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

TLDR

Presynaptic CaV2.2 (N‑type) calcium channels are modulated by interaction with syntaxin 1 and a syntaxin‑1‑sensitive Gαo G‑protein pathway. The study employed biochemical analysis of neuronal lysates and intensity‑correlation colocalization at the chick ciliary ganglion calyx to investigate the association of CaV2.2 with syntaxin 1 and Gαo. CaV2.2 localizes to release‑site puncta, syntaxin 1 co‑immunoprecipitates with it and with MUNC18‑1, Gαo shows punctate staining that correlates with CaV2.2 and syntaxin 1 but does not co‑immunoprecipitate, indicating syntaxin 1 forms two distinct presynaptic complexes—one minor at the release site with CaV2.2 and Gαo and one major remote with MUNC18‑1—that may serve different functions.

Abstract

Presynaptic Ca V 2.2 (N-type) calcium channels are subject to modulation by interaction with syntaxin 1 and by a syntaxin 1-sensitive Gα 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 Gα 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. Gα 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 Gα 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.