Long C terminal splice variant Cav2.2 identified in presynaptic membrane by mass spectrometric analysis
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1
Toronto Western Research Institute, Canada
Cav2.2 voltage gated calcium channels play a key role in the gating of transmitter release at presynaptic terminals. Recently we used mass spectrometry (MS) to analyze the protein complex associated with Cav2.2 in purified presynaptic terminal membranes (Khanna et al., J Biochem Mol Biol 40: 302, 2007). A number of known and new Cav2.2-associated proteins were identified, but not the channel itself.
Objective: We set out to explore the above mentioned anomaly and tested the hypothesis that the channel was missed due to high concentrations of co-precipitated proteins. As previously, antibody Ab571was used to capture the channel from purified synaptosome membrane lysate.
Materials and Methods: Cav2.2 specific antibody, Ab571, (Li et al., J Neurosci 24:4074, 2004) was immobilized onto the Protein A agarose gel and incubated with purified synaptosome membrane lysates. Protein complexes captured by the immobilized Ab571 were subjected to a high salt treatment and the remaining proteins were eluted with high concentration urea. Urea eluted protein complexes were analyzed by Liquid chromatography tandem mass spectrometry (LC MS/MS).
Results: LC MS/MS analysis of protein complexes that remained bound after the high salt treatment identified 12 distinct Cav2.2 peptides. These peptides were all from intracellular domains of the protein but otherwise their origins span almost the full-length of the protein, including 2 in the N terminus, 4 in the II-III loop, and 6 in the C terminus. One Cav2.2 peptide heralded from the alternatively spliced, long C terminal, region.
Conclusions: This finding is the first conclusive detection of a presynaptic Cav2-family calcium channel by mass spectrometry and the first detection of this particular channel type in any analysis. Furthermore, one peptide derived from the alternatively spliced, long-C terminal region, confirmed that this Cav2.2 splice variant is targeted to the presynaptic terminal.
Conference:
B.R.A.I.N. platform in Physiology poster day 2009, Toronto, ON, Canada, 16 Dec - 16 Dec, 2009.
Presentation Type:
Poster Presentation
Topic:
Poster presentations
Citation:
Gardezi
SR,
Taylor
P,
Khanna
R and
Stanley
EF
(2009). Long C terminal splice variant Cav2.2 identified in presynaptic membrane by mass spectrometric analysis.
Front. Neurosci.
Conference Abstract:
B.R.A.I.N. platform in Physiology poster day 2009.
doi: 10.3389/conf.neuro.03.2009.17.017
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Received:
17 Dec 2009;
Published Online:
17 Dec 2009.
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Correspondence:
Sabiha R Gardezi, Toronto Western Research Institute, Toronto, Canada, sabihag@gmail.com