A Syntaxin 1, Gαo, and N-Type Calcium Channel Complex at a Presynaptic Nerve Terminal: Analysis by Quantitative Immunocolocalization

• DOI: 10.1523/jneurosci.0346-04.2004
• Publication Date: 2004-04-21T20:57:52Z
• Authors: Qi Li; Anthony Lau; Terence J. Morris; Lin Guo; Christopher B. Fo...; Elise F. Stanley
• Keywords: Brain Chemistry; 0303 health sciences; Macromolecular Substances; Blotting, Western; Presynaptic Terminals; Vesicular Transport Proteins; Syntaxin 1; Nerve Tissue Proteins; Chick Embryo; GTP-Binding Protein alpha Subunits, Gi-Go; Immunohistochemistry; Rats; 03 medical and health sciences; Calcium Channels, N-Type; Munc18 Proteins; Antibody Specificity; Antigens, Surface; Animals; Cells, Cultured

Presynaptic CaV2.2 (N-type) calcium channels are subject to modulation by interaction with syntaxin 1 and by a syntaxin 1-sensitive GαOG-protein pathway. We used biochemical analysis of neuronal tissue lysates and a new quantitative test of colocalization by intensity correlation analysis at the giant calyx-type presynaptic terminal of the chick ciliary ganglion to explore the association of CaV2.2 with syntaxin 1 and GαO. CaV2.2 could be localized by immunocytochemistry (antibody Ab571) in puncta on the release site aspect of the presynaptic terminal and close to synaptic vesicle clouds. Syntaxin 1 coimmunoprecipitated with CaV2.2 from chick brain and chick ciliary ganglia and was widely distributed on the presynaptic terminal membrane. A fraction of the total syntaxin 1 colocalized with the CaV2.2 puncta, whereas the bulk colocalized with MUNC18-1. GαO,whether in its trimeric or monomeric state, did not coimmunoprecipitate with CaV2.2, MUNC18-1, or syntaxin 1. However, the G-protein exhibited a punctate staining on the calyx membrane with an intensity that varied in synchrony with that for both Ca channels and syntaxin 1 but only weakly with MUNC18-1. Thus, syntaxin 1 appears to be a component of two separate complexes at the presynaptic terminal, a minor one at the transmitter release site with CaV2.2 and GαO, as well as in large clusters remote from the release site with MUNC18-1. These syntaxin 1 protein complexes may play distinct roles in presynaptic biology.