BIOLOGICAL OVERVIEW

Membrane fusion is a Ca++ dependent process in which neurotransmitter-filled synaptic vesicles undergo docking and fusion with the presynaptic membrane to release neurotransmitter into the synaptic cleft. This secretion process involves the docking protein syntaxin and other synaptic protein components. Membrane fusion results from the interaction of two components of the presynaptic membrane, syntaxin and Snap 25, with two components of the vesicular membrane, Synaptotagmin and synaptobrevin/Vamp. Mediating this interaction are yet other proteins, among them a fusion protein called the N-ethylmaleimide-sensitive fusion protein (NSF) and the soluble NSF attachment proteins (SNAPs), all essential components of the intracellular membrane fusion apparatus. The four membrane components (syntaxin, Snap 25, synaptotagmin and synaptobrevin/Vamp) are termed SNAP receptors (SNAREs). The two vesicular SNAREs are known as v-SNAREs and the two target SNAREs are referred to as t-SNAREs (Söllner, 1993).

Many components of the membrane fusion apparatus have been identified in Drosophila. The t-SNARE Syntaxin 1A has been cloned as well as the v-SNARE Synaptobrevin. The known cytoplasmic components include Rop, N-ethymaleimide-sensitive fusion protein (dNSF) and the soluble NSF attachment protein (dalphaSNAP). Two proteins thus far identified are unique to the synapse: Synaptotagmin, a protein that senses Ca++ levels, and a Cysteine string protein.

All these identified components are coded for by single genes in Drosophila, with the exception of synaptobrevin, which (as in vertebrates) is present in both a neural-specifIc form and a ubiquitous form. Rop is the fly homolog of yeast Sec1p, a syntaxin interacting protein essential for late stages of secretion. dNSF is the product of comatose, a gene originally isolated on the basis of mutations that cause temperature-sensitive paralysis in the adult (Bate, 1995 and references).

Analysis of the subcellular distribution of Drosophila SYX-1A indicates both the presence of protein in presynaptic membranes and a synaptic vesicle fraction. Syntaxin 1A was originally believed to be strictly associated with the presynaptic membrane. In Drosophila, Syntaxin 1A is also associated with synaptic vesicles or possibly other small vesicles such as endosomes (Schulze, 1995).

Binding of the synaptic vesicle v-SNARE, synaptotagmin, to the plasma membrane t-SNARE, SNAP-25, can explain docked vesicles at neurotoxin-treated synapses

Genetic studies with Drosophila challenge the SNARE hypothesis, at least the view that syntaxin functions solely or exclusively as a docking protein. The SNARE hypothesis proposes, in its simplest form, that vesicles dock at presynaptic active zones by the association of synaptobrevin (v-SNARE) with syntaxin (t-SNARE). Ultrastructural studies show the vesicles are targeted to the presynaptic membrane and dock normally at specialized release sites even in the absence of neural synaptobrevin or syntaxin. These vesicles are mature and functional since spontaneous vesicle fusion persists in the absence of neural synaptobrevin or syntaxin (Broadie, 1995). Studies in mammals, also indicate that the simplified SNARE hypothesis is not completely valid. The synaptic vesicle protein and likely calcium sensor synaptotagmin interacts with the plasma membrane t-SNARE SNAP-25. This interaction appears to resolve the apparent paradox that synaptic vesicles are capable of docking even when VAMP (vesicle-associated membrane protein) or syntaxin is cleaved or deleted and suggests that two species of v-SNAREs (VAMP and synaptotagmin) and two species of t-SNAREs (SNAP-25 and syntaxin) interact to functionally dock synaptic vesicles (Schiavo, 1997).

SNAREpins: minimal machinery for membrane fusion

In addition to docking, SNARE proteins are implicated as the minimal components responsible for membrane fusion. A role for fusion, as opposed to docking, would resolve much of the ambiguity concerning syntaxin function in Drosophila. Recombinant v- and t-SNARE proteins reconstituted into separate lipid bilayer vesicles assemble into SNAREpins: SNARE complexes linking two membranes. This leads to spontaneous fusion of the docked membranes at physiological temperatures. Docked and unfused intermediates can accumulate at lower temperatures and can fuse when brought to physiological temperatures. A supply of unassembled v- and t-SNAREs is needed for these intermediates to form, but not for the fusion that follows. These data imply that SNAREpins are the minimal machinery for cellular membrane fusion and do not serve exclusively as docking proteins (Weber, 1998).

A pair of cognate proteins, capable of pairing membranes and fusing their bilayers appears to be the simplest possible means, and one utilized by cells, to link a mechanism for specifically pairing membranes to a mechanism for fusing their bilayer. Such linking not only embodies the crux of how subcellular compartments are maintained and propagated, but also suggests a natural way that the many and diverse compartments in eukaryotic cells could have evolved in the first place. It is easy to imagine how a primitive pair of v- and t-SNARE genes could give rise to a family of such genes by duplication and mutational variation, each v-SNARE evolving in tandem with its cognate t-SNARE because they are structurally and functionally linked. Cognate v- and t-SNAREs bind each other via membrane-proximal heptad repeat regions that are predicted to form coiled-coils or closely related helical bundles. In the electron microscope, SNARE complexes are seen to be rods, 13-14 nm long and approximately 2 nm wide, as is appropriate for such a coiled-coil structure. The membrane anchors of v- and t-SNAREs emerge at the same end of the rod, implying that the rod must lie approximately in the plane of contact between the vesicles paired by a complex of a v-SNARE in one vesicle with a t-SNARE in the other vesicle, a structure termed a "SNAREpin" (Weber, 1998).

These finding cast new light on the roles of SNAPS and ATPase NSFs in membrane fusion. Following fusion, dissociation of the now thermodynamically stable SNARE complexes (which will enable later rounds of fusion), requires investment of energy and therefore additional core machinery. The need for SNAPs and the ATPase NSF as core fusion machinery can most readily be understood from this perspective, as the minimal "add-on" needed for repeated use of a bilayer-fusing helical hairpin. By separating v-SNAREs from t-SNAREs at the expense of energy from ATP, SNAPs and NSF can make them available for subsequent interbilayer-partnering. Indeed, recent evidence implies that in some cases this may be the only role for NSF and SNAPs (Weber, 1998 and references).

Drosophila syntaxin is required for cell viability and may function in membrane formation and stabilization

SYX1A may mediate membrane assembly events throughout Drosophila development. Mutations in syx1A have been isolated that reveal roles for syntaxin outside the nervous system. In the ovary, SYX1A is present in the germarium, but it is predominantly localized to nurse cell membranes. Mitotic recombination experiments in the germline show SYX1A is essential for oogenesis and may participate in membrane biogenesis in the nurse cells. Mutant ovaries are rudimentary in their appearance and indistinguishable from that of ovoD1 heterozygotes. syx1a is also required in larval imaginal discs, as certain hypomorphic mutant combinations exhibit rough eyes defects indicative of cell death. The most severly affected eyes appear to have fewer ommatidia than wild type. The ommatidia are sometimes fused and often are improperly rotated and misaligned with respect to one another. Recombinant clones that lack syx1A cause cell lethality in the developing eye. Another defect in temperature sensitive alleles is notching along the posterior margin of the wing. In mutants, incomplete compaction of the ventral nerve cord is observed. In some mutants incomplete fasciculation of the intersegmental and segmental nerves is found. The staining of the intersegmental and segmental nerve bundles in mutants is quite diffuse, suggesting that the individual axons cannot adhere to one another, perhaps due to inconsistencies in the membrane constituents. Similar defects are observed in the longitudinal tracts of the CNS when visualized with anti-fasciclin II antibody. In the wild-type CNS, three longitudinal tracts are observed on either side of the midline, and each set of axon fibers exhibits a tight arrangement. In mutants, axon bundles are irregular and slightly defasciculated (Schulze, 1996).

