Direct interaction between synaptotagmin and the intracellular loop I-II of neuronal voltage-sensitive sodium channels.
Synaptotagmin, a synaptic vesicle protein involved in Ca(2+)-regulated exocytosis, displayed direct high affinity interaction with neuronal sodium channels. Monoclonal antibodies directed against synaptotagmins I and II adsorbed in a concentration-dependent and -specific manner [(3)H]saxitoxin prelabeled sodium channels extracted with detergent from nerve endings. Conversely, co-immunoprecipitation of synaptotagmin was achieved by antibodies against sodium channel subunits. Consistent with the co-immunoprecipitation assays, solubilized [(3)H]saxitoxin-prelabeled sodium channels were trapped on immobilized maltose binding protein (MBP)-synaptotagmin I. In vitro recombinant protein assays were employed to identify the interaction site of synaptotagmin I, which was located on the cytoplasmic loop between domains I and II of the sodium channel alphaIIA subunit. The co-immunoprecipitated synaptotagmin-sodium channel complexes were found to be Ca(2+)-dependent; this effect was mimicked by Ba(2+) and Sr(2+) but not Mg(2+). Finally the complex was shown to be distinct from the synaptotagmin-SNARE protein complex that can selectively interact with presynaptic calcium channels (N and P/Q types). Thus, our findings demonstrate an unexpected and direct interaction between sodium channels and synaptotagmin. The Ca(2+)-regulated association between sodium channels and a protein implicated in vesicular fusion may have intriguing consequences for the establishment and regulation of neuronal excitability.
Sampo
B
B
Institut National de la Santé et de la Recherche Médicale, U464, Institut Jean Roche, Université de la Méditerranée, Boulevard P. Dramard, 13916 Marseille Cedex 20, France.
Tricaud
N
N
Leveque
C
C
Seagar
M
M
Couraud
F
F
Dargent
B
B
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins; D015640:Ion Channel Gating; D008562:Membrane Glycoproteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism / Q000502:physiology*; D011233:Precipitin Tests; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D015222:Sodium Channels / Q000737:chemistry / Q000378:metabolism*; D050863:Synaptotagmin I; D050857:Synaptotagmins
D002135:Calcium-Binding Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D015222:Sodium Channels; D050863:Synaptotagmin I; C494312:Syt1 protein, rat; D050857:Synaptotagmins; D002118:Calcium