Direct interaction between synaptotagmin and the intracellular loop I-II of neuronal voltage-sensitive sodium channels

Abstract

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 ␣IIA 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 P͞Q 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. O ne of the major physiological roles of voltage-gated sodium channels is to initiate and propagate action potentials in excitable cells. In neurons, after the generation of a large transient Na ϩ current at the axon hillock͞initial segment or at the first of node Ranvier (reviewed in ref. 1), sodium channels ensure conduction along myelinated or unmyelinated fibers to nerve terminals. Sodium channels also participate in the integration of synaptic input and the modulation of firing properties and mediate the backpropagation of action potentials into the dendritic arborization, in certain types of neurons (reviewed in ref. 1). In addition, sodium channels can produce a non-inactivating Na ϩ current that only constitutes a small fraction of the total Na ϩ current but strongly affects neuronal firing properties (for a review, see ref.2).At the molecular level, sodium channels purified from rat brain nerve endings are composed of a heterotrimeric complex. The highly glycosylated ␣ subunit (260 kDa), which is the pore-forming protein, is associated noncovalently with the ␤1 subunit (36 kDa), and with the ␤2 subunit (33 kDa) via disulfide bonds (for a review, see ref.3). At least four genes encoding distinct ␣ subtypes that are mainly expressed in the central nervous system have been identified: ␣I and ␣II͞␣IIA (4, 5), ␣III (6), and ␣6 (7). In contrast, each auxiliary subunit is encoded by a single gene (8,9).In nerve terminals, the arrival of the depolarizing wave triggers the opening of presynaptic N-and P͞Q type calcium channels, produ...

Proteomic analysis of native cerebellar iFGF14 complexes

“…No significant differences in the amounts of Navb1, Navb2 or Navb4 subunits immunoprecipitating from WT and Fgf14 ¡/¡ with the maPanNav antibody were observed. Consistent with previous reports that Syt2 directly interacts with Nav a subunits, 25 there was no apparent difference in the amount of Syt2 that could be precipitated from WT versus Fgf14 ¡/¡ cerebellar lysates with the maPanNav antibody (Fig. 5B).…”

“…In addition to iFGF14, the Nav a subunits, Nav1.1, Nav1.2, Nav1.4, and Nav1.6, as well as several proteins previously shown to interact with Nav a subunits, including Navb1, Navb2, Navb4, calmodulin, synaptotagmin-2, ankyrin G, casein kinase II and 14.3.3, [22][23][24][25][26][27] were also identified ( Fig. 3 and Table 1).…”

Synaptotagmin III/VII Isoforms Mediate Ca2+-induced Insulin Secretion in Pancreatic Islet β-Cells

“…For example, Syt III may sense an increase in intracellular Ca 2ϩ due to influx of extracellular Ca 2ϩ , whereas Syt VII may be relevant when intracellular Ca 2ϩ elevation is induced by intracellular mobilization through inositol 1,4,5-trisphosphate receptors. It has been demonstrated that synaptotagmin directly interacts with N-type (49 -57) or P/Q-type (19,54,55) Ca 2ϩ channels in neurons and Lc-type Ca 2ϩ channels (20). Inositol 1,4,5-trisphosphate receptors are present in pancreatic ␤-cells; however, it is currently unclear whether they associate with synaptotagmin.…”

Fibroblast Growth Factor Homologous Factor 1B Binds to the C Terminus of the Tetrodotoxin-resistant Sodium Channel rNav1.9a (NaN)

“…More recently, the C-terminal polypeptide of rNa v 1.2 (brain type II) was shown to bind calmodulin, suggesting modulation of these channels by Ca 2ϩ (23), and the rNa v 1.2-L1 was shown to interact with synaptotagmin in a Ca 2ϩ -regulated manner (46). An interaction with synaptotagmin may modulate sodium channel activity by controlling the accessibility of a protein kinase A phosphorylation site located inside the binding region of this complex or may play a role in the internalization of sodium channels (46). A more intriguing possibility is that this interaction may result in Na ϩ -dependent exocytosis (46).…”

“…An interaction with synaptotagmin may modulate sodium channel activity by controlling the accessibility of a protein kinase A phosphorylation site located inside the binding region of this complex or may play a role in the internalization of sodium channels (46). A more intriguing possibility is that this interaction may result in Na ϩ -dependent exocytosis (46). The TTX-R sodium channel rNa v 1.9a is preferentially expressed in small diameter C-type sensory neurons of DRG and trigeminal ganglia (14).…”