Synaptotagmin (syt) 7 is one of three syt isoforms found in all metazoans; it is ubiquitously expressed, yet its function in neurons remains obscure. Here, we resolved Ca2+-dependent and Ca2+-independent synaptic vesicle (SV) replenishment pathways, and found that syt 7 plays a selective and critical role in the Ca2+-dependent pathway. Mutations that disrupt Ca2+-binding to syt 7 abolish this function, suggesting that syt 7 functions as a Ca2+-sensor for replenishment. The Ca2+-binding protein calmodulin (CaM) has also been implicated in SV replenishment, and we found that loss of syt 7 was phenocopied by a CaM antagonist. Moreover, we discovered that syt 7 binds to CaM in a highly specific and Ca2+-dependent manner; this interaction requires intact Ca2+-binding sites within syt 7. Together, these data indicate that a complex of two conserved Ca2+-binding proteins, syt 7 and CaM, serve as a key regulator of SV replenishment in presynaptic nerve terminals.DOI: http://dx.doi.org/10.7554/eLife.01524.001
SUMMARY Synaptotagmin-I (syt) is a Ca2+ sensor that triggers synchronous neurotransmitter release. The first documented biochemical property of syt was its ability to aggregate membranes in response to Ca2+. However, the mechanism and function of syt-mediated membrane aggregation are poorly understood. Here, we demonstrate that syt-mediated vesicle aggregation is driven by trans interactions between syt molecules bound to different membranes. We observed a strong correlation between the ability of Ca2+-syt to aggregate vesicles and to stimulate SNARE-mediated membrane fusion. Moreover, artificial aggregation of membranes - using non-syt proteins - also efficiently promoted fusion of SNARE-bearing liposomes. Finally, using a modified fusion assay, we observed that syt drives the assembly of otherwise non-fusogenic individual t-SNARE proteins into fusion competent heterodimers, in an aggregation-independent manner. Thus, membrane aggregation and t-SNARE assembly appear to be two key aspects of Ca2+-syt-regulated, SNARE-catalyzed fusion reactions.
Different synaptotagmin isoforms (syt I, VII, and IX) sort to populations of dense-core vesicles with different sizes. These isoforms differ in their sensitivities to divalent cations and trigger different modes of exocytosis. Exocytosis triggered by these isoforms also differs in its sensitivity to inhibition by another isoform, syt IV.
Synaptotagmin is the major calcium sensor for fast synaptic transmission which requires the synchronous fusion of synaptic vesicles. Synaptotagmin contains two calcium binding domains: C2A and C2B. Mutation of a positively charged residue (R233Q in rat) showed that Ca2+-dependent interactions between the C2A domain and membranes play a role in the electrostatic switch that initiates fusion. Surprisingly, aspartate to asparagine mutations in C2A that inhibit Ca2+ binding support efficient synaptic transmission, suggesting that Ca2+ binding by C2A is not required for triggering synchronous fusion. Based on a structural analysis, we generated a novel mutation of a single Ca2+-binding residue in C2A (D229E in Drosophila) that inhibited Ca2+ binding, but maintained the negative charge of the pocket. This C2A aspartate to glutamate mutation resulted in ~80% decrease in synchronous transmitter release and a decrease in the apparent Ca2+ affinity of release. Previous aspartate to asparagine mutations in C2A partially mimicked Ca2+ binding by decreasing the negative charge of the pocket. We now show that the major function of Ca2+ binding to C2A is to neutralize the negative charge of the pocket, thereby unleashing the fusion-stimulating activity of synaptotagmin. Our results demonstrate that Ca2+ binding by C2A is a critical component of the electrostatic switch that triggers synchronous fusion. Thus, Ca2+ binding by C2B is necessary and sufficient to regulate the precise timing required for coupling vesicle fusion to Ca2+ influx, but Ca2+ binding by both C2 domains is required to flip the electrostatic switch that triggers efficient synchronous synaptic transmission.
In vitro reconstitution fusion assays incorporating full-length membrane-anchored synaptotagmin I clarify its role in several steps in the secretory pathway.
