Edwin R. Chapman

Botulinum neurotoxin C1 blocks neurotransmitter release by means of cleaving HPC-1/syntaxin.

The anaerobic bacterium Clostridium botulinum produces several related neurotoxins that block exocytosis of synaptic vesicles in nerve terminals and that are responsible for the clinical manifestations of botulism. Recently, it was reported that botulinum neurotoxin type B as well as tetanus toxin act as zinc‐dependent proteases that specifically cleave synaptobrevin, a membrane protein of synaptic vesicles (Link et al., Biochem. Biophys. Res. Commun., 189, 1017‐1023; Schiavo et al., Nature, 359, 832‐835). Here we report that inhibition of neurotransmitter release by botulinum neurotoxin type C1 was associated with the proteolysis of HPC‐1 (= syntaxin), a membrane protein present in axonal and synaptic membranes. Breakdown of HPC‐1/syntaxin was selective since no other protein degradation was detectable. In vitro studies showed that the breakdown was due to a direct interaction between HPC‐1/syntaxin and the toxin light chain which acts as a metallo‐endoprotease. Toxin‐induced cleavage resulted in the generation of a soluble fragment of HPC‐1/syntaxin that is 2‐4 kDa smaller than the native protein. When HPC‐1/syntaxin was translated in vitro, cleavage occurred only when translation was performed in the presence of microsomes, although a full‐length product was obtained in the absence of membranes. However, susceptibility to toxin cleavage was restored when the product of membrane‐free translation was subsequently incorporated into artificial proteoliposomes. In addition, a translated form of HPC‐1/syntaxin, which lacked the putative transmembrane domain at the C‐terminus, was soluble and resistant to toxin action. We conclude that HPC‐1/syntaxin is involved in exocytotic membrane fusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Synaptotagmin: A Ca 2+ sensor that triggers exocytosis?

It has been fifty years since the discovery that Ca2+ triggers the rapid exocytosis of neurotransmitters from neurons. One of the proteins that has a crucial role in this secretion event is synaptotagmin I, an abundant constituent of synaptic vesicles that binds Ca2+ ions through two C2 domains. These properties prompted the idea that synaptotagmin I might function as a Ca2+-sensor that triggers neurotransmitter release. So does synaptotagmin trigger exocytosis in a Ca2+-dependent manner, and, if so, how does it operate?

How Does Synaptotagmin Trigger Neurotransmitter Release

Neurotransmitter release at synapses involves a highly specialized form of membrane fusion that is triggered by Ca(2+) ions and is optimized for speed. These observations were established decades ago, but only recently have the molecular mechanisms that underlie this process begun to come into view. Here, we summarize findings obtained from genetically modified neurons and neuroendocrine cells, as well as from reconstituted systems, which are beginning to reveal the molecular mechanism by which Ca(2+)-acting on the synaptic vesicle (SV) protein synaptotagmin I (syt)-triggers rapid exocytosis. This work sheds light not only on presynaptic aspects of synaptic transmission, but also on the fundamental problem of membrane fusion, which has remained a puzzle that has yet to be solved in any biological system.

PIP2 increases the speed of response of synaptotagmin and steers its membrane-penetration activity toward the plasma membrane.

Synaptotagmin-1 (syt), the putative Ca2+ sensor for exocytosis, is anchored to the membrane of secretory organelles. Its cytoplasmic domain is composed of two Ca2+-sensing modules, C2A and C2B. Syt binds phosphatidylinositol 4,5-bisphosphate (PIP2), a plasma membrane lipid with an essential role in exocytosis and endocytosis. We resolved two modes of PIP2 binding that are mediated by distinct surfaces on the C2B domain of syt. A novel Ca2+-independent mode of binding predisposes syt to penetrate PIP2-harboring target membranes in response to Ca2+ with submillisecond kinetics. Thus, PIP2 increases the speed of response of syt and steers its membrane-penetration activity toward the plasma membrane. We propose that syt-PIP2 interactions are involved in exocytosis by facilitating the close apposition of the vesicle and target membrane on rapid time scales in response to Ca2+.

Synaptotagmins I and II mediate entry of botulinum neurotoxin B into cells

Botulinum neurotoxins (BoNTs) cause botulism by entering neurons and cleaving proteins that mediate neurotransmitter release; disruption of exocytosis results in paralysis and death. The receptors for BoNTs are thought to be composed of both proteins and gangliosides; however, protein components that mediate toxin entry have not been identified. Using gain-of-function and loss-of-function approaches, we report here that the secretory vesicle proteins, synaptotagmins (syts) I and II, mediate the entry of BoNT/B (but not BoNT/A or E) into PC12 cells. Further, we demonstrate that BoNT/B entry into PC12 cells and rat diaphragm motor nerve terminals was activity dependent and can be blocked using fragments of syt II that contain the BoNT/B-binding domain. Finally, we show that syt II fragments, in conjunction with gangliosides, neutralized BoNT/B in intact mice. These findings establish that syts I and II can function as protein receptors for BoNT/B.