What might be Syntaxin's role in membrane integrity or development? Consideration of membrane formation during cellularization may provide clues to an answer. Precellular embryos exhibit ubiquitous distribution of SYX1A mRNA reflecting a maternal contribution. In young embryos during mitotic cycle 11, the plasma membrane partially envelops each dividing nucleus, forming "buds". SYX1A protein accumulates beneath these buds as they undergo cytokinesis. "Somatic buds" form around dividing somatic nuclei but undergo several rounds of formation and collapse as the nuclei beneath the buds divide. At metaphase for each mitotic division, the membrane of the bud invaginates markedly and "pseudocleavage furrows" form between the buds. SYX expression appears to follow the outline of the buds as they partially enwrap the dividing nuclei during mitotic cycles 10-14. Finally during cycle 14, the somatic nuclei become completely surrounded by the elongating membranes of the cytoplasmic buds, and cellularization is completed. SYX1A localization outlines these membranes as they form. SYX1A continues to be expressed in the membranes of each cell of the fully cellularized embryo (Schulze, 1996).

It is estimated that more than 23 times the amount of membrane overlying the syncytium of nuclei at mitotic cycle 14 is needed to completely cellularize the 6000 blastoderm cells of the Drosophila embryo. Although the mechanism by which membrane addition occurs during cellularization is unknown, the process has been divided into two phases: a slow phase involving saltatory transport along astral microtubules and a fast phase in which apical microvilli disappear. The additions of constituents to cell membranes during the fast phase is believed to involve a flattening of the microvilli and hence may not require the synthesis and addition of new membrane. The membrane constituents have simply been stored in the microvilli, and they add to the membrane as they disappear. However, during the earlier slow phase, particles have been observed to move along the microtubules with anterograde (toward the yolk) and retrograde (toward the nucleus at the apex) speeds resembling those observed in axoplasmic flow. Hence, similar to the way that vesicles are transported to the presynaptic terminals of neurons, phospholipid vesicles may be transported further away from the periphery of the cellularizing embryo to sites of membrane addition. SYX1A may play a role in the fusion of the vesicles at these sites, as it is enriched within the cytoplasmic buds that envelop the dividing blastoderm nuclei. This localization corresponds to that of the cytoskeletal elements and products of such genes as nullo, alpha Spectrin and serendipity-alpha, genes believed to form the structural foundation for somatic membrane invagination. The approximate localization of Syx to the apical and lateral walls at cycle 14 places it where it may perform this putative function in the fusion of membranous vesicles to achieve cellularization. It is concluded that syntaxin is involved not just in release of vesicular contents during synaptic function, but is also involved in building cell membranes early in embryogenesis, and perhaps throughout embryonic and larval development (Schultze, 1996 and references).

A mutational study of Syntaxin 1A interactions

Biochemical studies suggest that Syntaxin 1A participates in multiple protein-protein interactions in the synaptic terminal, but the in vivo significance of these interactions is poorly understood. A targeted mutagenesis approach was used to eliminate specific syntaxin binding interactions. Drosophila Syntaxin 1A has been shown to play multiple regulatory roles in neurotransmission in vivo. Syntaxin mutations that eliminate ROP/Munc-18 binding display increased neurotransmitter release, suggesting that ROP inhibits neurosecretion through its interaction with syntaxin. Syntaxin mutations that block Ca²⁺ channel binding also cause an increase in neurotransmitter release, suggesting that syntaxin normally functions by inhibiting Ca²⁺ channel opening. Additionally identified and characterized were a syntaxin Ca²⁺ effector domain, which may spatially organize the Ca²⁺ channel, a cysteine string protein, and synaptotagmin, all necessary for effective excitation-secretion coupling in the presynaptic terminal (Wu, 1999).

To dissect the putative functions of Syntaxin in vivo, partial loss-of-function mutations in syntaxin 1A (syx) were generated. Since this gene is refractory to standard mutagenesis with EMS (ethylmethylsulfonate), a site-directed mutagenesis approach was taken to interfere with specific protein-protein interactions. Syntaxin 1A has four coiled-coil domains (H1/HA, H2/HB, HC, and H3). All binding partners, with one exception (Munc-13), bind the C-terminal third of Syntaxin, including the H3 domain. Focus was thus placed on a structure-function analysis of the H3 domain (Wu, 1999).

Four mutations in the syx H3 domain were generated: two deletions (syx^H3-N and syx^H3-C) and two point mutations (syx² and syx³). The syx^H3-N deletion removes the majority of the N-terminal region of the H3 domain (amino acids 204-250), whereas the syx^H3-C deletion removes the highly basic C-terminal 14 amino acids of the H3 domain (amino acids 253-267). For the syx² and syx³ constructs, point mutations were made in the hydrophobic layers, which appear to be important for specific protein-protein interactions and for core complex stability. Specifically, the syx² point mutation falls in a region thought to be required for SNAP-25 binding, and the amino acid targeted by the syx³ mutation has been shown to be important for binding to n-Sec1/Munc-18 (Wu, 1999).

To define the binding defects caused by the syx mutations, GST pulldown assays were performed. In this assay, GST alone, wild-type GST-Syx, or mutant GST-Syntaxins were immobilized on glutathione-Sepharose beads and incubated with target proteins. The binary Syb-Syx interaction is easily disrupted, because every syx mutation reveals a strong reduction if not complete absence of Syb binding. This finding is consistent with studies using vertebrate proteins and does not appear physiologically relevant, since certain mutants display robust neurotransmission. Core complex formation was tested by assaying Syb binding in the presence of SNAP-25, one of the three core complex components. The syx^H3-N deletion abolishes formation of the core complex, while GST-Syx^H3-C can form a core complex, although less efficiently than wild-type GST-Syx. The syx² and syx³ point mutants are both capable of forming core complexes (Wu, 1999).

Next to be examined were the abilities of the GST-Syx mutant proteins to bind ROP, Synaptotagmin (Syt), and the synprint site of the N-type Ca²⁺ channel. GST-Syx^H3-N shows an abolishment of ROP binding, but binds Synaptotagmin and synprint. Conversely, the GST-Syx^H3-C deletion can bind ROP, but shows a severe reduction in Synaptotagmin and synprint binding, relative to wild-type GST-Syx. Therefore, these findings suggest that the N-terminal region of the H3 domain is essential for core complex formation and ROP binding, while the C-terminal 14 amino acids of the H3 domain are required for efficient binding to Synaptotagmin and synprint (Wu, 1999).

Binding analysis of the point mutations show that the syx² and syx³ mutations selectively disrupt binding to different Syntaxin partners. The syx³ mutation specifically abolishes ROP binding to Syntaxin. Surprisingly, the most striking binding defect caused by the syx² point mutation is an abolishment of binding to synprint. This effect is specific for syx², as GST-Syx³ and GST-Syx^H3-N are capable of binding synprint. Curiously, syx² lies within a domain not required for synprint binding, as demonstrated by GST-Syx^H3-N, suggesting that the point mutation induces a conformational change in the C-terminal region of the H3 domain. Although unexpected, structural studies have shown that amino acid substitutions can cause long-range effects on the binding properties of a protein. In contrast, the point mutations do not significantly affect Synaptotagmin binding, when compared to wild-type GST-Syx. These data confirm that, similar to vertebrate syntaxin 1A, the H3 domain of Drosophila Syntaxin is important for core complex formation and binding to ROP, Synaptotagmin, and synprint. Further, in vitro findings show that specific Syntaxin protein interactions can be selectively blocked by point mutations in the H3 domain (Wu, 1999).

To address the in vivo effects of the syx^H3-N, syx^H3-C, syx², and syx³ mutations, these mutations were introduced into flies. Since a 13.5 kb genomic fragment containing syx (syx^wt) can rescue a null allele (syx²²⁹) to adult viability, each mutation was introduced in the 13.5 kb fragment, sequenced, and used to generate transgenic flies. The wild-type genomic rescue construct was used as control (syx^wt). Transgenic flies bearing the mutant and wild-type constructs were then crossed into syx^P1697 (hypomorph) and syx²²⁹ (null) backgrounds. To control for position effects, several independent transgenic lines were established for each mutant construct. Single embryo Westerns showed that Syx^H3-N, Syx², and Syx³ mutant proteins are produced at levels similar to Syx^wt controls. However, Syx^H3-C mutant protein is expressed relatively poorly, compared to other mutants. Different transgenic lines for the same mutation expressed similar amounts of mutant Syntaxin protein, indicating that position effects do not significantly affect protein levels. Immunocytochemical stainings indicate that the mutant Syntaxins are expressed in a spatial and temporal pattern indistinguishable from wild-type (Wu, 1999).

