A syntaxin 1, Galpha(o), and N-type calcium channel complex at a presynaptic nerve terminal: analysis by quantitative immunocolocalization

J Neurosci. 2004 Apr 21;24(16):4070-81. doi: 10.1523/JNEUROSCI.0346-04.2004.

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 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibody Specificity
  • Antigens, Surface / chemistry
  • Antigens, Surface / metabolism*
  • Blotting, Western
  • Brain Chemistry
  • Calcium Channels, N-Type / chemistry
  • Calcium Channels, N-Type / metabolism*
  • Cells, Cultured
  • Chick Embryo
  • GTP-Binding Protein alpha Subunits, Gi-Go / chemistry
  • GTP-Binding Protein alpha Subunits, Gi-Go / metabolism*
  • Immunohistochemistry / methods*
  • Macromolecular Substances
  • Munc18 Proteins
  • Nerve Tissue Proteins / chemistry
  • Nerve Tissue Proteins / metabolism*
  • Presynaptic Terminals / metabolism*
  • Rats
  • Syntaxin 1
  • Vesicular Transport Proteins / metabolism

Substances

  • Antigens, Surface
  • Calcium Channels, N-Type
  • Macromolecular Substances
  • Munc18 Proteins
  • Nerve Tissue Proteins
  • Stx1a protein, rat
  • Stxbp1 protein, rat
  • Syntaxin 1
  • Vesicular Transport Proteins
  • GTP-Binding Protein alpha Subunits, Gi-Go