Description
- Abstract:
- CaV2.2 calcium channels are critical regulators of cellular calcium entry in neurons and couple electrical stimulation to neurotransmitter release. These channels are responsible for mediating the signals that support neuronal communication. Many agents, both endogenous and exogenous, are known to inhibit CaV2.2 channels, leading to modulation of neuronal signaling. Alternative spicing is also employed by the cell to regulate channel function. Alternative splicing is known to regulate CaV2.2 channel function by modifying biophysics, protein-protein interactions, as well as responses to signaling pathways. In this thesis I explore the alternative splicing of CaV2.2 and subsequent splicing-dependent effects on channel function. In Chapter 2 I employed a heterologous expression system and determined that two CaV2.2 isoforms (CaV2.2e[37a] and CaV2.2e[37b]) are differentially ubiquitinated. We also determined that active (surface) channels are differentially regulated by the proteasome in an isoform-specific manner. CaV2.2e[37b] channels are more robustly ubiquitinated and upregulated after MG132-mediated inhibition of the proteasome. This work was performed in a heterologous expression system, and in dorsal root ganglion (DRG) neurons of mice genetically engineered to express only one of the two channel isoforms. In Chapter 3 we show that CaV2.2e[37a] channels are more robustly inhibited by morphine through the �-opioid receptor pathway. We also showed via behavioral assays that wild type and CaV2.2e[37a]-only expressing mice respond more highly to morphine. Upon morphine treatment, these mice are more resistant to acute thermal pain than CaV2.2e[37b]-only expressing mice. This work extends from previous studies in our lab showing splicing-dependent effects on channel inhibition by G proteins. In Chapter 4 we aimed to determine if CaV2.2 channels can be inhibited by carbon nanoparticles, as was recently shown for potassium channels. In these experiments I performed electrophysiology on heterologously expressed CaV2.2 while applying suspensions containing carbon nanotubes. Through this work I determined that carbon nanotubes did in-fact inhibit CaV2.2 channels, however not through direct blockage of the pore as in potassium channels. The block in our case was mediated by catalyst-derived yttrium released from carbon nanotubes into the surrounding solution. This thesis documents and explores these three independent mechanisms of CaV2.2 downregulation.
- Notes:
- Thesis (Ph.D. -- Brown University (2011)
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Citation
Marangoudakis, Spiro A.,
"Molecular Mechanisms of CaV2.2 Calcium Channel Regulation"
(2011).
Molecular Biology, Cell Biology, and Biochemistry Theses and Dissertations.
Brown Digital Repository. Brown University Library.
https://doi.org/10.7301/Z0610XK2
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Molecular Biology, Cell Biology, and Biochemistry Theses and Dissertations
Theses and Dissertations for the Molecular Biology, Cell Biology, and Biochemistry department....