Mutants and wild-type controls were first assessed for their ability to rescue the lethality of syx^P1697 and syx²²⁹. The deletion mutations are the most severe, since syx^H3-N and syx^H3-C mutants are embryonic lethal in both the null and partial loss-of-function background. In contrast, the point mutations cause milder phenotypes, allowing animals to live to adulthood in the hypomorphic background, but not in the null background (Wu, 1999).

Wild-type Drosophila embryos undergo peristaltic muscular contractions prior to hatching. syx^H3-N and syx^H3-C mutants do not undergo spontaneous muscle contractions, suggesting that neurotransmission is severely affected. However, in response to tactile stimulation, syx^H3-C mutants are capable of weak, local contractions, whereas syx^H3-N mutants fail to respond. In contrast, syx² and syx³ mutant animals are capable of spontaneous peristaltic contractions and robust touch response. These data suggest that synaptic transmission is more severely affected in syx^H3-N and syx^H3-C mutants than in syx² or syx³ mutants (Wu, 1999).

In wild-type Drosophila embryos, epidermal cells secrete a cuticle apically. The presence of cuticle can easily be visualized by the presence of cuticular denticle belts, which is a convenient assay for nonneuronal secretion. syx null mutants (syx²²⁹) fail to secrete cuticle and completely lack denticles, indicating an essential role for Syntaxin in nonneuronal secretion. The ability of these different syx mutants to carry out nonneuronal secretion was examined. All mutants except syx^H3-N are capable of secreting cuticle normally, suggesting that the inability to form core complexes blocks nonneuronal secretion. Moreover, perturbations of specific syntaxin protein interactions, most importantly the Syntaxin-ROP interaction, does not appear to affect nonneuronal secretion (Wu, 1999).

Regulated exocytosis at the synaptic terminal was assayed in syntaxin mutants. The syx^H3-N deletion abolishes core complex formation. Synaptic transmission was examined in syx^H3-N mutants using whole-cell patch-clamp analysis at the embryonic neuromuscular junction (NMJ). Three different wild-type transgenic controls (syx^wt-1, syx^wt-2, syx^wt-3) showed robust evoked neurotransmission. However, evoked and spontaneous neurotransmission is completely abolished in syx^H3-N animals, showing that the syx^H3-N mutant phenotype is identical to that of syntaxin null mutants. As shown for the syx null mutant, direct glutamate pressure ejection at the synapse of syx^H3-N mutants reveals that the postsynaptic muscles respond normally to neurotransmitter, indicating that the block in neurotransmission is presynaptic in nature. The complete absence of neurotransmitter release in syx^H3-N mutants, taken together with the absence of nonneuronal secretion in these mutants, suggests that the core complex is essential for both neuronal and nonneuronal vesicular fusion (Wu, 1999).

To determine the function of the Ca²⁺ effector domain in neurotransmission, the electrophysiological phenotype of syx^H3-C deletion mutants was analyzed. In the majority of mutant animals, evoked transmission is observed, but EJC amplitude is strongly reduced, compared to syx^wt controls. In the remaining mutants, evoked secretion is abolished. In contrast to the complete absence of neurotransmission in syx^H3-N deletion mutants, syx^H3-C deletion mutants reveal strongly reduced evoked responses but an increase in the number of spontaneous quantal events. Both features have been previously associated with synaptotagmin mutants (Wu, 1999).

In addition to a severe reduction of EJC amplitude in syx^H3-C mutants, the mutants also exhibit decreased reliability of excitation-secretion coupling. Specifically, evoked neurotransmission in syx^H3-C mutants is characterized by asynchronous release, low fidelity of release, and a high failure rate. Normally, synchronized fusion of synaptic vesicles is tightly coupled to the Ca²⁺ influx resulting from a single nerve stimulation, and the amount of neurotransmitter released in response to a single stimulation is relatively consistent at the NMJ. However, a single stimulation at syx^H3-C synapses can elicit multiple, nonsynchronized EJCs at variable latencies. Furthermore, the variability of release in syx^H3-C mutants is significantly increased compared to syx^wt controls. Finally, whereas the NMJs of syx^wt animals always release neurotransmitter in response to a nerve stimulation in 1.8 mM extracellular Ca²⁺, syx^H3-C mutants fail to respond approximately 25% of the time. Collectively, these phenotypes suggest that loss of the Ca²⁺ effector domain causes defects in the ability of the fusion machinery to properly regulate synchronized release of synaptic vesicles in response to a Ca²⁺ influx. These features are hallmarks of synaptotagmin null mutations, suggesting that syx^H3-C mutants reveal defects in either the sensing of or response to Ca²⁺ in synaptic vesicle fusion (Wu, 1999).

The Syntaxin-Rop complex

One intriguing interaction is the binding of syntaxin 1A to members of the Munc-18/n-Sec1/ROP family. Genetic studies of Sec1 homologs have shown that these proteins perform a positive function in exocytosis, because mutants show severe defects in both neuronal and nonneuronal secretion. Therefore, it has been suggested that the syntaxin-Munc-18/n-Sec1 interaction positively regulates secretion. However, more recent studies of ROP, the Drosophila Sec1 homolog, have demonstrated that this protein also performs an inhibitory role in exocytosis in vivo. Hence, it is unclear whether the positive or inhibitory function of ROP/n-sec1 is mediated by its interaction with Syntaxin or by interaction with other proteins (Wu, 1999).

To define the function of the Syntaxin-ROP complex, a phenotypic analysis was carried out of the syx³ mutant, since the syx³ mutation selectively disrupts the Syntaxin-ROP interaction. Since the syx³ mutation affects a hydrophobic residue that may be important for core complex stability, the heat lability of SDS-resistant core complexes containing GST-Syx³ was tested. The syx³ mutation does not significantly alter core complex stability relative to wild-type control, although the levels of SDS-resistant core complexes are somewhat reduced at 54°C for GST-Syx³, compared to wild-type GST-Syx. Thus, syx³ mutants are capable of forming stable core complexes but specifically fail to bind to ROP (Wu, 1999).

To address the effects of disrupting the Syntaxin-ROP interaction on synaptic transmission, whole-cell patch-clamp analysis at the NMJ was performed. syx³ mutants demonstrate a very significantly enhanced evoked junctional current (EJC) amplitude when compared to syx^wt controls. Two independent transgenic lines show similarly increased evoked responses. To rule out the possibility that the enhancement of evoked response results from a postsynaptic change, the amplitude of spontaneous miniature excitatory junctional currents (mEJCs) was measured. These data show that quantal size is not significantly altered in syx³ mutants, indicating that the increase in evoked response results from an increase in the number of quanta released. Hence, these data demonstrate that the ROP-Syntaxin complex is not essential for neurotransmission and likely inhibits it (Wu, 1999).

The Syntaxin-Ca²⁺ channel interaction inhibits synaptic transmission

One particularly interesting regulatory interaction examined in this study was the binding of syntaxin 1A to synaptic N-, P-, and Q-type calcium channels, at the cytoplasmic loop between domains II and III, called the synprint site. A functional correlate of this binding was proposed on the basis of synprint peptide competition experiments, which suggested that inhibiting the syntaxin-Ca²⁺ channel interaction caused a reduction in evoked synaptic transmission, with an increase in asynchronous release. Thus, this interaction was proposed to tether the core complex at Ca²⁺ channels in order to localize the fusion machinery near the site of Ca²⁺ influx and potentiate neurotransmission. However, studies in which syntaxin 1A was expressed in Xenopus oocytes have shown that the protein functions to inhibit Ca²⁺ channels. In this context, syntaxin may function to reduce random or unregulated Ca²⁺ channel openings, and loss of this function would be postulated to lead to enhanced neurotransmitter release. Therefore, a syntaxin-Ca²⁺ channel interaction has been suggested to play at least two roles in neurotransmitter release: a potentiating tethering function and an inhibitory regulatory function (Wu, 1999 and references).

