Alternative splicing in the nervous system is an important regulator of neuronal function. CaV2.2 calcium channels control neurotransmission and are extensively alternatively spliced in a cell-type specific manner. Splice isoforms of these channels have distinct basic biophysical properties and unique responses to G protein-coupled receptors. RNA-binding proteins called splicing factors regulate the inclusion of alternative exons. There are several methods that can be used to identify the splicing factors that regulate inclusion of a particular exon. In this dissertation, I explore the factors that regulate the inclusion of functionally important alternative exons within CaV2.2. In Chapter 2, I show that Nova-2 represses inclusion of exons 31a in CaV2.1 and CaV2.2 in the central nervous system and present evidence that Nova-2 enhances inclusion of an exon which I discovered, exon 24a in CaV2.1. In Chapter 3, I show that Fox proteins repress inclusion of exon 18a in CaV2.2 and that this splicing regulation controls the voltage-independent inhibition of channel proteins by Gs-coupled receptor agonists. In Chapter 4, I present results from minigene and bioinformatics analyses that were used to attempt to identify the splicing factors that regulate the splicing of mutually exclusive exons 37a and 37b in CaV2.2. Inclusion of exon 37a in CaV2.2 channels in neurons of dorsal root ganglia allows Gi/o coupled-receptor agonists, like morphine, to inhibit CaV2.2 channels in a voltage-independent manner. Although I have yet to identify the splicing factors involved, I present evidence from minigene studies that suggests a model including a repressor of exon 37a and an enhancer of exon 37b. Bioinformatic analyses suggest that hnRNP-A/B and hnRNP-F may regulate inclusion of these exons. In the discussion chapter I examine how studies such as mine can provide important insight into cell-specific optimization of protein function. I also discuss models of splicing regulation and exciting avenues for future research.
Allen, Summer E.,
"Cell-specific splicing factors that optimize calcium channel function"
Neuroscience Theses and Dissertations.
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