Abstract
Cysteine string proteins (Csps) are J-domain chaperone proteins anchored at the surface of synaptic vesicles. Csps are involved in neurotransmitter release and may modulate presynaptic calcium channel activity, although the molecular mechanisms are unknown. Interactions between Csps, proteins of the synaptic core (SNARE) complex, and P/Q-type calcium channels were therefore explored. Co-immunoprecipitation suggested that Csps occur in complexes containing synaptobrevin (VAMP), but not syntaxin 1, SNAP-25, nor P/Q-type calcium channels labeled with 125I-omega-conotoxin MVIIC. However binding experiments with 35S-labeled Csp1 demonstrated an interaction (apparent KD = 700 nM at pH 7.4 and 4 degreesC) with a fusion protein containing a segment of the cytoplasmic loop linking homologous domains II-III of the alpha1A calcium channel subunit (BI isoform, residues 780-969). Binding was specific as it was displaced by unlabeled Csp1, and no interactions were detected with fusion proteins containing other calcium channel domains, VAMP, or syntaxin 1A. A Csp binding site on the P/Q-type calcium channel is thus located within the 200 residue synaptic protein interaction site that can also bind syntaxin I, SNAP-25, and synaptotagmin I. Csp may act as a molecular chaperone to direct assembly or disassembly of exocytotic complexes at the calcium channel.
Highlights
Cysteine string proteins (Csps)1 were initially discovered in Drosophila as neuronal antigens predominantly localized in synaptic regions [1]
First we performed co-immunoprecipitation experiments to examine the hypothesis that Csps may associate with components of the trimeric synaptic core (SNARE) complex and/or presynaptic P/Q-type calcium channels
Co-immunoprecipitation Experiments Reveal Protein Complexes Containing Csp and VAMP—Rat brain membranes were solubilized with CHAPS, and detergent extracts were incubated with non-immune immunoglobulins, polyclonal antibodies raised against Csp1 in the absence and presence of MBPCsp1, and polyclonal anti-VAMP antibodies
Summary
Csp(s), cysteine string protein(s); Hsc or Hsp, heat shock proteins; MBP, maltose-binding protein; GST, glutathione S-transferase; VAMP, vesicle-associated membrane protein or synaptobrevin; TBS, Tris-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; SNAP-25, synaptosomal associated 25-kDa protein; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis. The association of Csps with synaptic vesicles [7, 10] suggests that they are involved in membrane trafficking and/or exocytosis of neurotransmitters. Recent findings suggest that the default involves either a deficit in calcium entry or in the ability of calcium to trigger exocytosis [16]. These data are consistent with earlier results indicating that Csps act as positive modulators of -conotoxin GVIA-sensitive calcium channels from Torpedo electric organ, heterologously expressed in Xenopus oocytes [2]. Fusion competent synaptic vesicles are thought to be docked at the active zone in close proximity to the voltage-gated calcium channels that trigger release. We have examined the molecular interactions of Csp with proteins of the synaptic core (SNARE) complex [17] and P/Q-type calcium channels, which support a major fraction of transmitter release in many synaptic fields of the mammalian brain [18]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
