ORCID

http://orcid.org/0000-0002-9847-0531

Date of Award

2020

Document Type

Thesis

Degree Name

MS in Biology

Department

Biological Sciences

First Advisor

Matteo Ruggiu

Second Advisor

Dianella Howarth

Third Advisor

Javier Juarez

Abstract

AL-type voltage-gated calcium channel Cav1.3 contains a pore-forming α-1 subunit that is encoded by Cacna1d, a gene which has been shown to undergo alternative splicing (AS). Mutations in CACNA1D have been associated with neurological diseases such as spontaneous seizure disorder and epilepsy. Previous studies have shown that AS of the C-terminus of Cav1.3 results in a truncated variant of exon 42 (E42) that, when compared to the full-length exon, lowers the voltage threshold needed to activate the calcium channel. Therefore, AS of the a1-subunit can modulate the physiological properties of Cav1.3. As a lower voltage threshold of calcium channels increases neural excitability, AS of Cav1.3 could have many clinical implications.

Previous studies have also linked the alternatively spliced N-terminus of Cav1.3 to autism spectrum disorder (ASD). In a human patient study, naturally occurring missense mutations in mutually exclusive exons 8a and 8b have been correlated with various neurological disorders. These results highlight the important role that AS plays in Cav1.3 function. We were able to confirm four alternatively spliced regions, E8-E11, E12-E15, E32-36, and E46-E48 in Cacna1d. By semi-quantitative RT-PCR analysis, we found that these alternatively spliced variants are expressed at higher levels in brain-derived tissues when compared to non-brain derived tissues. We were also able to construct minigenes of E13, E33-E34, E35, and E47 to test their regulation by common splicing factors (SF) Nova- 1, Nova-2, Ptbp-2, Rbfox-1, and Mbln2. While AS of Cav1.3 has been shown to play important roles in electrophysiological properties and neurological disorders, how AS of Cav1.3 is regulated remains poorly understood. Our results will serve to characterize the mechanism of AS in Cacna1d for future studies aimed at determining whether or not certain splice isoforms lead to changes in the channel’s electrophysiological properties.

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