Targeted mutations in the syntaxin H3 domain specifically disrupt SNARE complex function in synaptic transmission

T. Fergestad, M. N. Wu, K. L. Schulze, T. E. Lloyd, H. J. Bellen, K. Broadie

Research output: Contribution to journalArticlepeer-review

Abstract

The cytoplasmic H3 helical domain of syntaxin is implicated in numerous protein-protein interactions required for the assembly and stability of the SNARE complex mediating vesicular fusion at the synapse. Two specific hydrophobic residues (Ala240, Val-244) in H3 layers 4 and 5 of mammalian syntaxin 1A have been suggested to be involved in SNARE complex stability and required for the inhibitory effects of syntaxin on N-type calcium channels. We have generated the equivalent double point mutations in Drosophila syntaxin 1A (A243V, V247A;syx4 mutant) to examine their significance in synaptic transmission in vivo. The syx4 mutant animals are embryonic lethal and display severely impaired neuronal secretion, although nonneuronal secretion appears normal. Synaptic transmission is nearly abolished, with residual transmission delayed, highly variable, and nonsynchronous, strongly reminiscent of transmission in null synaptotagmin I mutants. However, the syx4 mutants show no alterations in synaptic protein levels in vivo or syntaxin partner binding interactions in vitro. Rather, syx4 mutant animals have severely impaired hypertonic saline response in vivo, an assay indicating loss of fusion-competent synaptic vesicles, and in vitro SNARE complexes containing Syx4 protein have significantly compromised stability. These data suggest that the same residues required for syntaxin-mediated calcium channel inhibition are required for the generation of fusion-competent vesicles in a neuronal-specific mechanism acting at synapses.

Original languageEnglish (US)
Pages (from-to)9142-9150
Number of pages9
JournalJournal of Neuroscience
Volume21
Issue number23
DOIs
StatePublished - Dec 1 2001
Externally publishedYes

Keywords

  • Calcium channel
  • Core complex
  • Drosophila
  • SNARE complex
  • Synaptotagmin
  • Syntaxin

ASJC Scopus subject areas

  • Neuroscience(all)

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