Somatostatin modulation of initial fusion pores in Ca2+-triggered exocytosis from mouse chromaffin cells.

Somatostatin, a peptide hormone that activates G-protein-coupled receptors, inhibits the secretion of many hormones. This study investigated the mechanisms of this inhibition using amperometry recording of Ca2+-triggered catecholamine secretion from mouse chromaffin cells. Two distinct stimulation protocols, high-KCl depolarization and caffeine, were used to trigger exocytosis, and confocal fluorescence imaging was used to monitor the rise in intracellular free Ca2+. Analysis of single-vesicle fusion events (spikes) resolved the action of somatostatin on fusion pores at different stages. Somatostatin reduced spike frequency, and this reduction was accompanied by prolongation of pre-spike feet and slowing of spike rise times. This indicates that somatostatin stabilizes initial fusion pores and slows their expansion. This action on the initial fusion pore impacted the release mode to favour kiss-and-run over full-fusion. During a spike the permeability of a fusion pore peaks, declines and then settles into a plateau. Somatostatin had no effect on the plateau, suggesting no influence on late-stage fusion pores. These actions of somatostatin were indistinguishable between exocytosis triggered by high-KCl and caffeine, and fluorescence imaging showed that somatostatin had no effect on stimulus-induced rises in cytosolic Ca2+. Our findings thus demonstrate that the signalling cascades activated by somatostatin target the exocytotic machinery that controls the initial and expanding stages of fusion pores, while having no effect on late-stage fusion pores. As a result of its stronger inhibition of full-fusion compared to kiss-and-run, somatostatin will preferentially inhibit the secretion of large peptides over the secretion of small catecholamines. KEY POINTS: Somatostatin inhibits the secretion of various hormones by activating G-protein-coupled receptors. In this study, we used amperometry to investigate the mechanism by which somatostatin inhibits catecholamine release from mouse chromaffin cells. Somatostatin increased pre-spike foot lifetime and slowed fusion pore expansion. Somatostatin inhibited full-fusion more strongly than kiss-and-run. Our results suggest that the initial fusion pore is the target of somatostatin-mediated regulation of hormone release. The stronger inhibition of full-fusion by somatostatin will result in preferential inhibition of peptide release.

Synergistic regulation of fusion pore opening and dilation by SNARE and synaptotagmin-1.

Fusion pore opening is a transient intermediate state of synaptic vesicle exocytosis, which is highly dynamic and precisely regulated by the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex and synaptotagmin-1 (Syt1). Yet, the regulatory mechanism is not fully understood. In this work, using single-channel membrane fusion electrophysiology, we determined that SNAREpins are important for driving fusion pore opening and dilation but incapable of regulating the dynamics. When Syt1 was added, the closing frequency of fusion pores significantly increased, while the radius of fusion pores mildly decreased. In response to Ca2+, SNARE/Syt1 greatly increased the radius of fusion pores and reduced their closing frequency. Moreover, the residue F349 in the C2B domain of Syt1, which mediates Syt1 oligomerization, was required for clamping fusion pore opening in the absence of Ca2+, probably by extending the distance between the two membranes. Finally, in Ca2+-triggered fusion, the primary interface between SNARE and Syt1 plays a critical role in stabilizing and dilating the fusion pore, while the polybasic region of Syt1 C2B domain has a mild effect on increasing the radius of the fusion pore. In summary, our results suggest that Syt1, SNARE, and the anionic membrane synergically orchestrate the dynamics of fusion pore opening in synaptic vesicle exocytosis.

The role of α-synuclein in exocytosis.

The pathogenesis of degeneration in Parkinson's disease (PD) remains poorly understood but multiple lines of evidence have converged on the presynaptic protein α-synuclein (αsyn). αSyn has been shown to regulate several cellular processes, however, its normal function remains poorly understood. In this review, we will specifically focus on its role in exocytosis.

The Art of Viral Membrane Fusion and Penetration.