A phenotypic analysis of syx², which specifically impairs synprint binding was conducted to study the function of the Syntaxin-Ca²⁺ channel complex in vivo. To quantitate the reduction of synprint binding to GST-Syx², bound synprint was detected using ¹²⁵I-labeled secondary antibodies and phosphorimaging. Binding of the synprint peptide to wild-type GST-Syx is dose dependent and saturable with half-maximal binding at approximately 0.4 µM under these conditions. GST-Syx² binding to synprint shows an approximately 67% reduction in binding at 1.5 µM synprint, compared to wild-type GST-Syx. To rule out the possibility that the syx² mutation impairs core complex stability, the heat lability of SDS-resistant core complexes containing GST-Syx² was tested: results indicate that in this assay the syx² mutation does not affect core complex stability (Wu, 1999).

syx² mutants reveal very significantly increased EJC amplitude compared to syx^wt controls. Two independent lines, syx^2-1 and syx^2-2, have similarly increased EJC amplitudes. To ensure that this increase in evoked response represents a presynaptic phenomenon, mEJC amplitude was measured. mEJC amplitude is not significantly altered in syx^2-1 or syx^2-2 mutants, compared to syx^wt. Therefore, syx² mutants, in which synprint binding is specifically impaired, reveal a dramatic increase of evoked neurotransmitter release (Wu, 1999).

In addition to an increase in evoked response, the mEJC frequency is also greatly increased in both syx^2-1 and syx^2-2 lines. At the Drosophila embryonic NMJ, a population of mEJC events is Ca²⁺ dependent, and these Ca²⁺-dependent spontaneous fusions are thought to be triggered by random openings of Ca²⁺ channels. Thus, the increase in mEJC frequency in syx² mutants may represent an inability of these mutant Syntaxins to prevent random openings of Ca²⁺ channels. If additional quantal events observed in syx² mutants are due to increased openings of Ca²⁺ channels, then, in the absence of Ca²⁺, the mEJC frequency of syx² mutants should be reduced. mEJC frequency in syx² mutants is significantly reduced in the absence of Ca²⁺. In addition, even in zero Ca²⁺, syx² mutants show significantly more quantal events than syx^wt, suggesting that the Syntaxin-Ca²⁺ channel interaction also physically inhibits spontaneous fusion in a manner independent of Ca²⁺. These data suggest that the Syntaxin-Ca²⁺ channel interaction inhibits evoked and spontaneous neurotransmission in vivo (Wu, 1999).

Taken together, these observations suggest that Syntaxin binding to Ca²⁺ channels reduces both spontaneous and evoked openings of the channels. The findings do not support an essential function for Syntaxin in tethering the fusion machinery near Ca²⁺ channels, since loss of this function should lead to a reduction in synchronized evoked release. An attractive alternative candidate for such a tethering function is SNAP-25, which has also been shown to bind the synprint site. How are these data reconciled with the observation that injection of synprint peptides into cultured neurons inhibits neurotransmission and causes asynchronous release? In addition to syntaxin, synaptotagmin and CSP have been shown to bind synprint peptides, and the data suggest that the synprint peptide binds to the Ca²⁺ effector domain of Syntaxin. Therefore, one interpretation of these results is that injection of synprint peptides may inhibit the function of these proteins or their interaction with the syntaxin Ca²⁺ effector domain, resulting in a phenotype similar to that observed in syx^H3-C mutants. The syx³ and syx² mutations do not appear to alter the stability of their core complexes, suggesting that the structure of the core complex bundle is intact. However, the possibility that these mutations cause additional defects, such as affecting the conformation or function of the N-terminal domain of Syntaxin, which has been suggested to regulate core complex assembly, cannot be excluded (Wu, 1999).

CSP binds to the Ca²⁺ effector domain of Syntaxin and is an effective competitor of synprint binding

The binding of Syntaxin to presynaptic Ca²⁺ channels inhibits random opening of Ca²⁺ channels. Cysteine string protein (CSP), a synaptic vesicle associated protein, has also been shown to functionally and biochemically interact with Ca²⁺ channels. Genetic studies in Drosophila suggest that CSP, in concert with other proteins, is required for efficient presynaptic Ca²⁺ entry. It is therefore possible that CSP and Syntaxin interact to coordinate Ca²⁺ entry. However, a direct interaction between CSP and Syntaxin has not been reported (Wu, 1999).

To investigate whether CSP could regulate the Syntaxin-Ca²⁺ channel interaction, an examination was performed to see if CSP can be found in a complex with Syntaxin. Syntaxin complexes were immunoprecipitated from Drosophila head extracts with anti-Syntaxin. As shown for vertebrate proteins, Drosophila Synaptotagmin coimmunoprecipitates with Syntaxin. In addition, CSP also coimmunoprecipitates with Syntaxin. Since coimmunoprecipitation does not demonstrate direct binding, an examination was carried out of whether CSP interacts with Syx in a GST pulldown assay. CSP binds to wild-type GST-Syx, but not to GST alone. These data show that CSP can bind directly to Syntaxin in vitro and is present in a complex with Syntaxin in vivo (Wu, 1999).

The region deleted in GST-Syx^H3-N is essential for core complex formation and ROP binding, but not Synaptotagmin or synprint binding. Conversely, the region deleted in GST-Syx^H3-C is required for efficient binding to Synaptotagmin and to synprint, but not ROP. To determine if these regions are also important for CSP binding, an examination was made of the ability of CSP to interact with GST-Syx^H3-N and GST-Syx^H3-C, as well as GST-Syx² and GST-Syx³. While CSP can interact with GST-Syx^H3-N, CSP binding to GST-Syx^H3-C is reduced. In addition, CSP binds to GST-Syx² and GST-Syx³. To quantitate the amount of CSP, Synaptotagmin, and synprint binding to wild-type GST-Syx and GST-Syx^H3-C, binding experiments were performed. Binding of CSP to Syntaxin is dose dependent and saturable. The apparent stoichiometry of this interaction is approximately 0.3:1 (CSP:GST-Syx), when Syntaxin is immobilized on beads. Maximal binding of CSP to GST-Syx^H3-C is reduced approximately 80%. Maximal Synaptotagmin binding to GST-Syx^H3-C is severely reduced using these conditions, compared to wild-type GST-Syx. In addition, synprint binding to GST-Syx^H3-C is also reduced, relative to wild-type GST-Syx. The syx^H3-C deletion causes approximately 85% reduction in maximal synprint binding. Hence, these binding data suggest that the region deleted in syx^H3-C is required for binding of three proteins implicated in Ca²⁺ signaling: CSP, synprint, and Synaptotagmin. It is proposed that the N-terminal domain of Syntaxin acts as a core domain, while the highly basic C-terminal region of the H3 domain functions as a Ca²⁺ effector domain, spatially localizing proteins required for the production, regulation, and sensing of the Ca²⁺ signal (Wu, 1999).

A possible model to explain CSP function and its in vitro binding to Syntaxin is that the role of CSP is to alleviate the Syntaxin-mediated inhibition of Ca²⁺ channels, thereby signaling the presence of synaptic vesicles and preparing the machinery for fusion. This model makes a simple prediction, namely that CSP should be able to compete with Syntaxin for the binding to synprint. To test this possibility, immobilized wild-type GST-Syntaxin protein was incubated with synprint peptide in the presence of increasing amounts of CSP or Synaptotagmin. CSP effectively competes the Syntaxin-synprint interaction in a dose-dependent manner. Synaptotagmin can also compete the Syntaxin-synprint interaction, but somewhat less effectively. Therefore, while both CSP and Synaptotagmin can interact with the Ca²⁺ effector domain of Syntaxin, CSP appears to be a more effective competitor of the Syntaxin-synprint interaction than Synaptotagmin. These data further suggest that CSP, Synaptotagmin, and synprint bind to the Ca²⁺ effector domain of Syntaxin. Additionally, these data support the hypothesis that these proteins participate in competitive interactions to regulate Ca²⁺ entry and are consistent with the idea that CSP may disassemble Syntaxin-Ca²⁺ channel complexes by interacting with Syntaxin, the synprint site, or both (Wu, 1999).

