A Neuronal Sec1 Homolog Regulates Neurotransmitter Release at the Squid Giant Synapse

Abstract

Sec1-related proteins are essential for membrane fusion at distinct stages of the constitutive and regulated secretory pathways in eukaryotic cells. Studies of neuronal isoforms of the Sec1 protein family have yielded evidence for both positive and negative regulatory functions of these proteins in neurotransmitter release. Here, we have identified a squid neuronal homolog (s-Sec1) of Sec1 proteins and examined its function in neurotransmitter release at the squid giant synapse. Microinjection of s-Sec1 into the presynaptic terminal of the giant synapse inhibited evoked neurotransmitter release, but this effect was prevented by coinjecting the cytoplasmic domain of squid syntaxin (s-syntaxin), one of the binding partners of s-Sec1. A 24 amino acid peptide fragment of s-Sec1, which inhibited the binding of s-Sec1 to s-syntaxin in vitro, completely blocked release, suggesting an essential function of the s-Sec1/s-syntaxin interaction in transmitter release. Electron microscopy showed that injection of s-Sec1 did not change the spatial distribution of synaptic vesicles at presynaptic release sites ("active zones"), whereas the inhibitory peptide increased the number of docked vesicles. These distinct morphological effects lead us to conclude that Sec1 proteins function at different stages of synaptic vesicle exocytosis, and that an interaction of s-Sec1 with syntaxin-at a stage blocked by the peptide-is necessary for docked vesicles to fuse.

Evidence of a role for Munc18-2 and microtubules in mast cell granule exocytosis

“…The absence of inhibition by Munc18-3 in mast cells was somewhat surprising given that it binds to syntaxin 4, which is known to play a role in mast cell exocytosis (Paumet et al, 2000). However, we know from previous studies that overexpression of Munc18 isoforms does not always result in inhibition of exocytosis and could even be stimulatory in some cases (Dresbach et al, 1998;Graham et al, 1997;Schulze et al, 1994;Voets et al, 2001). Further studies are necessary to establish the role of Munc18-3.…”

Munc18: A Presynaptic Transmitter Release Site N type (CaV2.2) Calcium Channel Interacting Protein

“…The most direct demonstration was the finding that although injection of a (Squid)-UNC18 peptide into the squid giant synapse presynaptic terminal led to a pronounced inhibition of transmitter release, calcium influx was unaffected. 12 When Munc18 was coexpressed with CaV2.2 in a cell line the protein induced a G protein like, voltage sensitive inhibition of the calcium channels, 37 an effect that was not explored here. However, no study has carried out a systematic analysis of possible CaV2.2 current modulation by Munc18.…”

“…9,10 While the high-affinity interaction of Munc18 with syntaxin 1 must reflect an important biological function, this is unlikely to be its only role in neurosecretion. Indeed, Munc18 has been suggested to also be involved in the docking of secretory vesicles, 4,11 supported both by peptide injection into the squid giant synapse 12 and observations on knockouts and mutants of Unc-18 in c.elegans 4 or Munc18 in mouse anterior pituitary 13 and chromaffin cells. 14 Several studies have concluded that Munc18 is not involved in the initial contact of the vesicle with the membrane but in subsequent prefusion steps [5][6][7] that lead to the formation of a stable prefusion attachment.…”

Munc18-1 expression levels control synapse recovery by regulating readily releasable pool size

“…Genetic deletion of Munc18-1 and most other SM genes involved in synaptic-vesicle release across species results in a complete block of neurotransmitter release (3)(4)(5), which shows that Munc18-1 and probably all SM proteins are indispensable factors that promote vesicle secretion (2,6,7). However, identifying where SM proteins act in the cascade of events leading to the release of neurotransmitter has proven to be difficult and has generated apparently conflicting data (8)(9)(10).…”