Phosphorylation of synaptotagmin-1 controls a post-priming step in PKC-dependent presynaptic plasticity

Abstract

Presynaptic activation of the diacylglycerol (DAG)/protein kinase C (PKC) pathway is a central event in short-term synaptic plasticity. Two substrates, Munc13-1 and Munc18-1, are essential for DAGinduced potentiation of vesicle priming, but the role of most presynaptic PKC substrates is not understood. Here, we show that a mutation in synaptotagmin-1 (Syt1 T112A ), which prevents its PKCdependent phosphorylation, abolishes DAG-induced potentiation of synaptic transmission in hippocampal neurons. This mutant also reduces potentiation of spontaneous release, but only if alternative Ca 2+ sensors, Doc2A/B proteins, are absent. However, unlike mutations in Munc13-1 or Munc18-1 that prevent DAG-induced potentiation, the synaptotagmin-1 mutation does not affect pairedpulse facilitation. Furthermore, experiments to probe vesicle priming (recovery after train stimulation and dual application of hypertonic solutions) also reveal no abnormalities. Expression of synaptotagmin-2, which lacks a seven amino acid sequence that contains the phosphorylation site in synaptotagmin-1, or a synaptotagmin-1 variant with these seven residues removed (Syt1 Δ109-116 ), supports normal DAG-induced potentiation. These data suggest that this seven residue sequence in synaptotagmin-1 situated in the linker between the transmembrane and C2A domains is inhibitory in the unphosphorylated state and becomes permissive of potentiation upon phosphorylation. We conclude that synaptotagmin-1 phosphorylation is an essential step in PKC-dependent potentiation of synaptic transmission, acting downstream of the two other essential DAG/PKC substrates, Munc13-1 and Munc18-1.P resynaptic strength changes rapidly during repetitive stimulation [short-term plasticity (STP)] and activation of intracellular signal transduction pathways (1, 2). The diacylglycerol (DAG)/protein kinase C (PKC) cascade is one of the most potent pathways at the presynaptic terminal. Its activation leads to 50-100% potentiation of spontaneous and action potential (AP)-evoked release (3-5), and is critical for multiple forms of presynaptic plasticity (6-8). DAG directly activates the vesicle priming factor Munc13-1 (9, 10) and indirectly activates downstream effectors via PKC. Activation of both Munc13-1 and PKC is essential for this pathway to operate (Fig. 1A) (8, 11). We previously identified Munc18-1 as an essential PKC substrate, because a nonphosphorylatable Munc18-1 mutant completely inhibits PKC-dependent STP (8, 12). Importantly, a phosphomimetic mutation of Munc18-1 cannot fully bypass the requirement for PKC activation, indicating that other PKC substrates must contribute to this form of plasticity (8). These substrates have not been identified to date.Among other presynaptic PKC substrates is synaptotagmin-1 (Syt1, ref. 13). Syt1 is the vesicular Ca 2+ sensor that mediates fast AP-evoked release in the hippocampus (14) and drives a large fraction of spontaneous release (15). In the latter case, Syt1 competes with alternative sensors, in particular Doc2s, for SNARE binding ...

Tyrosine phosphorylation of Munc18‐1 inhibits synaptic transmission by preventing SNARE assembly

“…However, the inability of M18 Y473D to stimulate fusion in our reconstituted membrane fusion assay, which lacks CAPS, Munc13, and RIM‐BP, suggests that an inherent Munc18‐1 function is (also) inhibited (Fig E and F). Diacylglycerol (DAG) activates Munc13 (Rhee et al , ; Basu et al , ) and PKC, which phosphorylates Munc18‐1 (Wierda et al , ; Genc et al , ) and Synaptotagmin1 (de Jong et al , ) and increases release rates probably by lowering the energy barrier for fusion (Basu et al , ; Schotten et al , ). Our results suggest that tyrosine phosphorylation of Munc18‐1 does the opposite: In the phosphorylated state, the energy barrier for fusion appears to be increased, leading to lower vesicular release probability and low spontaneous fusion rates.…”

Synaptic UNC13A protein variant causes increased neurotransmission and dyskinetic movement disorder

“…Application of PDBU leads to a rapid (<5 seconds) augmentation of the evoked EPSC amplitude, due to an increase in p r that is mediated by direct activation of Munc13 proteins (32,36,63,64) and by PKC-mediated phosphorylation of Munc18-1 (64, 65) and synaptotagmin 1 (66). The PDBU augmentation ratio of hippocampal neurons expressing Munc13-1 WT was significantly higher than that measured for neurons expressing Munc13-1 P827L (P < 0.001, 5 seconds after PDBU application; Table 2).…”

C2‐domain containing calcium sensors in neuroendocrine secretion

“…Recently, PKC phosphorylation at T112 within the linker region of syt-1 (Hilfiker et al 1999) was identified as a prerequisite for phorbol ester stimulation of excitatory postsynaptic current amplitudes in autaptic cultured hippocampal neurons (de Jong et al 2016). In contrast, in embryonic mouse, chromaffin cells mutating this residue was without effect .…”