Trapping of Syntaxin1a in presynaptic nanoclusters by a clinically relevant general anesthetic

Propofol is the most commonly used general anesthetic in humans. Understanding of its mechanism of action has focused on its capacity to potentiate inhibitory systems in the brain. However, it is unknown whether other neural mechanisms are involved in general anesthesia. This study demonstrates that the synaptic release machinery is also a target. Using single-particle tracking photoactivation localization microscopy, it was shown that clinically relevant concentrations of propofol and etomidate restrict syntaxin1A mobility on the plasma membrane, whereas non-anesthetic analogs produce the opposite effect and increase syntaxin1A mobility. Removing the interaction with the t-SNARE partner SNAP-25 abolishes propofol-induced Syntaxin1A confinement, indicating that Syntaxin1A and SNAP-25 together form an emergent drug target. Impaired Syntaxin1A mobility and exocytosis under propofol are both rescued by co-expressing a truncated Syntaxin1A construct that interacts with SNAP-25. These results suggest that propofol interferes with a step in SNARE complex formation, resulting in non-functional Syntaxin1A nanoclusters (Bademosi, 2018).

This study demonstrates that clinical concentrations of a commonly used GABA-acting general anesthetic, propofol, also restrict syntaxin1A mobility on the plasma membrane. The contrast seen with the effect of propofol analogs is particularly striking, with the non-anesthetic analogs significantly increasing syntaxin1A mobility instead. These results indicate that propofol acts like its non-anesthetic analogs when the interaction between syntaxin1A and SNAP-25 is lost, suggesting that propofol targets this interaction to immobilize syntaxin1A. It seems plausible that syntaxin1A confinement to nanoclusters could lead to impaired neurotransmission, which was also observed under propofol. However, more work is needed to establish causality here. How exactly propofol impairs syntaxin1A mobility remains unclear, although the requirement for SNAP-25 interaction suggests the nanoclusters are composed of syntaxin1A/SNAP-25 heterodimers that have been blocked from proceeding to a subsequent step in SNARE complex formation due to the presence of the general anesthetic. It is also unclear how a truncated syntaxin1A protein might preserve this process against the effects of propofol on syntaxin1A mobility and exocytosis. The finding that the truncated syntaxin1A molecule simultaneously interacts with both SNAP-25 and wild-type syntaxin1A suggests occupancy of a site that might otherwise be targeted by propofol. In this regard, future experiments with other truncation constructs employing propofol resistance as a readout will be helpful toward determining whether the effects on syntaxin1A mobility and exocytosis are indeed correlated (Bademosi, 2018).

In addition to identifying an alternative target process for this widely used sedative, the current findings may provide a more complete understanding of general anesthesia. Every neuron communicates with other neurons by way of syntaxin1A-mediated neurotransmission, which is highly conserved from worms to humans. Although these experiments were focused on the intravenous drugs propofol and etomidate, it will be interesting to see in future studies whether other general anesthetics have the same effect on syntaxin1A mobility. There is already considerable evidence that a broader range of general anesthetics affect synaptic release mechanisms, and a previous study using nuclear magnetic resonance found that clinical concentrations of these drugs interact with syntaxin1A and SNAP-25, but not VAMP2, which is consistent with the conclusion that propofol acts before completed SNARE formation. One hypothesis consistent with these findings would be that a general anesthetic target emerges only when syntaxin1A and SNAP-25 interact on the plasma membrane and that the association of propofol with this emergent target interferes with subsequent steps in SNARE formation. This would lead to a 'traffic jam' of syntaxin1A/SNAP-25 heterodimers (or another pre-SNARE moiety), which would manifest as syntaxin1A nanoclusters in this analysis. Another explanation for the decrease in syntaxin1A mobility could be that propofol promotes its recruitment into nonfunctional SNARE complexes that do not promote vesicle fusion. Whereas the data suggest interactions in the membrane, this need not be the only explanation for altered syntaxin1A mobility. An alternative possibility is that anesthetics might alter syntaxin1A mobility by more specifically interfering with other key protein interactions leading to SNARE formation, such as between syntaxin1A/SNAP-25 and Munc-13, which is a crucial mediator in forming the final tetrameric complex with VAMP2. Future experiments testing the effects of mutating candidate residues in the syntaxin1A SNARE motif should reveal the exact nature of this propofol-binding target, as has been revealed for other propofol targets, such as GABAA receptors (Bademosi, 2018).

Like sleep, general anesthesia resembles a reversible switch, and the search for mechanisms of anesthesia has justifiably focused on proteins that exert major effects on neuronal excitability, such as inhibitory GABAA receptors, which are indeed targets of many general anesthetics. However, the current results and the work of others show that clinically relevant concentrations of general anesthetics also compromise neurotransmitter release, and the current set of results with intravenous drugs suggests this may be consequence of effects on syntaxin1A mobility in the plasma membrane. However, general anesthetics do not abolish neurotransmission; they only decrease quantal content. So how could this be relevant to the behavioral endpoint that is general anesthesia? With most animal brains comprising anywhere between millions and trillions of synapses, it seems plausible that normal brain functions would be compromised if syntaxin1A mobility became globally restricted across a variety of synapses following exposure to general anesthetics. While a decrease in quantal content may not significantly impair some muscular (or spinal cord) functions, it is likely that a similar effect on central synapses would dramatically change temporal dynamics in the brain, leading to a loss of functional connectivity. In support of this view, recent electroencephalogram (EEG) and fMRI studies have shown that functional connectivity throughout the brain is significantly altered in patients undergoing general anesthesia. Thus, other manipulations that compromise presynaptic communication, including effects on presynaptic excitability , might fall into the same category of anesthetic mechanisms as the syntaxin1A-mediated effects described in this study, that may be considered a class of effects that is distinct from GABAergic sleep-related mechanisms. One possibility, which has been raised previously, is that GABAergic processes (e.g., sedation and loss of consciousness) are induced at lower drug doses (e.g., < 1 µM propofol), while the presynaptic processes discussed in this study are affected at the slightly higher concentrations required for surgery. It remains unknown however whether other general anesthetics target presynaptic mechanisms. A recent study using hippocampal cultures found that isoflurane inhibits synaptic vesicle exocytosis through reduced Ca2+ influx rather than Ca2+-exocytosis coupling. In contrast, the current results suggest that propofol and etomidate-mediated presynaptic effects might be directly coupled to the exocytosis machinery. Whether this is a difference between intravenous and volatile anesthetics is unclear. Nevertheless, a set of distinct presynaptic mechanisms linked to exocytosis might explain why recovery from general anesthesia appears to involve a different process than anesthesia induction; re-establishing functional connectivity after neurotransmission has returned to normal levels across the brain would likely involve a different process than falling asleep or waking up. It will be interesting in future research to use transgenic syntaxin1A animals to link the local effects found at the presynapse with consequent changes in global readouts, such as whole-brain connectivity and coherence (Bademosi, 2018).

Syntaxin1A neomorphic mutations promote rapid recovery from isoflurane anesthesia in Drosophila melanogaster

Syntaxin1A is a presynaptic molecule that plays a key role in vesicular neurotransmitter release. Mutations of syntaxin1A result in resistance to both volatile and intravenous anesthetics. Truncated syntaxin1A isoforms confer drug resistance in cell culture and nematode models of anesthesia Resistance to isoflurane anesthesia can be produced by transiently expressing truncated syntaxin1A proteins in adult Drosophila flies. Electrophysiologic and behavioral studies in Drosophila show that mutations in syntaxin1A facilitate recovery from isoflurane anesthesia. These observations suggest that presynaptic mechanisms, via syntaxin1A-mediated regulation of neurotransmitter release, are involved in general anesthesia maintenance and recovery Mutations in the presynaptic protein syntaxin1A modulate general anesthetic effects in vitro and in vivo. Coexpression of a truncated syntaxin1A protein confers resistance to volatile and intravenous anesthetics, suggesting a target mechanism distinct from postsynaptic inhibitory receptor processes. Hypothesizing that recovery from anesthesia may involve a presynaptic component, whether synatxin1A mutations facilitated recovery from isoflurane anesthesia in Drosophila melanogaster was tested. The same neomorphic syntaxin1A mutation that confers isoflurane resistance in cell culture and nematodes also produces isoflurane resistance in Drosophila. Resistance in Drosophila is, however, most evident at the level of recovery from anesthesia, suggesting that the syntaxin1A target affects anesthesia maintenance and recovery processes rather than induction. The absence of truncated syntaxin1A from the presynaptic complex suggests that the resistance-promoting effect of this molecule occurs before core complex formation (Troup, 2019).

