The long-term aim of this project is to better understand the ubiquitination and regulation of Cav2.2e[37a] and Cav2.2e[37b]- two splice isoforms of the Cav2.2 N-type channel. These two isoforms differ in only fourteen amino acid residues, but have been shown to be ubiquitinated at different levels. These two isoforms also undergo an interesting interplay in the nociceptive neurons of the dorsal root ganglia (DRG). All neurons contain Cav2.2e[37b], however Cav2.2e[37a] has very limited expression, and is enriched in nociceptive neurons of the DRG. Since the Cav2.2 channel is already one of the most efficacious and specific targets to treat chronic pain, understanding the regulation of these two types of channels is likely to aid in understanding and treating chronic pain mediated through nociceptive neurons. The research proposed here aims to determine the mechanisms involved in regulating Cav2.2e[37a] and Cav2.2e[37b] function through ubiquitination. This proposal has three aims.
Aim one will utilize novel mouse lines that express only one isoform of Cav2.2- Cav2.2e[37a] or Cav2.2e[37b], to determine ubiquitination levels of each isoform in vivo. Immunoprecipitation (IP) of the channel will be performed from these mouse lines and run on SDS-PAGE gel. Afterward a western blot will be performed where ubiquitin and Cav2.2 are probed. Ubiquitination levels of the two isoforms will be compared.
The second aim will utilize mutants of ubiquitin to determine the type of ubiquitin attachment to the two channel isoforms. These experiments will utilize at least three mutants of ubiquitin (KOR, K48R, and K63R) that are deficient in forming ubiquitin chains. The mutants will be expressed in primary DRG neuronal cultures of the mice proposed to be used in aim one. The channel will then be immunoprecipitated, and probed for ubiquitin to determine if either isoform is modified with ubiquitin chains differentially.
For aim three, mutants of the channel will be created in which stretches of amino acids from Cav2.2e[37a] will be replaced with those from Cav2.2e[37b], and vice versa, to determine which of the fourteen amino acids are responsible for ubiquitination of Cav2.2e[37b]. After this aim is completed, point mutants of Cav2.2 may also be created to determine the exact amino acid residue(s) responsible for the differential ubiquitination. This work is relevant to public health because it will lead to better understanding of the pathways underlying the degradation and modulation of N-type calcium channels. If these pathways can be better understood, then treatments for chronic pain can likely be created that are more potent and cause fewer side-effects than those that currently exist.

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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Marangoudakis, Spiro; Andrade, Arturo; Helton, Thomas D et al. (2012) Differential ubiquitination and proteasome regulation of Ca(V)2.2 N-type channel splice isoforms. J Neurosci 32:10365-9
Andrade, Arturo; Denome, Sylvia; Jiang, Yu-Qiu et al. (2010) Opioid inhibition of N-type Ca2+ channels and spinal analgesia couple to alternative splicing. Nat Neurosci 13:1249-56