Activity-dependent phosphorylation of Ser187 is required for SNAP-25-negative modulation of neuronal voltage-gated calcium channels

Abstract

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a SNARE protein that regulates neurotransmission by the formation of a complex with syntaxin 1 and synaptobrevin/VAMP2. SNAP-25 also reduces neuronal calcium responses to stimuli, but neither the functional relevance nor the molecular mechanisms of this modulation have been clarified. In this study, we demonstrate that hippocampal slices from Snap25 ؉/؊ mice display a significantly larger facilitation and that higher calcium peaks are reached after depolarization by Snap25 ؊/؊ and Snap25 ؉/؊ cultured neurons compared with wild type. We also show that SNAP-25b modulates calcium dynamics by inhibiting voltage-gated calcium channels (VGCCs) and that PKC phosphorylation of SNAP-25 at ser187 is essential for this process, as indicated by the use of phosphomimetic (S187E) or nonphosphorylated (S187A) mutants. Neuronal activity is the trigger that induces the transient phosphorylation of SNAP-25 at ser187. Indeed, enhancement of network activity increases the levels of phosphorylated SNAP-25, whereas network inhibition reduces the extent of protein phosphorylation. A transient peak of SNAP-25 phosphorylation also is detectable in rat hippocampus in vivo after i.p. injection with kainate to induce seizures. These findings demonstrate that differences in the expression levels of SNAP-25 impact on calcium dynamics and neuronal plasticity, and that SNAP-25 phosphorylation, by promoting inhibition of VGCCs, may mediate a negative feedback modulation of neuronal activity during intense activation. S ynaptosomal-associated protein of 25 KDa (SNAP-25) belongs to the SNARE superfamily of membrane proteins that participate in the regulation of neuronal exocytosis. SNAP-25 is present in two isoforms, a and b, resulting from alternative splicing of the exon 5 of the gene, which is differentially expressed during development. SNAP-25a is expressed at the embryonic stage, and SNAP-25b becomes the major isoform postnatally (1-3). SNAP-25 is anchored to the cytosolic face of membranes by palmitoyl side chains located in the central region of the molecule and contributes two ␣-helices to the exocytotic fusion complex, together with syntaxin-1 and synaptobrevin/ VAMP2 (4-5). SNAP-25 also interacts with the synaptic vesicle protein, synaptotagmin I (6), a major calcium sensor that regulates neurotransmitter release (7,8). Interaction of synaptotagmin with SNAP-25 is essential for the calcium-dependent triggering of membrane fusion (9) and for the control of fusion pore during the final steps of exocytosis (10). Furthermore, the C terminus of SNAP-25 is a target of G protein ␤-and ␥-subunits that mediate presynaptic inhibition (11). Therefore, SNAP-25 represents a multifunctional protein involved in the control of secretion by multiple interactions. Besides its well characterized role in regulating exocytosis, there is increasing evidence that SNAP-25 modulates various ion channels (12, 13). In particular, SNAP-25 physically interacts with different types of voltagegated calcium chann...

Intrinsic calcium dynamics control botulinum toxin A susceptibility in distinct neuronal populations

“…Conversely, downregulation of SNAP-25, which increases evoked calcium transients [5], makes glutamatergic neurons more resistant to BoNT/A. These data suggest that susceptibility of neurons to BoNT/A results from the intrinsic calcium responsiveness to depolarization, which, among different factors, is modulated by the level of expression of SNAP-25 [5,6,28]. Interestingly, different intracellular calcium dynamics do not affect toxin penetration [11].…”

“…In addition, the C-terminal region of SNAP-25 between BoNT/A and BoNT/E cleavage sites is also required for SNAP-25 modulation of voltage gated calcium currents [5,6].…”

“…It contributes two alpha-helices in the formation of the exocytotic fusion complex together with syntaxin-1 and synaptobrevin/VAMP [1,2]. Besides acting as a fundamental component of the SNARE complex, SNAP-25 has been shown to negatively modulate voltage gated calcium channels in various overexpression systems [3,4] and in cultured hippocampal neurons [5,6].…”

Early Exposure to Alcohol Leads to Permanent Impairment of Dendritic Excitability in Neocortical Pyramidal Neurons

“…It is known that voltagegated channel trafficking is regulated by neuronal activity (Kim et al, 2007). Pozzi et al (2008) demonstrated that the network activity can modulate neuronal voltage-gated calcium channels through the induction of protein phosphorylation. Interestingly, Servais et al (2007) observed that calcium entry into cerebellar Purkinje cells was reduced after early exposure to alcohol.…”

Native Presynaptic Metabotropic Glutamate Receptor 4 (mGluR4) Interacts with Exocytosis Proteins in Rat Cerebellum

“…This activation induces inhibition of presynaptic P/Q-type voltage-gated calcium channels, leading to reduction of glutamatergic synaptic transmission (32). Recently, it appears that this inhibition would involve the phosphorylation of SNAP-25 on Ser-187 by PKC (33,34). These results show the tight link between mGluR7 and scaffold/exocytosis proteins in the control of neurotransmission regulation via voltage-gated calcium channels.…”