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PMID: 11717347 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

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

The Journal of neuroscience : the official journal of the Society for Neuroscience ·Vol. 21 ·No. 23 ·2001-12-01 ·Pages 9142-50

Fergestad T, Wu MN, Schulze KL, Lloyd TE, Bellen HJ, Broadie K

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 (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.

MeSH Terms
Amino Acid Substitution Animals Animals, Genetically Modified Antigens, Surface/genetics,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,metabolism Neurons/metabolism Neurotransmitter Agents/genetics,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
Chemicals
Antigens, Surface Macromolecular Substances Membrane Proteins Nerve Tissue Proteins Neurotransmitter Agents SNARE Proteins Saline Solution, Hypertonic Syntaxin 1 Vesicular Transport Proteins
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Fergestad T
Department of Biology, University of Utah, Salt Lake City, Utah 84112-0840, USA.
Wu M N
Schulze K L
Lloyd T E
Bellen H J
Broadie K
References (54)
54 references, click to expand
  1. The synaptic vesicle protein synaptotagmin associates with calcium channels and is a putative Lambert-Eaton myasthenic syndrome antigen.
    Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3625-9 PMID: 1314395
  2. SNARE complex formation is triggered by Ca2+ and drives membrane fusion.
    Cell. 1999 Apr 16;97(2):165-74 PMID: 10219238
  3. Distinct domains of syntaxin are required for synaptic vesicle fusion complex formation and dissociation.
    Neuron. 1995 May;14(5):991-8 PMID: 7748566
  4. Syntaxin: a synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones.
    Science. 1992 Jul 10;257(5067):255-9 PMID: 1321498
  5. Syntaxin and synaptobrevin function downstream of vesicle docking in Drosophila.
    Neuron. 1995 Sep;15(3):663-73 PMID: 7546745
  6. Interactions between proteins implicated in exocytosis and voltage-gated calcium channels.
    Philos Trans R Soc Lond B Biol Sci. 1999 Feb 28;354(1381):289-97 PMID: 10212477
  7. Synaptotagmin restores kinetic properties of a syntaxin-associated N-type voltage sensitive calcium channel.
    FEBS Lett. 1997 Mar 10;404(2-3):203-7 PMID: 9119064
  8. A conformational switch in syntaxin during exocytosis: role of munc18.
    EMBO J. 1999 Aug 16;18(16):4372-82 PMID: 10449403
  9. Expression of synaptotagmin in Drosophila reveals transport and localization of synaptic vesicles to the synapse.
    Development. 1993 Aug;118(4):1077-88 PMID: 8269841
  10. Identification of a minimal core of the synaptic SNARE complex sufficient for reversible assembly and disassembly.
    Biochemistry. 1998 Jul 21;37(29):10354-62 PMID: 9671503
  11. The synaptic protein syntaxin1 is required for cellularization of Drosophila embryos.
    J Cell Biol. 1997 Aug 25;138(4):861-75 PMID: 9265652
  12. Membrane fusion. SNAREs line up in new environment.
    Nature. 1998 May 7;393(6680):14-5 PMID: 9590682
  13. Structure and function of neuronal Ca2+ channels and their role in neurotransmitter release.
    Cell Calcium. 1998 Nov-Dec;24(5-6):307-23 PMID: 10091001
  14. The Caenorhabditis elegans unc-64 locus encodes a syntaxin that interacts genetically with synaptobrevin.
    Mol Biol Cell. 1998 Jun;9(6):1235-52 PMID: 9614171
  15. Functional impact of syntaxin on gating of N-type and Q-type calcium channels.
    Nature. 1995 Dec 7;378(6557):623-6 PMID: 8524397
  16. Effect of mutations in vesicle-associated membrane protein (VAMP) on the assembly of multimeric protein complexes.
    J Neurosci. 1997 Mar 1;17(5):1596-603 PMID: 9030619
  17. Inhibition of neurotransmission by peptides containing the synaptic protein interaction site of N-type Ca2+ channels.
    Neuron. 1996 Oct;17(4):781-8 PMID: 8893034
  18. Synaptic vesicle fusion complex contains unc-18 homologue bound to syntaxin.
    Nature. 1993 Nov 25;366(6453):347-51 PMID: 8247129
  19. Interaction of the synprint site of N-type Ca2+ channels with the C2B domain of synaptotagmin I.
    Proc Natl Acad Sci U S A. 1997 May 13;94(10):5405-10 PMID: 9144250
  20. Vectors for P-mediated transformation in Drosophila.
    Biotechnology. 1988;10:437-56 PMID: 2850048
  21. Physical link and functional coupling of presynaptic calcium channels and the synaptic vesicle docking/fusion machinery.
    J Bioenerg Biomembr. 1998 Aug;30(4):335-45 PMID: 9758330
  22. Definition of the readily releasable pool of vesicles at hippocampal synapses.
    Neuron. 1996 Jun;16(6):1197-207 PMID: 8663996
  23. Genetic transformation of Drosophila with transposable element vectors.
    Science. 1982 Oct 22;218(4570):348-53 PMID: 6289436
  24. Syntaxin modulation of calcium channels in cortical synaptosomes as revealed by botulinum toxin C1.
    J Neurosci. 2000 Jun 15;20(12):4368-78 PMID: 10844005
  25. Alteration of Ca2+ dependence of neurotransmitter release by disruption of Ca2+ channel/syntaxin interaction.