Synaptotagmin-Mediated Bending of the Target Membrane Is a Critical Step in Ca2+-Regulated Fusion

Decades ago it was proposed that exocytosis involves invagination of the target membrane, resulting in a highly localized site of contact between the bilayers destined to fuse. The vesicle protein synaptotagmin-I (syt) bends membranes in response to Ca(2+), but whether this drives localized invagination of the target membrane to accelerate fusion has not been determined. Previous studies relied on reconstituted vesicles that were already highly curved and used mutations in syt that were not selective for membrane-bending activity. Here, we directly address this question by utilizing vesicles with different degrees of curvature. A tubulation-defective syt mutant was able to promote fusion between highly curved SNARE-bearing liposomes but exhibited a marked loss of activity when the membranes were relatively flat. Moreover, bending of flat membranes by adding an N-BAR domain rescued the function of the tubulation-deficient syt mutant. Hence, syt-mediated membrane bending is a critical step in membrane fusion.

Synaptophysin Regulates the Kinetics of Synaptic Vesicle Endocytosis in Central Neurons

Despite being the most abundant synaptic vesicle membrane protein, the function of synaptophysin remains enigmatic. For example, synaptic transmission was reported to be completely normal in synaptophysin knockout mice; however, direct experiments to monitor the synaptic vesicle cycle have not been carried out. Here, using optical imaging and electrophysiological experiments, we demonstrate that synaptophysin is required for kinetically efficient endocytosis of synaptic vesicles in cultured hippocampal neurons. Truncation analysis revealed that distinct structural elements of synaptophysin differentially regulate vesicle retrieval during and after stimulation. Thus, synaptophysin regulates at least two phases of endocytosis to ensure vesicle availability during and after sustained neuronal activity.

Temperature-Sensitive Paralytic Mutations Demonstrate that Synaptic Exocytosis Requires SNARE Complex Assembly and Disassembly

The neuronal SNARE complex is formed via the interaction of synaptobrevin with syntaxin and SNAP-25. Purified SNARE proteins assemble spontaneously, while disassembly requires the ATPase NSF. Cycles of assembly and disassembly have been proposed to drive lipid bilayer fusion. However, this hypothesis remains to be tested in vivo. We have isolated a Drosophila temperature-sensitive paralytic mutation in syntaxin that rapidly blocks synaptic transmission at nonpermissive temperatures. This paralytic mutation specifically and selectively decreases binding to synaptobrevin and abolishes assembly of the 7S SNARE complex. Temperature-sensitive paralytic mutations in NSF (comatose) also block synaptic transmission, but over a much slower time course and with the accumulation of syntaxin and SNARE complexes on synaptic vesicles. These results provide in vivo evidence that cycles of assembly and disassembly of SNARE complexes drive membrane trafficking at synapses.

Different domains of synaptotagmin control the choice between kiss-and-run and full fusion

Exocytosis—the release of the contents of a vesicle—proceeds by two mechanisms. Full fusion occurs when the vesicle and plasma membranes merge. Alternatively, in what is termed kiss-and-run, vesicles can release transmitter during transient contacts with the plasma membrane. Little is known at the molecular level about how the choice between these two pathways is regulated. Here we report amperometric recordings of catecholamine efflux through individual fusion pores. Transfection with synaptotagmin (Syt) IV increased the frequency and duration of kiss-and-run events, but left their amplitude unchanged. Endogenous Syt IV, induced by forskolin treatment, had a similar effect. Full fusion was inhibited by mutation of a Ca2+ ligand in the C2A domain of Syt I; kiss-and-run was inhibited by mutation of a homologous Ca2+ ligand in the C2B domain of Syt IV. The Ca2+ sensitivity for full fusion was 5-fold higher with Syt I than Syt IV, but for kiss-and-run the Ca2+ sensitivities differed by a factor of only two. Syt thus regulates the choice between full fusion and kiss-and-run, with Ca2+ binding to the C2A and C2B domains playing an important role in this choice.

Direct Interaction of a Ca2+-binding Loop of Synaptotagmin with Lipid Bilayers

Synaptotagmin 1 binds Ca2+ and membranes via its C2A-domain and plays an essential role in excitation-secretion coupling. In this study, we sought to identify Ca2+- and membrane-induced local conformational changes in the C2A-domain of synaptotagmin and to delineate the C2A-lipid binding interface. To address these questions native phenylalanine residues were replaced, at each face of the domain, with tryptophan reporters. Changes in tryptophanyl fluorescence indicated that Ca2+induced long range conformational changes throughout C2A, including regions distant from an established Ca2+-binding site. Addition of liposomes resulted in Ca2+-dependent increases in the fluorescence of tryptophans 193, 231, and 234. Only the tryptophan residues at positions 234 and 231, which lie within a Ca2+-binding loop of C2A, exhibited liposome-induced blue shifts in their emission spectra. Quenching experiments, using membrane-imbedded doxyl spin labels, revealed that tryptophan residues 231 and 234 penetrated lipid bilayers. These data delineate the lipid binding interface of C2A and provide the first evidence for adjacent Ca2+- and lipid-binding sites within a C2-domain. The penetration of C2A into membranes may function to bring components of the fusion machinery into contact with the lipid bilayer to initiate exocytosis.