Functional analysis of the C2A domain of synaptotagmin 1: implications for calcium-regulated secretion.
Synaptotagmin 1 is proposed to function as a low affinity calcium sensor for calcium-triggered exocytosis from neural and neuroendocrine cells. Because of the calcium-binding properties of the C2A domain of synaptotagmin 1, calcium-dependent interactions through this domain may modulate neurotransmitter release. We addressed this question by using alanine-scanning mutagenesis to generate a series of mutations within the C2A domain of synaptotagmin 1. The effects of these mutations on synaptotagmin 1 C2A function were analyzed for (1) calcium-dependent phospholipid binding, (2) calcium-dependent binding to syntaxin 1A, a plasma membrane protein critical for vesicle docking or fusion, and (3) calcium-regulated secretion after microinjection into neuroendocrine pheochromocytoma (PC12) cells. Our analyses reveal that a polylysine motif at residues 189-192 confers an inhibitory effect on secretion by recombinant synaptotagmin C2A fragments. The synaptotagmin 1 C2A polylysine motif functions independently of calcium-mediated interactions with phospholipids and syntaxin 1A. Furthermore, alpha-latrotoxin reverses the inhibitory effect of injected recombinant C2A fragments, suggesting that they perturb the cellular calcium-sensing machinery by interfering with synaptotagmin 1 activity in vivo. Our results indicate that novel calcium-independent interactions mediated through the C2A polylysine motif of synaptotagmin 1 function to modulate neurotransmitter release.
Thomas
D M
DM
Wayne State University, Department of Biological Sciences, Detroit, Michigan 48202, USA.
Elferink
L A
LA
D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000235:genetics*; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D008239:Lysine; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics*; D008565:Membrane Proteins / Q000378:metabolism; D016296:Mutagenesis / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D018377:Neurotransmitter Agents / Q000378:metabolism; D016716:PC12 Cells; D010446:Peptide Fragments / Q000235:genetics; D010743:Phospholipids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D050765:Qa-SNARE Proteins; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics; D013111:Spider Venoms / Q000494:pharmacology; D050863:Synaptotagmin I; D050857:Synaptotagmins; D050827:Syntaxin 1; D014966:Xanthenes / Q000493:pharmacokinetics
D002135:Calcium-Binding Proteins; D005456:Fluorescent Dyes; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D018377:Neurotransmitter Agents; D010446:Peptide Fragments; D010743:Phospholipids; D050765:Qa-SNARE Proteins; D011994:Recombinant Proteins; D013111:Spider Venoms; C494213:Stx1a protein, rat; D050863:Synaptotagmin I; D050827:Syntaxin 1; C494312:Syt1 protein, rat; D014966:Xanthenes; D050857:Synaptotagmins; C020763:alpha-latrotoxin; C034657:Texas red; D008239:Lysine; D002118:Calcium