Interaction of the Nav1.2a subunit of the voltage-dependent sodium channel with nodal ankyrinG. In vitro mapping of the interacting domains and association in synaptosomes.
Voltage-dependant sodium channels at the axon initial segment and nodes of Ranvier colocalize with the nodal isoforms of ankyrin(G) (Ank(G) node). Using fusion proteins derived from the intracellular regions of the Nav1.2a subunit and the Ank repeat domain of Ank(G) node, we mapped a major interaction site in the intracellular loop separating alpha subunit domains I-II. This 57-amino acid region binds the Ank repeat region with a K(D) value of 69 nm. We identified another site in intracellular loop III-IV, and we mapped both Nav1.2a binding sites on the ankyrin repeat domain to the region encompassing repeats 12-22. The ankyrin repeat domain did not bind the beta(1) and beta(2) subunit cytoplasmic regions. We showed that in cultured embryonic motoneurons, expression of the beta(2) subunit is not necessary for the colocalization of Ank(G) node with functional sodium channels at the axon initial segment. Antibodies directed against the beta(1) subunit intracellular region, alpha subunit loop III-IV, and Ank(G) node could not co-immunoprecipitate Ank(G) node and sodium channels from Triton X-100 solubilisates of rat brain synaptosomes. Co-immunoprecipitation of sodium channel alpha subunit and of the 270- and 480-kDa AnkG node isoforms was obtained when solubilization conditions that maximize membrane protein extraction were used. However, we could not find conditions that allowed for co-immunoprecipitation of ankyrin with the sodium channel beta(1) subunit.
Bouzidi
Mourad
M
INSERM U 464 Neurobiologie des Canaux Ioniques, Institut Jean Roche, Université de la Méditerranée, 13916 Marseille Cedex 20, France.
Tricaud
Nicolas
N
Giraud
Pierre
P
Kordeli
Ekaterini
E
Caillol
Ghislaine
G
Deleuze
Charlotte
C
Couraud
Francois
F
Alcaraz
Gisele
G
D000818:Animals; D017487:Ankyrins / Q000378:metabolism*; D001369:Axons / Q000378:metabolism; D001665:Binding Sites; D001921:Brain / Q000196:embryology / Q000378:metabolism; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D050356:Lipid Metabolism; D008856:Microscopy, Fluorescence; D062551:NAV1.2 Voltage-Gated Sodium Channel; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D017830:Octoxynol / Q000494:pharmacology; D011233:Precipitin Tests; D011485:Protein Binding; D020033:Protein Isoforms; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012964:Sodium / Q000378:metabolism*; D015222:Sodium Channels / Q000378:metabolism*; D013574:Synaptosomes / Q000378:metabolism
C490614:Ank3 protein, rat; D017487:Ankyrins; D018076:DNA, Complementary; D062551:NAV1.2 Voltage-Gated Sodium Channel; D009419:Nerve Tissue Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; C568253:Scn2A protein, rat; D015222:Sodium Channels; D017830:Octoxynol; D012964:Sodium