The sizes of mammalian proteomes are vast compared to the sizes of mammalian genomes. This is because the majority of genes encode for multiple protein variations. Cell controlled alternative pre-mRNA processing is the mechanism that generates diverse sets of proteins from a single gene. Surprisingly, little is known about the factors that regulate the splicing of specific genes, including voltage-gated calcium channels. Two sites of alternative splicing in the N-type (CaV2.2) calcium channel gene have exceptional biological interest. They regulate channel function and their expression patterns vary with neuron-type and development. An unaddressed question of outstanding importance is: what factors regulate the tissue-specific expression patterns of these exons? The goal of this proposal is to identify the proteins that regulate splicing of these two biologically important exons. Different methods will be used to answer this question, including siRNA, overexpressing protein by cDNA plasmids. Western blotting of proteins isolated from cell lines and primary neurons, and analysis of CaV2.2 calcium channel RNA and protein levels in tissue isolated from Nova-2 knockout mice.
The first aim i s to identify splicing factors that control exon 18a (el 8a) inclusion in CaV2.2 during splicing. This exon controls closed-state inactivation of the N-type channel and may regulate inhibition of the channel by Gq/11 G-proteins Intriguingly, levels of e18a-containing mRNAs vary by tissue-type and with age. The main goal of this first aim is to test if the Fox family of splicing factors represses inclusion of exon 18a in certain cells and if, conversely, the Nova family of splicing factors enhances its inclusion in specific cells.
The second aim i s to identify splicing factors that regulate exon 37a (e37a) inclusion during CaV2.2 pre-mRNA splicing. This exon controls the sensitivity of N-type calcium channels to inhibition by Gi/o G-proteins. The pattern of e37a and 37b expression is controlled at the single cell level. Nociceptors of dorsal root ganglia express channels containing e37a at a higher rate than other neurons, including non-nociceptors of dorsal root ganglia. The main goal of the second aim is to test the hypotheses that Nova-1/2 proteins repress inclusion of e37a and that hnRNP-F proteins enhance inclusion of e37b.

Public Health Relevance

This project will fill a gap in the knowledge of cellular factors that control the activity of neuronal voltage-gated calcium ion channels. It will also be relevant to understanding how certain neurons, including those that mediate pain, can express a specific calcium channel isoform. Ultimately, this work could have relevance to understanding how splicing patterns are altered in the chronic pain state and how they might be normalized.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Predoctoral Individual National Research Service Award (F31)
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Special Emphasis Panel (ZRG1-F03B-H (20))
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Silberberg, Shai D
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Brown University
Schools of Medicine
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Lipscombe, Diane; Andrade, Arturo; Allen, Summer E (2013) Alternative splicing: functional diversity among voltage-gated calcium channels and behavioral consequences. Biochim Biophys Acta 1828:1522-9
Allen, Summer E; Darnell, Robert B; Lipscombe, Diane (2010) The neuronal splicing factor Nova controls alternative splicing in N-type and P-type CaV2 calcium channels. Channels (Austin) 4:483-9