Synaptotagmin (syt) 1 is localized to synaptic vesicles, binds Ca 2؉ , and regulates neuronal exocytosis. Syt 1 harbors two Ca 2؉ -binding motifs referred to as C2A and C2B. In this study we examine the function of the isolated C2 domains of Syt 1 using a reconstituted, SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor)-mediated, fusion assay. We report that inclusion of phosphatidylethanolamine into reconstituted SNARE vesicles enabled isolated C2B, but not C2A, to regulate Ca 2؉ -triggered fusion. The isolated C2B domain had a 6-fold lower EC 50 for Ca 2؉ -activated fusion than the intact cytosolic domain of Syt 1 (C2AB). Phosphatidylethanolamine increased both the rate and efficiency of C2AB-and C2B-regulated fusion without affecting their abilities to bind membraneembedded syntaxin-SNAP-25 (t-SNARE) complexes. At equimolar concentrations, the isolated C2A domain was an effective inhibitor of C2B-, but not C2AB-regulated fusion; hence, C2A has markedly different effects in the fusion assay depending on whether it is tethered to C2B. Finally, scanning alanine mutagenesis of C2AB revealed four distinct groups of mutations within the C2B domain that play roles in the regulation of SNARE-mediated fusion. Surprisingly, substitution of Arg-398 with alanine, which lies on the opposite end of C2B from the Ca 2؉ /membrane-binding loops, decreases C2AB t-SNARE binding and Ca 2؉-triggered fusion in vitro without affecting Ca 2؉ -triggered interactions with phosphatidylserine or vesicle aggregation. In addition, some mutations uncouple the clamping and stimulatory functions of syt 1, suggesting that these two activities are mediated by distinct structural determinants in C2B.In nerve terminals, synaptic vesicles (SVs) 2 are loaded with neurotransmitter, docked at active zones, and primed for exocytosis. Increases in intracellular [Ca 2ϩ ] then trigger the synchronous fusion of docked SVs with the plasma membrane, releasing transmitters into the synaptic cleft (1). The fusion step is thought to be mediated by SNARE proteins that form the core of a conserved membrane fusion machine. The SV SNARE complex consists of the vesicle protein (v-SNARE) synaptobrevin (syb), and the target membrane proteins (t-SNAREs) syntaxin 1 and SNAP-25 (2). A current challenge is to understand how the SNARE complex is regulated such that it drives fusion, in response to Ca 2ϩ influx, on the microsecond to millisecond time scale.Syt 1 is localized to synaptic vesicles and large dense-core granules in neurons and neuroendocrine cells and is thought to function as a Ca 2ϩ sensor for rapid synchronous release of neurotransmitter (3). Structurally, syt 1 consists of a short luminal tail, a single transmembrane domain, and a cytosolic region comprising two C2 domains, C2A and C2B, connected by a short linker (4). In response to binding Ca 2ϩ , both C2 domains of syt 1 partially penetrate into lipid bilayers that contain anionic phospholipids such as phosphatidylserine (PS) (5-7); these interactions play a critical role during mem...
The purpose of the study was to determine whether novel, selective antagonists of human A 3 adenosine receptors (ARs) derived from the A 3 -selective agonist Cl-IB-MECA lower intraocular pressure (IOP) and act across species. IOP was measured invasively with a micropipette by the ServoNull Micropipette System (SNMS) and by non-invasive pneumotonometry during topical drug application. Antagonist efficacy was also assayed by measuring inhibition of adenosine-triggered shrinkage of native bovine nonpigmented ciliary epithelial (NPE) cells. Five agonist-based A 3 AR antagonists lowered mouse IOP measured with SNMS tonometry by 3-5 mm Hg within minutes of topical application. Of the five agonist derivatives, LJ 1251 was the only antagonist to lower IOP measured by pneumotonometry. No effect was detected pneumotonometrically over 30 min following application of the other four compounds, consonant with slower, smaller responses previously measured non-invasively following topical application of A 3 AR agonists and the dihydropyridine A 3 AR antagonist MRS 1191. Latanoprost similarly lowered SNMS-measured IOP, but not IOP measured non-invasively over 30 minutes. Like MRS 1191, agonist-based A 3 AR antagonists applied to native bovine NPE cells inhibited adenosine-triggered shrinkage. In summary, the results indicate that antagonists of human A 3 ARs derived from the potent, selective A 3 agonist Cl-IB-MECA display efficacy in mouse and bovine cells, as well. When intraocular delivery was enhanced by measuring mouse IOP invasively, five derivatives of the A 3 AR agonist Cl-IB-MECA lowered IOP but only one rapidly reduced IOP measured non-invasively after topical application. We conclude that derivatives of the highly selective A 3 AR agonist Cl-IB-MECA can reduce IOP upon reaching their intraocular target, and that nucleoside-based derivatives are promising A 3 antagonists for study in multiple animal models.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.