As obligate pathogens, viruses have developed diverse mechanisms to deliver their genome across host cell membranes to sites of virus replication. While enveloped viruses utilize viral fusion proteins to accomplish fusion of their envelope with the cellular membrane, non-enveloped viruses rely on machinery that causes local membrane ruptures and creates an opening through which the capsid or viral genome is released. Both membrane fusion and membrane penetration take place at the plasma membrane or in intracellular compartments, often involving the engagement of the cellular machinery and antagonism of host restriction factors. Enveloped and non-enveloped viruses have evolved intricate mechanisms to enable virus uncoating and modulation of membrane fusion in a spatiotemporally controlled manner. This chapter summarizes and discusses the current state of understanding of the mechanisms of viral membrane fusion and penetration. The focus is on the role of lipids, viral scaffold uncoating, viral membrane fusion inhibitors, and host restriction factors as physicochemical modulators. In addition, recent advances in visualizing and detecting viral membrane fusion and penetration using cryo-electron microscopy methods are presented.

α-Synuclein is required for sperm exocytosis at a post-fusion stage.

The sperm acrosome is a large dense-core granule whose contents are secreted by regulated exocytosis at fertilization through the opening of numerous fusion pores between the acrosomal and plasma membranes. In other cells, the nascent pore generated when the membrane surrounding a secretory vesicle fuses with the plasma membrane may have different fates. In sperm, pore dilation leads to the vesiculation and release of these membranes, together with the granule contents. α-Synuclein is a small cytosolic protein claimed to exhibit different roles in exocytic pathways in neurons and neuroendocrine cells. Here, we scrutinized its function in human sperm. Western blot revealed the presence of α-synuclein and indirect immunofluorescence its localization to the acrosomal domain of human sperm. Despite its small size, the protein was retained following permeabilization of the plasma membrane with streptolysin O. α-Synuclein was required for acrosomal release, as demonstrated by the inability of an inducer to elicit exocytosis when permeabilized human sperm were loaded with inhibitory antibodies to human α-synuclein. The antibodies halted calcium-induced secretion when introduced after the acrosome docked to the cell membrane. Two functional assays, fluorescence and transmission electron microscopies revealed that the stabilization of open fusion pores was responsible for the secretion blockage. Interestingly, synaptobrevin was insensitive to neurotoxin cleavage at this point, an indication of its engagement in cis SNARE complexes. The very existence of such complexes during AE reflects a new paradigm. Recombinant α-synuclein rescued the inhibitory effects of the anti-α-synuclein antibodies and of a chimeric Rab3A-22A protein that also inhibits AE after fusion pore opening. We applied restrained molecular dynamics simulations to compare the energy cost of expanding a nascent fusion pore between two model membranes and found it higher in the absence than in the presence of α-synuclein. Hence, our results suggest that α-synuclein is essential for expanding fusion pores.

Ca2+ and cAMP open differentially dilating synaptic fusion pores.

Neuronal dense-core vesicles (DCVs) contain neuropeptides and much larger proteins that affect synaptic growth and plasticity. Rather than using full collapse exocytosis that commonly mediates peptide hormone release by endocrine cells, DCVs at the Drosophila neuromuscular junction release their contents via fusion pores formed by kiss-and-run exocytosis. Here, we used fluorogen-activating protein (FAP) imaging to reveal the permeability range of synaptic DCV fusion pores and then show that this constraint is circumvented by cAMP-induced extra fusions with dilating pores that result in DCV emptying. These Ca2+-independent full fusions require PKA-R2, a PKA phosphorylation site on Complexin and the acute presynaptic function of Rugose, the homolog of mammalian neurobeachin, a PKA-R2 anchor implicated in learning and autism. Therefore, localized Ca2+-independent cAMP signaling opens dilating fusion pores to release large cargoes that cannot pass through the narrower fusion pores that mediate spontaneous and activity-dependent neuropeptide release. These results imply that the fusion pore is a variable filter that differentially sets the composition of proteins released at the synapse by independent exocytosis triggers responsible for routine peptidergic transmission (Ca2+) and synaptic development (cAMP).

Intrinsic Disorder in α-Synuclein Regulates the Exocytotic Fusion Pore Transition.

Today, it is widely accepted that intrinsic disorder is strongly related to the cell cycle, during mitosis, differentiation, and apoptosis. Of particular interest are hybrid proteins possessing both structured and unstructured domains that are critical in human health and disease, such as α-synuclein. In this work, we describe how α-synuclein interacts with the nascent fusion pore as it evolves toward expansion. We unveil the key role played by its intrinsically disordered region as a thermodynamic regulator of the nucleation-expansion energy barrier. By analyzing a truncated variant of α-synuclein that lacks the disordered region, we find that the landscape of protein interactions with PIP2 and POPS lipids is highly altered, ultimately increasing the energy cost for the fusion pore to transit from nucleation to expansion. We conclude that the intrinsically disordered region in full-length α-synuclein recognizes and allocates pivotal protein:lipid interactions during membrane remodeling in the first stages of the fusion pore.

