Molecular Determinants of Syntaxin 1 Modulation of N-type Calcium Channels

• Published in: The Journal of biological chemistry 2002-11, Vol.277 (46), p.44399-44407
• Main Authors: Jarvis, Scott E, Barr, Wendy, Feng, Zhong-Ping, Hamid, Jawed, Zamponi, Gerald W
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Antigens, Surface - genetics; Antigens, Surface - metabolism; Antigens, Surface - physiology; Blotting, Western; Brain - metabolism; Calcium Channels, N-Type - metabolism; Cattle; Cloning, Molecular; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Glutathione Transferase - metabolism; Models, Biological; Models, Molecular; Molecular Sequence Data; Mutation; Nerve Tissue Proteins - genetics; Nerve Tissue Proteins - metabolism; Nerve Tissue Proteins - physiology; Patch-Clamp Techniques; Polymerase Chain Reaction; Protein Binding; Protein Conformation; Protein Structure, Tertiary; Rats; Recombinant Fusion Proteins - metabolism; Sequence Homology, Amino Acid; Syntaxin 1; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M206902200

We have previously reported that syntaxin 1A, a component of the presynaptic SNARE complex, directly modulates N-type calcium channel gating in addition to promoting tonic G-protein inhibition of the channels, whereas syntaxin 1B affects channel gating but does not support G-protein modulation (Jarvis, S. E., and Zamponi, G. W. (2001) J. Neurosci. 21, 2939–2948). Here, we have investigated the molecular determinants that govern the action of syntaxin 1 isoforms on N-type calcium channel function. In vitro evidence shows that both syntaxin 1 isoforms physically interact with the G-protein β subunit and the synaptic protein interaction (synprint) site contained within the N-type calcium channel domain II–III linker region. Moreover, in vitro evidence suggests that distinct domains of syntaxin participate in each interaction, with the COOH-terminal SNARE domain (residues 183–230) binding to Gβ and the N-terminal (residues 1–69) binding to the synprint motif of the channel. Electrophysiological analysis of chimeric syntaxin 1A/1B constructs reveals that the variable NH 2 -terminal domains of syntaxin 1 are responsible for the differential effects of syntaxin 1A and 1B on N-type calcium channel function. Because syntaxin 1 exists in both “open” and “closed” conformations during exocytosis, we produced a constitutively open form of syntaxin 1A and found that it still promoted G-protein inhibition of the channels, but it did not affect N-type channel availability. This state dependence of the ability of syntaxin 1 to mediate N-type calcium channel availability suggests that syntaxin 1 dynamically regulates N-type channel function during various steps of exocytosis. Finally, syntaxin 1A appeared to compete with Gγ for the Gβ subunit both in vitro and under physiological conditions, suggesting that syntaxin 1A may contain a G-protein γ subunit-like domain.