There is growing evidence that general anesthetics target presynaptic mechanisms in addition to postsynaptic receptors. For example, clinical concentrations of both intravenous and volatile anesthetics have been found to impair neurotransmission. Syntaxin1A plays a key role in neurotransmission, presenting a crucial endpoint for synaptic vesicle release, without which neurotransmission could not occur. Mutations in this protein produce both hypersensitivity and resistance to volatile anesthetics in nematode worms and Drosophila flies, suggesting that the protein may be proximal to a presynaptic target for these drugs. Coexpression of a truncated syntaxin1A protein has been shown to produce resistance to volatile anesthetics in nematode worms and mammalian neurosecretory cells,mas well as resistance to the intravenous anesthetic propofol in mammalian cells. How exactly this neomorphic syntaxin1A protein protects synaptic release from the effect of general anesthetics remains unclear, although an interaction with other presynaptic-related proteins seems likely (Troup, 2019).

Most explanations of general anesthesia relate to postsynaptic targets. However, should general anesthesia comprise at least two distinct target domains, one postsynaptic and one presynaptic, this is likely to be reflected in the different kinetics of anesthesia induction and recovery. Many general anesthetics have rapid induction and slower recovery kinetics. This recovery inertia has been proposed as evidence that different processes might be involved during induction and recovery. One idea that has been proposed is that induction is rapid because it primarily reflects the sedative properties of these drugs, and the loss of consciousness associated with sleep is a rapid process. However, recovery time can vary significantly, and some patients report incomplete recovery for days or even months after the procedure. It has been have proposed that recovery inertia reflects in part presynaptic processes, in contrast to the rapid induction kinetics which are understood to reflect postsynaptic processes. Therefore, manipulations of presynaptic proteins that preserve neurotransmission under isoflurane anesthesia in vitro should reduce the recovery time required after the procedure, in vivo. This study tested this in an animal model, Drosophila melanogaster (Troup, 2019).

General anesthesia is fundamentally a behavioral endpoint, and understanding its mechanisms of action requires methods to probe behavioral responsiveness in behaving animals. Because the sedative component of general anesthesia most likely engages sleep-promoting pathways in the brain, it was decided to use Drosophila, which have sleep-promoting neurons that have been found to be involved in isoflurane anesthesia (Troup, 2013). Drosophila is an established model to study general anesthesia. Assays for probing sleep intensity or behavioral responsiveness are also well developed for Drosophila, providing an effective way of assessing the role of syntaxin1A in isoflurane induction and recovery. A tagged, truncated version of syntaxin1A was developed could be expressed in fly neurons, to determine how this affected isoflurane induction and recovery for behavioral endpoints as well as for neurotransmission. It was hypothesized that syntaxin1A effects on recovery from isoflurane anesthesia would be reflected across these different levels of investigation (Troup, 2019).

This study found that deletion mutations in syntaxin1A, when coexpressed alongside wild-type syntaxin1A in Drosophila melanogaster, significantly reduce the recovery time after isoflurane anesthesia. Whereas resistance to isoflurane was also evident during anesthesia induction, this effect was weaker compared with effects on recovery, in adult flies. This suggests that recovery from isoflurane anesthesia depends at least in part on syntaxin1A function. It was surprising how long adult wild-type flies required to regain normal levels of behavioral responsiveness after the procedure, compared with regaining locomotion; behavioral responsiveness still remained impaired even after two hours. In contrast, the syntaxin1A mutants could recover behavioral responsiveness after 10min. Delayed recovery of behavioral responsiveness in Drosophila may therefore be a promising model for studying cognitive impairments following general anesthesia in humans, which also often follow a longer time course than simply regaining consciousness. A full restoration of presynaptic functions across the brain is probably a complex problem in any animal, and the extremely conserved nature of synaptic release mechanisms suggests this might be a common mechanism (Troup, 2019).

syntaxin1A manipulations improved anesthesia recovery times across entirely different levels of analysis. Examination of effects on neurotransmission at the fly neuromuscular junction corroborated behavioral findings: recovery of quantal content occurred within 5min in the syntaxin1A mutant animals. Although it is unknown what neurotransmission recovery dynamics might be like in adult brain synapses, it seems likely that similar effects on recovery might be present because the same syntaxin1A protein is involved in the brain as at the larval neuromuscular junction, or in all animal synapses. Mutant Drosophila larvae also recovered faster than controls behaviorally (within 5 min), indicating an effect that transcends brains at different levels of complexity (the larval brain has an order of magnitude fewer neurons than the adult fly brain). It remains unknown, however, whether adult brain synapses are affected in the same way as motor synapses. Behavioral recovery dynamics in adult mutant animals remain more sluggish than recovery of quantal content at the larval neuromuscular junction. This suggests that brain synapses might recover function differently than motor nerve terminals in larvae (Troup, 2019).

One limitation of this study was the use of only female flies for behavioral experiments in adults. Sex-specific effects in Drosophila have been observed during recovery from anesthesia, although these effects generalize to cold-shock and oxygen deprivation anesthesia, which are likely unrelated to the presynaptic mechanisms described in this study. Given the lack of sexual dimorphism in synaptic proteins, it is unlikely that the phenotypes described in this study would be different using male flies, although this remains to be tested experimentally. In the larval studies both male and female animals were used. Despite any potential sexual differentiation in larval motor nerve terminals, significant effects were still found in the larval isoflurane experiments with expression of the truncated syntaxin1A protein (Troup, 2019).

How might a coexpressed syntaxin1A truncation construct be conferring a rapid recovery from isoflurane anesthesia? This same manipulation has now been shown to produce resistance to diverse general anesthetics across a variety of systems, in vitro and in vivo. Because syntaxin1A is a key player in SNARE-mediated exocytosis, it was therefore surprising to find that the HA-tagged truncated protein syx²²⁷ was not present in soluble nethylmaleimide sensitive factor attachment protein receptor complexes, at least in Drosophila. Because syx²²⁷ has been shown to interact with synaptosomal associated protein 25 (SNAP25) and wild-type syntaxin1A, this suggests an effect immediately before SNARE formation, meaning the truncated protein is probably ejected upon full soluble n-ethylmaleimide sensitive factor attachment protein receptor formation (when vesicle-associated membrane protein 2 links with syntaxin1A and SNAP25 to form a release ready tetrameric complex). This would imply that the protective effect of this protein is required before SNARE formation, and accordingly that the anesthetic effect on syntaxin1A function is also prior to soluble nethylmaleimide sensitive factor attachment protein receptor formation. This view is consistent with recent findings using super-resolution microscopy to track the mobility of single syntaxin1A molecules under propofol anesthesia (Troup, 2019).

It was found that clustering of syntaxin1A caused by propofol was dependent on an interaction with SNAP25, but not with vesicle-associated membrane protein 2, thereby suggesting a mechanism of action (for propofol) immediately before full SNARE formation. Work in other systems also suggests an interaction between volatile anesthetics and syntaxin1A/SNAP25 suggesting that this might indeed by a general anesthetic target. On the other hand, work in Caenorhabditis elegans, where the effect of the truncated syntaxin1A was discovered, points to unc-13 as a likely mediator of this resistance-promoting mechanism. unc-13 is understood to be associated with presynaptic active zones, where the SNAREs ultimately reside, so one interpretation of these diverse findings is that the drug-mediated clustering of syntaxin1A/SNAP25 occurs at these active zones, and that these pre-SNARE complexes are prevented from transforming into full SNAREs because unc-13 is less able to catalyze the next step. In this regard, it will be especially interesting to investigate what role unc-13 plays in this process; unc-13 has been shown to keep syntaxin1A in a closed conformation, until interaction with unc-13 opens syntaxin1A to promote complete full SNARE formation. One hypothesis consistent with this model is that general anesthetics promote a closed syntaxin1A conformation, by for example impairing the capacity of unc-13 to catalyze SNARE formation. One hypothesis for how syx²²⁷ affords resistance then is that the truncated (or deletion) proteins might promote open syntaxin1A moieties, and in this way remove an emergent target (the closed syntaxin1A-unc-18 complex). Interactions with vesicle-bound vesicle-associated membrane protein 2 would then lead to an energetically more favorable ternary complex, effectively ejecting syx²²⁷ upon SNARE formation. Future biochemical experiments should determine whether syx²²⁷ promotes an open syntaxin1A conformation, and to what level unc-13 and unc-18 are involved in this process (Troup, 2019).

