Triggered Vesicle Fusion Research Articles

PIP2 Is An Electrostatic Catalyst for Vesicle Fusion by Lowering the Hydration Energy: Arresting Vesicle Fusion by Masking PIP2.

Lipids are key factors in regulating membrane fusion. Lipids are not only structural components to form membranes but also active catalysts for vesicle fusion and neurotransmitter release, which are driven by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. SNARE proteins seem to be partially assembled before fusion, but the mechanisms that arrest vesicle fusion before Ca2+ influx are still not clear. Here, we show that phosphatidylinositol 4,5-bisphosphate (PIP2) electrostatically triggers vesicle fusion as an electrostatic catalyst by lowering the hydration energy and that a myristoylated alanine-rich C-kinase substrate (MARCKS), a PIP2-binding protein, arrests vesicle fusion in a vesicle docking state where the SNARE complex is partially assembled. Vesicle-mimicking liposomes fail to reproduce vesicle fusion arrest by masking PIP2, indicating that native vesicles are essential for the reconstitution of physiological vesicle fusion. PIP2 attracts cations to repel water molecules from membranes, thus lowering the hydration energy barrier.

Membrane Capacitance Measurements of Stimulus-Evoked Exocytosis in Adrenal Chromaffin Cells.

Research using membrane capacitance (Cm) measurements in adrenal chromaffin cells has transformed our understanding of the molecular mechanisms controlling regulated exocytosis. This is in part due to the exquisite temporal resolution of the technique, and the possibility of combining quantification of exo-/endocytosis at the whole-cell level, with the ability to simultaneously monitor and control the calcium signals triggering vesicle fusion. In this regard, experiments performed with Cm measurements complement amperometry experiments that give a measure of secreted transmitter and the behavior of the fusion pore, and fluorescent microscopy studies used to monitor vesicle and protein dynamics in imaged regions of the cell. In this chapter, we provide a detailed account of the methodology used to perform whole-cell patch clamp measurements of Cm in combination with voltage-clamp recordings of voltage-gated calcium channels to quantify stimulus-secretion coupling in chromaffin cells. Stimulus protocols developed for investigation of functionally distinct releasable vesicle pools are also described.

Mixed Silane‐based Self‐assembled Monolayers Deposited on Fluorine Doped Tin Oxide as Model System for Development of Biosensors for Toxin Detection

AbstractIn this work mixed hybrid phospholipid bilayers (mhBLM) were deposited on fluorine doped tin oxide (FTO) films. Two component silane‐based self‐assembled monolayers (SAMs) formed on FTO surface trigger vesicle fusion and formation of mhBLMs which are stable, can be easily regenerated, and therefore, used for multiple experiments. We found that certain chemical and physical conditions under which mixed SAMs are fabricated translate into functional properties of mhBLMs. In all cases we observed interaction of melittin with mhBLMs demonstrating biological relevance of these biomimetic surface constructs and their possible application in biosensors for toxin detection.

Membrane Association and Functional Mechanism of Synaptotagmin-1 in Triggering Vesicle Fusion

Upon Ca2+ influx, synaptic vesicles fuse with the presynaptic plasma membrane (PM) to release neurotransmitters. Membrane fusion is triggered by synaptotagmin-1, a transmembrane protein in the vesicle membrane (VM), but the mechanism is under debate. Synaptotagmin-1 contains a single transmembrane helix (TM) and two tandem C2 domains (C2A and C2B). This study aimed to use molecular dynamics simulations to elucidate how Ca2+-bound synaptotagmin-1, by simultaneously associating with VM and PM, brings them together for fusion. Although C2A stably associates with VM via two Ca2+-binding loops, C2B has a propensity to partially dissociate. Importantly, an acidic motif in the TM-C2A linker competes with VM for interacting with C2B, thereby flipping its orientation to face PM. Subsequently, C2B readily associates with PM via a polybasic cluster and a Ca2+-binding loop. The resulting mechanistic model for the triggering of membrane fusion by synaptotagmin-1 reconciles many experimental observations.

The C2A domain of synaptotagmin is an essential component of the calcium sensor for synaptic transmission.