Barriers to exocytotic vesicle discharge.

Regulated exocytosis, a universal process of eukaryotic cells, involves the merging between the vesicle membrane and the plasma membrane, plays a key role in cell-to-cell communication, particularly in the release of hormones and neurotransmitters. There are a number of barriers a vesicle needs to pass to discharge vesicle content to the extracellular space. At the pre-fusion site vesicles need to be transported to the sites on the plasma membrane where the merger may begin. Classically cytoskeleton was considered an important barrier for vesicle translocation and was thought to be disintegrated to allow vesicle access to the plasma membrane [1]. However, it was considered later that cytoskeletal elements may also play a role at the post-fusion stage, promoting the vesicle merger with the plasma membrane and fusion pore expansion [4,22,23]. In this Special Issue of Cell Calcium entitled "Regulated Exocytosis", the authors address outstanding issues related to vesicle chemical messenger release by regulated exocytosis, including that related to the question whether vesicle content discharge is complete or only partial upon the merging of the vesicle membrane with the plasma membrane triggered by Ca2+. Among processes that limit vesicle discharge at the post-fusion stage is the accumulation of cholesterol in some vesicles [19], a process that has recently been associated with cell aging [20].

Forty years of the adrenal chromaffin cell through ISCCB meetings around the world.

This historical review focuses on the evolution of the knowledge accumulated during the last two centuries on the biology of the adrenal medulla gland and its chromaffin cells (CCs). The review emerged in the context of a series of meetings that started on the Spanish island of Ibiza in 1982 with the name of the International Symposium on Chromaffin Cell Biology (ISCCB). Hence, the review is divided into two periods namely, before 1982 and from this year to 2022, when the 21st ISCCB meeting was just held in Hamburg, Germany. The first historical period extends back to 1852 when Albert Kölliker first described the fine structure and function of the adrenal medulla. Subsequently, the adrenal staining with chromate salts identified the CCs; this was followed by the establishment of the embryological origin of the adrenal medulla, and the identification of adrenaline-storing vesicles. By the end of the nineteenth century, the basic morphology, histochemistry, and embryology of the adrenal gland were known. The twentieth century began with breakthrough findings namely, the experiment of Elliott suggesting that adrenaline was the sympathetic neurotransmitter, the isolation of pure adrenaline, and the deciphering of its molecular structure and chemical synthesis in the laboratory. In the 1950s, Blaschko isolated the catecholamine-storing vesicles from adrenal medullary extracts. This switched the interest in CCs as models of sympathetic neurons with an explosion of studies concerning their functions, i.e., uptake of catecholamines by chromaffin vesicles through a specific coupled transport system; the identification of several vesicle components in addition to catecholamines including chromogranins, ATP, opioids, and other neuropeptides; the calcium-dependence of the release of catecholamines; the underlying mechanism of exocytosis of this release, as indicated by the co-release of proteins; the cross-talk between the adrenal cortex and the medulla; and the emission of neurite-like processes by CCs in culture, among other numerous findings. The 1980s began with the introduction of new high-resolution techniques such as patch-clamp, calcium probes, marine toxins-targeting ion channels and receptors, confocal microscopy, or amperometry. In this frame of technological advances at the Ibiza ISCCB meeting in 1982, 11 senior researchers in the field predicted a notable increase in our knowledge in the field of CCs and the adrenal medulla; this cumulative knowledge that occurred in the last 40 years of history of the CC is succinctly described in the second part of this historical review. It deals with cell excitability, ion channel currents, the exocytotic fusion pore, the handling of calcium ions by CCs, the kinetics of exocytosis and endocytosis, the exocytotic machinery, and the life cycle of secretory vesicles. These concepts together with studies on the dynamics of membrane fusion with super-resolution imaging techniques at the single-protein level were extensively reviewed by top scientists in the field at the 21st ISCCB meeting in Hamburg in the summer of 2022; this frontier topic is also briefly reviewed here. Many of the concepts arising from those studies contributed to our present understanding of synaptic transmission. This has been studied in physiological or pathophysiological conditions, in CCs from animal disease models. In conclusion, the lessons we have learned from CC biology as a peripheral model for brain and brain disease pertain more than ever to cutting-edge research in neurobiology. In the 22nd ISCCB meeting in Israel in 2024 that Uri Asheri is organizing, we will have the opportunity of seeing the progress of the questions posed in Ibiza, and on other questions that undoubtedly will arise.

