, I took part in radio 4's Material World in Nov 2008 to highlight the use of Drosophila for research in to human diseases.
3. I also have a dedicated lab website which I use to advertise the type of research that we carry out
4. As an active member of the teaching staff at Manchester, I also use and advertise my research to undergraduates through lectures and final year projects in the hope of encouraging some to consider this area of research for their future careers. For example a student in my lab this year designed and presented a workshop to 6th form students on the utility of non-mammalian models for neurological research.

Collaboration

I have recently obtained funding, from The Wellcome Trust, to develop a core-Drosophila facility at Manchester to encourage a greater degree of interactivity and collaboration between existing fly groups and those wishing to exploit this model system for the first time. Many researchers using mammalian models could benefit by incorporating this fly into their research programmes and this facility will reduce the inertia to do so.

Exploitation & Application

Drosophila offers the opportunity to develop cheap, large-scale, drug screens that are a viable alternative to using rodents. Such screens have already been undertaken for a number of diseases, including epilepsy (by Cambria Biosciences, USA, with whom I have an active collaboration).