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

J Neurosci. 2001 Dec 1;21(23):9142-50. doi: 10.1523/JNEUROSCI.21-23-09142.2001.


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 (Ala-240, Val-244) in H3 layers 4 and 5 of mammalian syntaxin1A 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 syntaxin1A (A243V, V247A; syx(4) mutant) to examine their significance in synaptic transmission in vivo. The syx(4) mutant animals are embryonic lethal and display severely impaired neuronal secretion, although non-neuronal 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 syx(4) mutants show no alterations in synaptic protein levels in vivo or syntaxin partner binding interactions in vitro. Rather, syx(4) 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 Syx(4) 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Substitution
  • Animals
  • Animals, Genetically Modified
  • Antigens, Surface / genetics*
  • Antigens, Surface / metabolism*
  • Conserved Sequence / physiology
  • Drosophila
  • Embryo, Nonmammalian / physiology
  • Evoked Potentials / physiology
  • Gene Targeting
  • Genes, Lethal
  • Macromolecular Substances
  • Membrane Proteins / metabolism*
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Nerve Tissue Proteins / genetics*
  • Nerve Tissue Proteins / metabolism*
  • Neurons / metabolism
  • Neurotransmitter Agents / genetics
  • Neurotransmitter Agents / metabolism
  • Phenotype
  • Protein Binding / physiology
  • Protein Structure, Tertiary / physiology
  • SNARE Proteins
  • Saline Solution, Hypertonic / pharmacology
  • Sequence Homology, Amino Acid
  • Structure-Activity Relationship
  • Synapses / metabolism
  • Synaptic Transmission / physiology*
  • Syntaxin 1
  • Vesicular Transport Proteins*


  • Antigens, Surface
  • Macromolecular Substances
  • Membrane Proteins
  • Nerve Tissue Proteins
  • Neurotransmitter Agents
  • SNARE Proteins
  • Saline Solution, Hypertonic
  • Syntaxin 1
  • Vesicular Transport Proteins