Local PI(4,5)P2 signaling inhibits fusion pore expansion during exocytosis.

Phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) is an important signaling phospholipid that is required for regulated exocytosis and some forms of endocytosis. The two processes share a topologically similar pore structure that connects the vesicle lumen with the outside. Widening of the fusion pore during exocytosis leads to cargo release, while its closure initiates kiss&run or cavicapture endocytosis. We show here, using live-cell total internal reflection fluorescence (TIRF) microscopy of insulin granule exocytosis, that transient accumulation of PI(4,5)P2 at the release site recruits components of the endocytic fission machinery and stalls the late fusion pore expansion that is required for peptide release. The absence of clathrin differentiates this mechanism from clathrin-mediated endocytosis. Knockdown of phosphatidylinositol-phosphate-5-kinase-1c or optogenetic recruitment of 5-phosphatase reduces PI(4,5)P2 transients and accelerates fusion pore expansion, suggesting that acute PI(4,5)P2 synthesis is involved. Thus, local phospholipid signaling inhibits fusion pore expansion and peptide release through an unconventional endocytic mechanism.

Rat Pheochromocytoma PC12 Cells in Culture.

PC12 cells serve as a secretory cell model, especially suitable for studying the molecular mechanisms underlying fusion pore kinetics in regulated exocytosis of dense-core vesicles (DCVs). In this chapter, we describe a series of PC12 cell culture procedures optimized for real-time functional assays such as single-vesicle amperometry. In addition, these conditions have been widely used for single-cell biochemical assays such as the proximity ligation assay with immunostaining.

Visualization of Exo- and Endocytosis Membrane Dynamics with Super-Resolution STED Microscopy.

Recent advances in stimulated emission depletion (STED) microscopy offer an unparalleled avenue to study membrane dynamics of exo- and endocytosis, such as fusion pore opening, pore expansion, constriction, and closure, as well as the membrane transformation from flat-shaped to round-shaped vesicles in real time. Here we depict a method of using the state-of-the-art STED microscopy to image these membrane dynamics in bovine chromaffin cells. This method can potentially be applied to study other membrane structure dynamics in other cell model system.

Patch Amperometry and Intracellular Patch Electrochemistry.

Both patch amperometry (PA) and intracellular patch electrochemistry (IPE) take advantage of a recording configuration where an electrochemical detector-carbon fiber electrode (CFE)-is housed inside a patch pipette. PA, which is employed in cell-attached or excised inside-out patch clamp configuration, offers high-resolution patch capacitance measurements with simultaneous amperometric detection of catecholamines released during exocytosis. The method provides precise information on single-vesicle size and quantal content, fusion pore conductance, and permeability of the pore for catecholamines. IPE, on the other hand, measures cytosolic catecholamines that diffuse into the patch pipette following membrane rupture to achieve the whole-cell configuration. In amperometric mode, IPE detects total catechols, whereas in cyclic voltammetric mode, it provides more specific information on the nature of the detected molecules and may selectively quantify catecholamines, providing a direct approach to determine cytosolic concentrations of catecholaminergic transmitters and their metabolites. Here, we provide detailed instructions on setting up PA and IPE, performing experiments and analyzing the data.

Artificial Cells for Dissecting Exocytosis.

The fusion of vesicles and exocytosis release of neurotransmitters into the extracellular space for detection and chemical signal decoding by neighboring cells is the key process in neuronal communication. It is important to understand what regulates exocytosis because the amount of neurotransmitters released into the synaptic cleft has a direct impact on brain function such as cognition learning and memory as well as on brain malfunctions. Much success in molecular biology can be credited for the existence of simplified model systems. Therefore, for gaining deeper insights into the details of exocytosis and what controls vesicle-mediated neurotransmission, functional artificial cells for exocytosis have been developed that can be used for studying various biophysical aspects and roles of molecules affecting exocytosis, which is difficult to study in living cells. Here, we describe the design and fabrication of specific artificial cell models and how chemical measurements at these cells can be implemented for probing dynamics of the exocytosis fusion pore and its effect on the regulation of neurochemical release. We introduce bottom-up synthetic methods for constructing model cells using protein-free giant unilamellar vesicles (GUV) as starting material, which allows further tuning of molecular complexity in a manner that is not possible in living cells and therefore can be used for dissecting the role of essential molecular components affecting the exocytosis process. The experimental setup uses microscopy video recording, micromanipulation and microelectroinjection techniques, and amperometry detection to study neurotransmitter release from these cells mimicking exocytosis.