    J Neurosci. 1997 Sep 1;17(17):6647-56 PMID: 9254677
  26. Functional interaction of syntaxin and SNAP-25 with voltage-sensitive L- and N-type Ca2+ channels.
    EMBO J. 1996 Aug 15;15(16):4100-10 PMID: 8861939
  27. Ca2+-dependent and -independent interactions of the isoforms of the alpha1A subunit of brain Ca2+ channels with presynaptic SNARE proteins.
    Proc Natl Acad Sci U S A. 1997 Dec 23;94(26):14782-6 PMID: 9405690
  28. Mutational analysis of Drosophila synaptotagmin demonstrates its essential role in Ca(2+)-activated neurotransmitter release.
    Cell. 1993 Sep 24;74(6):1125-34 PMID: 8104705
  29. Development of the embryonic neuromuscular synapse of Drosophila melanogaster.
    J Neurosci. 1993 Jan;13(1):144-66 PMID: 8093713
  30. Kinetics of synaptotagmin responses to Ca2+ and assembly with the core SNARE complex onto membranes.
    Neuron. 1999 Oct;24(2):363-76 PMID: 10571230
  31. The C terminus of SNAP25 is essential for Ca(2+)-dependent binding of synaptotagmin to SNARE complexes.
    J Biol Chem. 2000 Mar 3;275(9):6328-36 PMID: 10692432
  32. Genetic and electrophysiological studies of Drosophila syntaxin-1A demonstrate its role in nonneuronal secretion and neurotransmission.
    Cell. 1995 Jan 27;80(2):311-20 PMID: 7834751
  33. Drosophila syntaxin is required for cell viability and may function in membrane formation and stabilization.
    Genetics. 1996 Dec;144(4):1713-24 PMID: 8978057
  34. Absence of synaptotagmin disrupts excitation-secretion coupling during synaptic transmission.
    Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10727-31 PMID: 7938019
  35. Reciprocal regulation of P/Q-type Ca2+ channels by SNAP-25, syntaxin and synaptotagmin.
    Nat Neurosci. 1999 Nov;2(11):939-41 PMID: 10526329
  36. Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 A resolution.
    Nature. 1998 Sep 24;395(6700):347-53 PMID: 9759724
  37. The biochemistry of neurotransmitter secretion.
    J Biol Chem. 1995 Feb 3;270(5):1971-4 PMID: 7836421
  38. N-type voltage-sensitive calcium channel interacts with syntaxin, synaptotagmin and SNAP-25 in a multiprotein complex.
    Receptors Channels. 1998;6(2):89-98 PMID: 9932286
  39. Molecular determinants of the functional interaction between syntaxin and N-type Ca2+ channel gating.
    Proc Natl Acad Sci U S A. 2000 Dec 5;97(25):13943-8 PMID: 11087812
  40. Structural insights into the molecular mechanism of Ca(2+)-dependent exocytosis.
    Curr Opin Neurobiol. 2000 Jun;10(3):293-302 PMID: 10851178
  41. Munc13-1 is essential for fusion competence of glutamatergic synaptic vesicles.
    Nature. 1999 Jul 29;400(6743):457-61 PMID: 10440375
  42. SNAREpins: minimal machinery for membrane fusion.
    Cell. 1998 Mar 20;92(6):759-72 PMID: 9529252
  43. Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly.
    EMBO J. 1994 Nov 1;13(21):5051-61 PMID: 7957071
  44. Drosophila UNC-13 is essential for synaptic transmission.
    Nat Neurosci. 1999 Nov;2(11):965-71 PMID: 10526334
  45. nSec1 binds a closed conformation of syntaxin1A.
    J Cell Biol. 2000 Jan 24;148(2):247-52 PMID: 10648557
  46. The voltage sensitive Lc-type Ca2+ channel is functionally coupled to the exocytotic machinery.
    Proc Natl Acad Sci U S A. 1999 Jan 5;96(1):248-53 PMID: 9874804
  47. Morphologically docked synaptic vesicles are reduced in synaptotagmin mutants of Drosophila.
    J Neurosci. 1998 Oct 1;18(19):7662-73 PMID: 9742137
  48. Synaptocanalins (N-type Ca channel-associated proteins) form a complex with SNAP-25 and synaptotagmin.
    Ann N Y Acad Sci. 1993 Dec 20;707:373-5 PMID: 9137572
  49. A cysteine-string protein is expressed in retina and brain of Drosophila.
    J Neurogenet. 1990 Nov;7(1):15-29 PMID: 2129171
  50. Mutations in the Drosophila Rop gene suggest a function in general secretion and synaptic transmission.
    Neuron. 1994 Sep;13(3):555-66 PMID: 7917291
  51. UNC-13 is required for synaptic vesicle fusion in C. elegans.
    Nat Neurosci. 1999 Nov;2(11):959-64 PMID: 10526333
  52. Temperature-sensitive paralytic mutations demonstrate that synaptic exocytosis requires SNARE complex assembly and disassembly.
    Neuron. 1998 Aug;21(2):401-13 PMID: 9728921
  53. synaptotagmin mutants reveal essential functions for the C2B domain in Ca2+-triggered fusion and recycling of synaptic vesicles in vivo.
    J Neurosci. 2001 Mar 1;21(5):1421-33 PMID: 11222632
  54. Syntaxin 1A interacts with multiple exocytic proteins to regulate neurotransmitter release in vivo.
    Neuron. 1999 Jul;23(3):593-605 PMID: 10433270
Article Info
Journal
The Journal of neuroscience : the official journal of the Society for Neuroscience
Abbr.
J Neurosci
ISSN
1529-2401
Published
2001-12-01
Pages
9142-50
Language
English
Region
United States
NLM ID
8102140
PMCID
PMC6763887
Subset
IM
Grants
NICHD NIH HHS · 5T32HD07491 · United States
NHLBI NIH HHS · 5T32HL07747 · United States
NIGMS NIH HHS · GM53571 · United States
NIGMS NIH HHS · GM54544 · United States
External Links
PubMed source PMC Full Text View journal details
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