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The Journal of Neuroscience, December 1, 2001, 21(23):9142-9150

Targeted Mutations in the Syntaxin H3 Domain Specifically Disrupt SNARE Complex Function in Synaptic Transmission

Tim Fergestad¹, Mark N. Wu², Karen L. Schulze³, Thomas E. Lloyd², Hugo J. Bellen^{2, 3, 4}, and Kendal Broadie¹

¹ Department of Biology, University of Utah, Salt Lake City, Utah 84112-0840, and Departments of ² Molecular and Cellular Biology and ³ Molecular and Human Genetics, ⁴ Howard Hughes Medical Institute, Baylor College of Medicine, Houston, Texas 77030

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; 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 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 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.

Key words: Drosophila; SNARE complex; core complex; syntaxin; synaptotagmin; calcium channel

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Copyright © 2001 Society for Neuroscience  0270-6474/01/21239142-09$05.00/0

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