High-speed imaging reveals the bimodal nature of dense core vesicle exocytosis.

During exocytosis, the fusion of secretory vesicle with plasma membrane forms a pore that regulates release of neurotransmitter and peptide. Heterogeneity of fusion pore behavior has been attributed to stochastic variation in a common exocytic mechanism, implying a lack of biological control. Using a fluorescent false neurotransmitter (FFN), we imaged dense core vesicle (DCV) exocytosis in primary mouse adrenal chromaffin cells by total internal reflection fluorescence microscopy at millisecond resolution and observed strikingly divergent modes of release, with fast events lasting <30 ms and slow events persisting for seconds. Dual imaging of slow events shows a delay in the entry of external dye relative to FFN release, suggesting exclusion by an extremely narrow pore <1 nm in diameter. Unbiased comprehensive analysis shows that the observed variation cannot be explained by stochasticity alone, but rather involves distinct mechanisms, revealing the bimodal nature of DCV exocytosis. Further, loss of calcium sensor synaptotagmin 7 increases the proportion of slow events without changing the intrinsic properties of either class, indicating the potential for independent regulation. The identification of two distinct mechanisms for release capable of independent regulation suggests a biological basis for the diversity of fusion pore behavior.

Calcium-dependent subquantal peptide release from single docked lawn-resident vesicles of pituitary lactotrophs.

Regulated exocytosis consists of the fusion between vesicles and the plasma membranes, leading to the formation of a narrow fusion pore through which secretions exit the vesicle lumen into the extracellular space. An increase in the cytosolic concentration of free Ca2+ ([Ca2+]i) is considered the stimulus of this process. However, whether this mechanism can be preserved in a simplified system of membrane lawns with docked secretory vesicles, devoid of cellular components, is poorly understood. Here, we studied peptide discharge from individual secretory vesicles docked at the plasma membrane, prepared from primary endocrine pituitary cells (the lactotrophs), releasing hormone prolactin. To label secretory vesicles, we transfected lactotrophs to express the fluorescent atrial natriuretic peptide (ANP.emd), previously shown to be expressed in and released from prolactin-containing vesicles. We used stimulating solutions containing different [Ca2+] to evoke vesicle peptide discharge, which appeared similar in membrane lawns and in intact stimulated lactotrophs. All vesicles examined discharged peptides in a subquantal manner, either exhibiting a unitary or sequential time course. In the membrane lawns, the unitary vesicle peptide discharge was predominant and slightly slower than that recorded in intact cells, but with a shorter delay with respect to the stimulation onset. This study revealed directly that Ca2+ triggers peptide discharge from docked single vesicles in the membrane lawns with a half-maximal response of ∼8 µM [Ca2+], consistent with previous whole-cell patch-clamp studies in endocrine cells where the rapid component of exocytosis, interpreted to represent docked vesicles, was fully activated at <10 µM [Ca2+]. Interestingly, the sequential subquantal peptide vesicle discharge indicates that fluctuations between constricted and dilated fusion pore states are preserved in membrane lawns and that fusion pore regulation appears to be an autonomously controlled process.

The beginning and the end of SNARE-induced membrane fusion.

Membrane fusion is not a spontaneous process. Physiologically, the formation of coiled-coil protein complexes, the SNAREpins, bridges the membrane of a vesicle and a target membrane, brings them in close contact, and provides the energy necessary for their fusion. In this review, we utilize results from in vitro experiments and simple physics and chemistry models to dissect the kinetics and energetics of the fusion process from the encounter of the two membranes to the full expansion of a fusion pore. We find three main energy barriers that oppose the fusion process: SNAREpin initiation, fusion pore opening, and expansion. SNAREpin initiation is inherent to the proteins and makes in vitro fusion kinetic experiments rather slow. The kinetics are physiologically accelerated by effectors. The energy barriers that precede pore opening and pore expansion can be overcome by several SNAREpins acting in concert.

Full-fusion and kiss-and-run in chromaffin cells controlled by irreversible vesicle size-dependent fusion pore transitions.