The synaptic vesicle protein, synaptotagmin, is the principle Ca2+ sensor for synaptic transmission. Ca2+ influx into active nerve terminals is translated into neurotransmitter release by Ca2+ binding to synaptotagmin's tandem C2 domains, triggering the fast, synchronous fusion of multiple synaptic vesicles. Two hydrophobic residues, shown to mediate Ca2+-dependent membrane insertion of these C2 domains, are required for this process. Previous research suggested that one of its tandem C2 domains (C2B) is critical for fusion, while the other domain (C2A) plays only a facilitatory role. However, the function of the two hydrophobic residues in C2A have not been adequately tested in vivo. Here we show that these two hydrophobic residues are absolutely required for synaptotagmin to trigger vesicle fusion. Using in vivo electrophysiological recording at the Drosophila larval neuromuscular junction, we found that mutation of these two key C2A hydrophobic residues almost completely abolished neurotransmitter release. Significantly, mutation of both hydrophobic residues resulted in more severe deficits than those seen in synaptotagmin null mutants. Thus, we report the most severe phenotype of a C2A mutation to date, demonstrating that the C2A domain is absolutely essential for synaptotagmin's function as the electrostatic switch.

An Electrostatic Energy Barrier for SNARE-Dependent Spontaneous and Evoked Synaptic Transmission

Information transfer across CNS synapses depends on the very low basal vesicle fusion rate and the ability to rapidly upregulate that rate upon Ca2+ influx. We show that local electrostatic repulsion participates in creating an energy barrier, which limits spontaneous synaptic transmission. The barrier amplitude is increased by negative charges and decreased by positive charges on the SNARE-complex surface. Strikingly, the effect of charges on the barrier is additive and this extends to evoked transmission, but with a shallower charge dependence. Action potential-driven synaptic release is equivalent to the abrupt addition of ∼35 positive charges to the fusion machine. Within an electrostatic model for triggering, the Ca2+ sensor synaptotagmin-1 contributes ∼18 charges by binding Ca2+, while also modulating the fusion barrier at rest. Thus, the energy barrier for synaptic vesicle fusion has a large electrostatic component, allowing synaptotagmin-1 to act as an electrostatic switch and modulator to trigger vesicle fusion.

Models of synaptotagmin-1 to trigger Ca2+ -dependent vesicle fusion.

Vesicles in neurons and neuroendocrine cells store neurotransmitters and peptide hormones, which are released by vesicle fusion in response to Ca2+ -evoking stimuli. Synaptotagmin-1 (Syt1), a Ca2+ sensor, mediates ultrafast exocytosis in neurons and neuroendocrine cells. After vesicle docking, Syt1 has two main groups of binding partners: anionic phospholipids and the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) complex. The molecular mechanisms by which Syt1 triggers vesicle fusion remain controversial. This Review introduces and summarizes six molecular models of Syt1: (a) Syt1 triggers SNARE unclamping by displacing complexin, (b) Syt1 clamps SNARE zippering, (c) Syt1 causes membrane curvature, (d) membrane bridging by Syt1, (e) Syt1 is a vesicle-plasma membrane distance regulator, and (f) Syt1 undergoes circular oligomerization. We discuss important conditions to test Syt1 activity in vitro and attempt to illustrate the possible roles of Syt1.

Formation and properties of phospholipid bilayers on fluorine doped tin oxide electrodes

