A proteomic screen for presynaptic terminal N-type calcium channel (CaV2.2) binding partners.
N type calcium channels (CaV2.2) play a key role in the gating of transmitter release at presynaptic nerve terminals. These channels are generally regarded as parts of a multimolecular complex that can modulate their open probability and ensure their location near the vesicle docking and fusion sites. However, the proteins that comprise this component remain poorly characterized. We have carried out the first open screen of presynaptic CaV2.2 complex members by an antibody-mediated capture of the channel from purified rat brain synaptosome lysate followed by mass spectroscopy. 589 unique peptides resulted in a high confidence match of 104 total proteins and 40 synaptosome proteome proteins. This screen identified several known CaV2.2 interacting proteins including syntaxin 1, VAMP, protein phosphatase 2A, G(O alpha), G beta and spectrin and also a number of novel proteins, including clathrin, adaptin, dynamin, dynein, NSF and actin. The unexpected proteins were classified within a number of functional classes that include exocytosis, endocytosis, cytoplasmic matrix, modulators, chaperones, and cell-signaling molecules and this list was contrasted to previous reports that catalogue the synaptosome proteome. The failure to detect any postsynaptic density proteins suggests that the channel itself does not exhibit stable trans-synaptic attachments. Our results suggest that the channel is anchored to a cytoplasmic matrix related to the previously described particle web.
Khanna
Rajesh
R
Cellular and Molecular Biology Division, Toronto Western Research Institute, University Health Network, Toronto, Ontario, Canada.
Zougman
Alexandre
A
Stanley
Elise F
EF
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D020864:Calcium Channels, N-Type / Q000378:metabolism*; D002352:Carrier Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D002853:Chromatography, Liquid; D047468:Immunoprecipitation; D008969:Molecular Sequence Data; D017729:Presynaptic Terminals / Q000378:metabolism*; D011485:Protein Binding; D020543:Proteome / Q000032:analysis* / Q000235:genetics; D040901:Proteomics / Q000379:methods; D051381:Rats; D017207:Rats, Sprague-Dawley; D013574:Synaptosomes / Q000378:metabolism; D053719:Tandem Mass Spectrometry
D020864:Calcium Channels, N-Type; D002352:Carrier Proteins; D020543:Proteome