Direct interaction of the calcium sensor protein synaptotagmin I with a cytoplasmic domain of the alpha1A subunit of the P/Q-type calcium channel.
Synaptotagmins are synaptic vesicle proteins containing two calcium-binding C2 domains which are involved in coupling calcium influx through voltage-gated channels to vesicle fusion and exocytosis of neurotransmitters. The interaction of synaptotagmins with native P/Q-type calcium channels was studied in solubilized synaptosomes from rat cerebellum. Antibodies against synaptotagmins I and II, but not IV co-immunoprecipitated [125I]omega-conotoxin MVIIC-labelled calcium channels. Direct interactions were studied between in vitro-translated [35S]synaptotagmin I and fusion proteins containing cytoplasmic loops of the alpha1A subunit (BI isoform). Gel overlay revealed the association of synaptotagmin I with a single region (residues 780-969) located in the intracellular loop connecting homologous domains II and III. Saturable calcium-independent binding occurred with equilibrium dissociation constants of 70 nM and 340 nM at 4 degrees C and pH 7.4, and association was blocked by addition of excess recombinant synaptotagmin I. Direct synaptotagmin binding to the pore-forming subunit of the P/Q-type channel may optimally locate the calcium-binding sites that initiate exocytosis within a zone of voltage-gated calcium entry.
Charvin
N
N
Institut National de la Sant'e et de la Recherche M'edicale, Unit'e 464, Institut Jean Roche, Facult'e de M'edecine Secteur Nord, Marseille, France.
L'evêque
C
C
Walker
D
D
Berton
F
F
Raymond
C
C
Kataoka
M
M
Shoji-Kasai
Y
Y
Takahashi
M
M
De Waard
M
M
Seagar
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001665:Binding Sites; D015220:Calcium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002135:Calcium-Binding Proteins; D002531:Cerebellum / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013574:Synaptosomes / Q000378:metabolism; D050863:Synaptotagmin I; D050861:Synaptotagmin II; D050857:Synaptotagmins; D020864:Calcium Channels, N-Type
D000906:Antibodies; D015220:Calcium Channels; D002135:Calcium-Binding Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D050863:Synaptotagmin I; D050861:Synaptotagmin II; D050857:Synaptotagmins; C491367:Cacna1a protein, rat; D020864:Calcium Channels, N-Type; C494312:Syt1 protein, rat; C494264:Syt2 protein, rat