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The Journal of Neuroscience, April 21, 2004, 24(16):4070-4081; doi:10.1523/JNEUROSCI.0346-04.2004

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

Qi Li, ^* Anthony Lau, ^* Terence J. Morris, ^* Lin Guo, Christopher B. Fordyce, and Elise F. Stanley

Cellular and Molecular Biology Division, Toronto Western Research Institute, University Health Network, Toronto, Ontario, M5T 2S8 Canada

Presynaptic Ca_V2.2 (N-type) calcium channels are subject to modulation by interaction with syntaxin 1 and by a syntaxin 1-sensitive G_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_V2.2 with syntaxin 1 and G_O. Ca_V2.2 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_V2.2 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_V2.2 puncta, whereas the bulk colocalized with MUNC18-1. G_O, whether in its trimeric or monomeric state, did not coimmunoprecipitate with Ca_V2.2, 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_V2.2 and G_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.

Key words: calcium channel; presynaptic; N-type; Ca_V2.2; 1B subunit; syntaxin; G-protein; G_O; transmitter release site; n-sec-1; MUNC18-1; intensity correlation analysis; intensity correlation quotient; colocalization; immunocytochemistry

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Received Jan 30, 2004; revised March 8, 2004; accepted March 12, 2004.

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