A syntaxin 1, Galpha(o), and N-type calcium channel complex at a presynaptic nerve terminal: analysis by quantitative immunocolocalization.
Presynaptic Ca(V)2.2 (N-type) calcium channels are subject to modulation by interaction with syntaxin 1 and by a syntaxin 1-sensitive Galpha(O) G-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 Ca(V)2.2 with syntaxin 1 and Galpha(O). Ca(V)2.2 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 Ca(V)2.2 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 Ca(V)2.2 puncta, whereas the bulk colocalized with MUNC18-1. Galpha(O,) whether in its trimeric or monomeric state, did not coimmunoprecipitate with Ca(V)2.2, 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 Ca(V)2.2 and Galpha(O), 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.
Li
Qi
Q
Cellular and Molecular Biology Division, Toronto Western Research Institute, University Health Network, Toronto, Ontario, M5T 2S8 Canada.
Lau
Anthony
A
Morris
Terence J
TJ
Guo
Lin
L
Fordyce
Christopher B
CB
Stanley
Elise F
EF
D000818:Animals; D000918:Antibody Specificity; D000954:Antigens, Surface / Q000737:chemistry / Q000378:metabolism*; D015153:Blotting, Western; D001923:Brain Chemistry; D020864:Calcium Channels, N-Type / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D002642:Chick Embryo; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry / Q000378:metabolism*; D007150:Immunohistochemistry / Q000379:methods*; D046911:Macromolecular Substances; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D017729:Presynaptic Terminals / Q000378:metabolism*; D051381:Rats; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins / Q000378:metabolism
D000954:Antigens, Surface; D020864:Calcium Channels, N-Type; D046911:Macromolecular Substances; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; C494213:Stx1a protein, rat; C497939:Stxbp1 protein, rat; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go