Exocytosis operates through two distinct modes. Full-fusion leads to rapid expulsion of the entire content of a vesicle; kiss-and-run leads to slow and partial expulsion. These two modes have important biological consequences for endocrine regulation and synaptic transmission. Amperometry recordings of catecholamine release from chromaffin cells reveal single-vesicle fusion events corresponding to both of these modes, but classification is often difficult. This study introduces a new method of analyzing amperometry data to improve this classification. The ratio of the average amplitude to the peak amplitude differs between full-fusion and kiss-and-run, and the probability distribution of this ratio is well fitted by a double-Gaussian. Kiss-and-run events identified by this method have fusion pores with kinetic properties different from pores associated with full-fusion. They have slower transition rates and lifetime distributions indicative of irreversible transitions. The total-charge of an amperometric spike is expected to scale with vesicle volume during a full-fusion event. The cube root of this quantity should therefore scale with diameter, but the distribution of this quantity differs from the distribution of vesicle diameter seen in the electron microscope. Fusion pore lifetimes associated with full-fusion depend on vesicle size, and this makes the choice of mode size dependent. The fusion pore thus bifurcates after opening, and vesicle size influences this choice. The secretory vesicle protein synaptophysin influences the size dependence of fusion pore lifetime and the choice of release mode. Incorporating vesicle size into an analysis of release mode reconciled the kinetics of fusion pores, as well as the distributions of vesicle diameter and catecholamine content. Thus, the initial fusion pore emerges as a critical focus in endocrine regulation. By modulating the size-dependence of the mode of exocytosis, changes in the molecular makeup of the exocytotic apparatus can impact the shape and size of an amperometric event, and the speed and composition of secretion.

Synaptic Integration of Subquantal Neurotransmission by Colocalized G Protein-Coupled Receptors in Presynaptic Terminals.

In presynaptic terminals, membrane-delimited Gi/o-mediated presynaptic inhibition is ubiquitous and acts via Gßγ to inhibit Ca2+ entry, or directly at SNARE complexes to inhibit Ca2+-dependent synaptotagmin-SNARE complex interactions. At CA1-subicular presynaptic terminals, 5-HT1B and GABAB receptors colocalize. GABAB receptors inhibit Ca2+ entry, whereas 5-HT1B receptors target SNARE complexes. We demonstrate in male and female rats that GABAB receptors alter Pr, whereas 5-HT1B receptors reduce evoked cleft glutamate concentrations, allowing differential inhibition of AMPAR and NMDAR EPSCs. This reduction in cleft glutamate concentration was confirmed by imaging glutamate release using a genetic sensor (iGluSnFR). Simulations of glutamate release and postsynaptic glutamate receptor currents were made. We tested effects of changes in vesicle numbers undergoing fusion at single synapses, relative placement of fusing vesicles and postsynaptic receptors, and the rate of release of glutamate from a fusion pore. Experimental effects of Pr changes, consistent with GABAB receptor effects, were straightforwardly represented by changes in numbers of synapses. The effects of 5-HT1B receptor-mediated inhibition are well fit by simulated modulation of the release rate of glutamate into the cleft. Colocalization of different actions of GPCRs provides synaptic integration within presynaptic terminals. Train-dependent presynaptic Ca2+ accumulation forces frequency-dependent recovery of neurotransmission during 5-HT1B receptor activation. This is consistent with competition between Ca2+-synaptotagmin and Gßγ at SNARE complexes. Thus, stimulus trains in 5-HT1B receptor agonist unveil dynamic synaptic modulation and a sophisticated hippocampal output filter that itself is modulated by colocalized GABAB receptors, which alter presynaptic Ca2+ In combination, these pathways allow complex presynaptic integration.SIGNIFICANCE STATEMENT Two G protein-coupled receptors colocalize at presynaptic sites, to mediate presynaptic modulation by Gßγ, but one (a GABAB receptor) inhibits Ca2+ entry whereas another (a 5-HT1B receptor) competes with Ca2+-synaptotagmin binding to the synaptic vesicle machinery. We have investigated downstream effects of signaling and integrative properties of these receptors. Their effects are profoundly different. GABAB receptors alter Pr leaving synaptic properties unchanged, whereas 5-HT1B receptors fundamentally change properties of synaptic transmission, modifying AMPAR but sparing NMDAR responses. Coactivation of these receptors allows synaptic integration because of convergence of GABAB receptor alteration on Ca2+ and the effect of this altered Ca2+ signal on 5-HT1B receptor signaling. This presynaptic convergence provides a novel form of synaptic integration.