Ca2+-dependent Synaptotagmin Binding to SNAP-25 Is Essential for Ca2+-triggered Exocytosis

Neuron. 2002 May 16;34(4):599-611. doi: 10.1016/s0896-6273(02)00671-2.

Abstract

Synaptotagmin is a proposed Ca2+ sensor on the vesicle for regulated exocytosis and exhibits Ca2+-dependent binding to phospholipids, syntaxin, and SNAP-25 in vitro, but the mechanism by which Ca2+ triggers membrane fusion is uncertain. Previous studies suggested that SNAP-25 plays a role in the Ca2+ regulation of secretion. We found that synaptotagmins I and IX associate with SNAP-25 during Ca2+-dependent exocytosis in PC12 cells, and we identified C-terminal amino acids in SNAP-25 (Asp179, Asp186, Asp193) that are required for Ca2+-dependent synaptotagmin binding. Replacement of SNAP-25 in PC12 cells with SNAP-25 containing C-terminal Asp mutations led to a loss-of-function in regulated exocytosis at the Ca2+-dependent fusion step. These results indicate that the Ca2+-dependent interaction of synaptotagmin with SNAP-25 is essential for the Ca2+-dependent triggering of membrane fusion.

Publication types

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

MeSH terms

  • Amino Acid Sequence / genetics
  • Animals
  • Aspartic Acid / genetics
  • Aspartic Acid / metabolism
  • Calcium / metabolism
  • Calcium Signaling / physiology*
  • Calcium-Binding Proteins*
  • Central Nervous System / metabolism
  • Exocytosis / physiology*
  • Membrane Glycoproteins / metabolism*
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Mutation / genetics
  • Nerve Tissue Proteins / genetics
  • Nerve Tissue Proteins / metabolism*
  • Neurotransmitter Agents / metabolism
  • PC12 Cells
  • Presynaptic Terminals / metabolism*
  • Protein Binding / physiology
  • Protein Structure, Tertiary / genetics
  • Rats
  • SNARE Proteins
  • Synaptic Membranes / metabolism*
  • Synaptic Transmission / physiology
  • Synaptic Vesicles / metabolism*
  • Synaptosomal-Associated Protein 25
  • Synaptotagmins
  • Vesicular Transport Proteins*

Substances

  • Calcium-Binding Proteins
  • Membrane Glycoproteins
  • Membrane Proteins
  • Nerve Tissue Proteins
  • Neurotransmitter Agents
  • SNARE Proteins
  • Snap25 protein, rat
  • Synaptosomal-Associated Protein 25
  • Vesicular Transport Proteins
  • Synaptotagmins
  • Aspartic Acid
  • Calcium