One of the most striking observations in this this study is the prolonged duration of recovery from isoflurane anesthesia in wild-type flies, and how syntaxin1A mutations significantly reduce this recovery time. If syx²²⁷ is acting before SNARE formation, then how might this lead to faster recovery? One possibility following from the hypothesis proposed above is that syx²²⁷ provides more efficient access to already open pre-SNARE complexes that are ready to be incorporated into fully formed SNAREs. If general anesthetics produce a traffic jam of nonfunctional pre-SNARE nanoclusters, as suggested by single-molecule imaging experiments, then the time required for dissolving this proteinaceous traffic jam might take longer than clearance of the anesthetic drugs themselves. Consistent with this view, imaging work showed that expression of syx²²⁷ in mammalian cells prevented the syntaxin1A clustering effects of another general anesthetic, propofol. In contrast to these sluggish presynaptic recovery effects, the postsynaptic effects of general anesthetics such as isoflurane and propofol are most likely rapid, as they primarily linked to gamma-aminobutyric acid receptor function. General anesthesia induction is a rapid process, as this probably engages potent inhibitory systems in the brain that are designed to promote a rapid loss of consciousness. However, a rapid reversal of the effect on gamma-aminobutyric acid receptors after removal of these drugs might have little consequence on recovery from anesthesia until presynaptic processes across the brain have been fully restored. The data on syntaxin1A fly mutants exposed to isoflurane support this view of general anesthesia, with the largest effects seen for recovery rather than induction. However, the fact that these mutants are also resistant to isoflurane upon induction suggests that presynaptic effects might play a role during anesthesia induction as well. It will be interesting in future experiments to combine genetic manipulations that promote anesthetic resistance at both a pre and postsynaptic levels, in animals that have both target mechanisms (i.e., sleep/wake pathways and SNAREs). Such experiments will allow better dissecting of the relative contributions of either target process, and to determine whether some circuits or neurotransmitter systems are more affected by the presynaptic mechanisms this study has uncovered (Troup, 2019).

Proportional downscaling of glutamatergic release sites by the general anesthetic propofol at Drosophila motor nerve terminals

Propofol is the most common general anesthetic used for surgery in humans, yet its complete mechanism of action remains elusive. In addition to potentiating inhibitory synapses in the brain, propofol also impairs excitatory neurotransmission. This study used electrophysiological recordings from individual glutamatergic boutons in male and female larval Drosophila melanogaster motor nerve terminals to characterize this effect. Recording was performed from two bouton types, which have distinct presynaptic physiology and different average numbers of release sites or active zones. A clinically relevant dose of propofol (3mμM) impairs neurotransmitter release similarly at both bouton types, by decreasing the number of active release sites by half, without affecting release probability. In contrast, an analog of propofol has no effect on glutamate release. Co-expressing a truncated syntaxin1A protein in presynaptic boutons completely blocked this effect of propofol. Overexpressing wild-type syntaxin1A in boutons also conferred a level of resistance, by increasing the number of active release sites to a physiological ceiling set by the number of active zones or T-bars, and in this way counteracting the effect of propofol. These results point to the presynaptic release machinery as a target for the general anesthetic. Proportionally equivalent effects of propofol on the number of active release sites across the different bouton types suggests that smaller glutamatergic boutons with fewer release sites may be more vulnerable to the presynaptic effects of the drug (Karunanithi, 2020).

In the 3 decades since its discovery, propofol has become the most commonly used general anesthetic for surgical procedures in humans. Like many other general anesthetics, propofol is understood to potentiate inhibitory pathways in the brain by acting on postsynaptic GABAA receptor subunits. Thus, an increasingly accepted idea for propofol-induced sedation is that it engages endogenous sleep pathways in the brain (Hemmings, 2019). However, sleep pathways cannot be the entire explanation, since-unlike sleep-surgical anesthesia is by definition not reversible as long as an adequate dose of the drug is applied. Additionally, there is growing evidence that general anesthesia more broadly is physiologically different from sleep, when investigated at the level of the neural circuitry involved and the associated brain activity measures. This suggests alternate target mechanisms that might complement the better understood processes relating to postsynaptic GABAA receptor function (Karunanithi, 2020).

Recent work has shown that GABA-acting drugs such as propofol target a number of proteins other than GABAA receptors, including TrpA1 channels (Ton, 2017) and kinesin (Woll, 2018), resulting in parallel consequences on neural functions alongside sleep induction. Another emerging target mechanism is the presynaptic release machinery. There is increasing evidence in animal models as well as cell culture preparations that general anesthetics also act presynaptically. Exactly how general anesthetics impair neurotransmission remains unclear, with isoflurane affecting presynaptic sodium and calcium channels (Koyanagi, 2019; Zhou, 2019) as well as SNARE formation (Xie, 2013; Troup, 2019); however, it is unknown whether intravenous and volatile agents might have common presynaptic effects. Work in rat neurosecretory PC12 cells showed that clinical concentrations of both propofol and isoflurane inhibit neurotransmission from these cells, and that coexpression of a mutant form of the presynaptic protein syntaxin1A preserves neurotransmission in these cells under anesthetic exposure. Syntaxin1A is a key membrane-bound component of the presynaptic release machinery, which is necessary to enable vesicular release of neurotransmitters. The effect of general anesthetics on syntaxin1A remained unclear, however, until the advent of super-resolution microscopy techniques that permitted visualizing and tracking single molecules through time. In a recent super-resolution imaging study of a similar PC12 cell preparation, a presynaptic effect of propofol was confirmed, and the intravenous anesthetic was shown to impair neurotransmission by restricting the mobility syntaxin1A on the cell membrane, resulting in nonfunctional nanoclusters (Bademosi, 2018). The same syntaxin1A clustering effect was also found in a live animal preparation, namely motonerve terminals of the fly larval neuromuscular junction (NMJ; Bademosi, 2018), suggesting a common presynaptic target mechanism across different cell types and animal species. Together, these results also suggested an anesthetic effect more proximal to the presynaptic release machinery, at least for some intravenous agents such as propofol. One compelling hypothesis emerging from these findings is that propofol-impaired neurotransmitter release could result from decreased availability of syntaxin1A at release sites on the plasma membrane. Release sites, also referred to as “active zones,” are where the presynaptic machinery comes together to facilitate the fusion of synaptic vesicles with the cell membrane. Although multiple studies have now shown that general anesthetics impair neurotransmission, what remains unclear is how exocytosis physiology is affected in 'real' synapses, in live animals. In the current study, the fly larval NMJ was used to test how a clinically relevant dose (3 μm) of propofol impairs glutamate release from individual motonerve terminals, or boutons. This study further assessed how coexpression of mutant and wild-type syntaxin1A protein modulate these presynaptic effects (Karunanithi, 2020).

This work provides evidence that a clinically relevant concentration of the intravenous general anesthetic propofol impairs excitatory neurotransmission by decreasing (by half) the availability of active release sites at presynaptic terminals. These findings are intriguing because they point to a likely mechanism whereby drugs such as propofol might be impairing presynaptic release: not by decreasing the release probability of already primed (i.e., active) release sites, but instead by altering the ratio between active and inactive sites. Most presynaptic terminals involve a dynamic balance of active and inactive release sites, and this balance can vary across synapse types. By recording from individual boutons at the Drosophila NMJ, this study has shown that knowing the n value of a bouton (the maximum number of quanta that can be released following an action potential) is key to understanding the effect of propofol on neurotransmission. Even within similar bouton types n can vary widely, so to capture the effect of propofol on n requires a bouton-by-bouton assessment of the number of active release sites that are available. This would have been missed by simply averaging all bouton recordings or by only measuring combined 1s and 1b potentials recorded from postsynaptic tissue. Similarly, previous studies investigating presynaptic effects of propofol at the frog NMJ or in rat cortical cultures did not acquire physiological data allowing estimates of release site numbers, as was done in this study. It is interesting to note, however, that propofol exposure increases quantal size in the both the fly and frog NMJ (Karunanithi, 2020).

Earlier work on the vertebrate NMJ showed that opioids and adenosine also inhibit neurotransmission by decreasing n. These compounds activate presynaptic autoreceptors, which then initiate a cascade of events leading to decreased neurotransmitter release via an estimated decrease in n. As in those earlier vertebrate studies, the current estimates of n in fly larval boutons are purely electrophysiological. However, electrophysiological estimates of n maxima match well with anatomic counts of T-bar numbers or BRP puncta in fly larval boutons, providing confidence that estimates of the effect of propofol on n are accurate. There are currently no reliable imaging or immunochemical techniques for disambiguating active from inactive release sites, although methods aimed at tracking the molecular dynamics and conformation of key vesicular priming proteins such as Munc13 provide a promising future research direction to verify the electrophysiological observations (Karunanithi, 2020).

One interpretation of the results is that propofol converts a proportion of active sites with a high release probability to inactive sites with low release probability. Alternatively, propofol could inhibit inactive sites from converting to active sites. The data do not reveal the directionality of this effect, only that propofol increases the number of inactive sites without affecting the release probability of remaining active sites. Thus, release sites unaffected by propofol presumably remain fully functional (rather than half-functional, for example), as evidenced by the finding that release probability at the fly NMJ is not impaired by propofol. In previous studies, it was suggested that propofol may act on components of the synaptic release machinery prior to SNARE formation (Bademosi, 2018), which is consistent with the current finding that n is affected rather than p. Additionally, this study has shown that the resistance-inducing truncated syntaxin1A protein is not incorporated into the SNARE complex (Troup, 2019), which again suggests a role prior to SNARE formation. One possibility that was raised before (Bademosi, 2018) is that propofol inhibits a step in the formation of functional release sites, thereby creating 'traffic jams' of inactive sites that fail to become fully functional. A level of resistance to propofol is thus afforded at some boutons, depending on the availability of additional inactive release sites, which could be recruited to become active, depending on the physiological demands of a neuron. Thus, overexpressing syntaxin1A in 1b boutons maintains glutamate release near wild-type levels in the presence of propofol because these larger boutons have additional release sites that can be recruited to become high-release probability (thus active) sites. On the other hand, coexpressing a truncated syntaxin1A in 1b boutons preserves n and thus QC from the effects of propofol specifically in these boutons, while n is again halved in unprotected 1s boutons in the same tissue. Together, these genetic manipulations provide compelling evidence that, alongside its known effects on postsynaptic receptors and ion channels, propofol impairs neurotransmission by decreasing the availability of active release sites. Genetic manipulations of different components of the SNARE assembly and priming processes should clarify whether propofol renders active sites inactive, or whether it prevents inactive sites from becoming active (Karunanithi, 2020).

Although synaptic release mechanisms are extremely conserved across animals, not all presynapses are the same. As this study has shown, even synapses releasing the same neurotransmitter (glutamate) onto the same muscle can be physiologically different. 1s boutons have a higher release probability, and all of their release sites are active, whereas 1b boutons are weaker but have a capacity to recruit more release sites. Yet, both types of boutons are impacted in a similar way by propofol, with the drug decreasing the number of active release sites by half. If propofol impairs neurotransmitter release by proportionally decreasing n by half, as was show in this study, then smaller boutons (e.g., 1s in this study) could be more vulnerable to the presynaptic effects of the drug, and this would especially be the case for synapses with only one active zone, which probably describes most excitatory synapses in the brain. Extrapolating to humans, this may explain why some circuits are more affected than others by surgical concentrations of general anesthetics such as propofol. Future work should reveal whether the presynaptic effect that this study has uncovered for propofol extends to other types of synapses (e.g., inhibitory) and neurotransmitter systems, and whether this effect may be a common presynaptic mechanism of other general anesthetics (Karunanithi, 2020).

GENE STRUCTURE

Three SYX-1A transcripts, 3.5- 4.2- and 8.0-kb are abundant in young embryos. At 3-9 hours of development the 3.5-kb transcript slowly disappears while 4.2-, 8.0- and 9.0-kb messages predominant until the end of embryogenesis. Between 9-15 hours, two additional transcripts (7.0 and 12 kb) appear. During first instar larval life, five messages are present, whereas second and third instars utilize three and two messages respectively. During pupariation, all five transcripts reappear, and the adult expression pattern resembles that of pupae. Three of the adult transcripts are the same as those found in young embryos, suggesting some or all of these messages represent the maternal contribution to the embryo. Since the cDNA is contained within a single exon, it is likely that alternative splicing, various promoters, and different polyadenylation signals are used to generate this heterogeneity (Schulze, 1996).

PROTEIN STRUCTURE

Amino Acids - 291

Structural Domains

The protein exhibits 70% amino acid identity with rat syntaxin-1A over its full length and 25% identity with yeast Sso1p. Identity with Rat syntaxin-5 is only 22% (Schulze, 1995).

A monoclonal antibody, mAb 44D5, has been used to identify and clone Drosophila syntaxin 1 (Dsynt1), a homolog of rat syntaxin 1. The deduced amino acid sequence of the Dsynt1 cDNA cloned is highly homologous to rat syntaxin 1A. Dsynt1 contains 291 amino acid residues and like other members of the syntaxin family is an integral membrane protein, with a transmembrane region at its carboxy-terminus and several regions of the molecule predicted to be in a coiled-coil conformation (Cerezo, 1995).

Conserved domains have been analyzed in t-SNAREs (soluble N-ethylmaleimide-sensitive factor [NSF] attachment protein [SNAP] receptors in the target membrane). These proteins are believed to be involved in the fusion of transport vesicles with their target membrane. A new homology domain has been identified that is common in the two protein families ( the syntaxin and SNAP-25 [synaptosome-associated protein of 25 kDa] families) previously identified to act as t-SNAREs, and which therefore constitute a new superfamily. This homology domain of approximately 60 amino acids is predicted to form a coiled-coil structure. The significance of this homology domain could be demonstrated by a partial suppression of the coiled-coil properties of the domain profile. In proteins belonging to the syntaxin family, a single homology domain is located near the transmembrane domain, whereas the members of the SNAP-25 family possess two homology domains. This domain has also been identified in several proteins that have been implicated in vesicular transport but do not belong to any of the t-SNARE protein families. Several new yeast, nematode, and mammalian proteins have been identified that belong to the new superfamily. The evolutionary conservation of the SNARE coiled-coil homology domain suggests that this domain has a similar function in different membrane fusion proteins (Weimbs, 1997).

The interaction between the proteins syntaxin 1A and SNAP-25 is a key step in synaptic vesicle docking and fusion. To define the SNAP-25 binding domain on syntaxin, peptides were prepared that span the syntaxin H3 domain (residues 191-266), the region previously shown to be important for binding to SNAP-25. The affinities of these peptides for binding to SNAP-25 were then determined. A minimal binding domain was identified within a region of 32 amino acids (residues 189-220). Its affinity for SNAP-25 is substantially enhanced by C-terminal extension (residues 221-266). Circular dichroism reveals the presence of substantial alpha-helicity in the H3 domain and in the 32-mer minimal binding domain, but not in H3 peptides that do not bind to SNAP-25. At temperatures that denature the alpha-helix of the minimal binding domain peptide, SNAP-25 binding is lost. Selected mutations in evolutionarily conserved residues of the amphiphilic alpha-helix within the minimal binding domain (e.g., residues 205 and 209) greatly reduce the affinity for SNAP-25 but have no major effect on secondary structure, suggesting that these residues may interact directly with SNAP-25. The H3 domain peptide and the minimal binding domain peptide inhibit norepinephrine release from PC12 cells. These results suggest that specific amino acid residues in the H3 domain, positioned by the underlying alpha-helical structure, are important for its binding to SNAP-25 and support the notion that this interaction is important for presynaptic vesicular exocytosis (Zhong, 1997).

date revised: 22 May 2023

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