Commercially available fluorine doped tin oxide (FTO) films on glass were functionalized by the octadecyltrichlorosilane (OTS) self-assembled monolayers (SAM), which upon exposure to a multilamellar phospholipid vesicle solutions trigger vesicle fusion and formation of a tethered phospholipid bilayer membrane (tBLM). The formation of tBLMs was monitored in real-time by the Fast Fourier Transform (FFT) electrochemical impedance spectroscopy (EIS), which attested for the completion of the fusion process within 2000 s. We established that highly insulating complete bilayers can be accomplished only in some specific cases of OTS functionalized FTO films. In particular, only OTS functionalized FTO films exhibiting double semicircular features on the Cole – Cole EIS plots produced tBLMs with the specific capacitance below 1 μF cm−2 comparable to capacitances of the free-standing phospholipid bilayers. The double-semicircular features of EIS spectra can be used as a diagnostic tool for predicting the suitability of silanized FTO substrates for the complete vesicles fusion leading to a formation of the tBLMs. The average value of the capacitance of DOPC bilayers containing 40% (mol) cholesterol was of 0.82 ± 0.10 μF cm−2 consistent with the 3.02 nm thick dielectric film with the relative dielectric constant of 2.8. In contrast to the gold substrates, tBLMs on OTS functionalized FTO films can be regenerated at least 3 times by washing off lipid layer and re-assembling tBLM via the vesicle fusion process. EIS provided direct evidence of the susceptibility of the tBLMs to phospholipase A2, which triggered degradation of the films. However, transmembrane reconstitution of the pore-forming toxin vaginolysin (VLY) proteins was not detected. Surface constructs described in this study may be utilized in peripheral protein studies or in applications in which planar phospholipid layers supported on a semiconductor metal oxide surfaces are required.

Synaptotagmin 7 Mediates Both Facilitation and Asynchronous Release at Granule Cell Synapses.

When an action potential invades a presynaptic terminal it evokes large, brief Ca2+ signals that trigger vesicle fusion within milliseconds that is followed by a small residual Ca2+ (Cares) signal. At many synapses Cares produces synaptic facilitation that lasts up to hundreds of milliseconds and, although less common, Cares can also evoke asynchronous release (AR) that persists for tens of milliseconds. The properties of facilitation and AR are very different, which suggests that they are mediated by distinct mechanisms. However, recently it has been shown that the slow calcium sensor synaptotagmin 7 (Syt7) mediates facilitation at many synapses where AR does not occur, and conversely Syt7 can mediate AR without mediating facilitation. Here we study cerebellar granule cell synapses onto stellate cells and Purkinje cells in mice of both sexes to assess the role of Syt7 in these phenomena at the same synapse. This is of particular interest at granule cell synapses where AR is much more calcium dependent and shorter-lived than facilitation. We find that Syt7 can mediate these two processes despite their divergent properties. In Syt7 knock-out animals, facilitation and AR are smaller and shorter lived than in wild-type animals, even though the initial probability of release and Cares signals are unchanged. Although there are short-lived Syt7-independent mechanisms that mediate facilitation and AR in Syt7 KO animals, we find that at granule cell synapses AR and facilitation are both mediated primarily by Syt7.SIGNIFICANCE STATEMENT At synapses made by cerebellar granule cells, presynaptic activity elevates calcium for tens of milliseconds, which in turn evokes both asynchronous release (AR) and synaptic facilitation. AR is more calcium sensitive and shorter-lived than facilitation at these synapses, suggesting that they are mediated by different mechanisms. However, we find that the slow calcium sensor synaptotagmin 7 mediates both of these phenomena. Small, rapidly decaying components of AR and facilitation are present in Syt7 KO animals, indicating that additional mechanisms can contribute to both AR and facilitation at these synapses.

Accessory and Central α-helices of Complexin Selectively Activate Ca2+ Triggering of Synaptic Exocytosis.

Complexins, binding to assembling soluble NSF-attachment protein receptor (SNARE) complexes, activate Ca2+ triggered exocytosis and clamp spontaneous release in the presynaptic terminal. Functions of complexin are structural dependent and mechanistically distinct. To further understand the functional/structural dependence of complexin, here we show that the accessory and central α-helices of complexin are sufficient in activation of Ca2+ triggered vesicle fusion but not in clamping spontaneous release. Targeting the two α-helices to synaptic vesicle suppresses spontaneous release, thus further emphasizing the importance of curvature membrane localization in clamping function.

Enzymatic cleavage of myoferlin releases a dual C2-domain module linked to ERK signalling

Myoferlin and dysferlin are closely related members of the ferlin family of Ca2+-regulated vesicle fusion proteins. Dysferlin is proposed to play a role in Ca2+-triggered vesicle fusion during membrane repair. Myoferlin regulates endocytosis, recycling of growth factor receptors and adhesion proteins, and is linked to the metastatic potential of cancer cells. Our previous studies establish that dysferlin is cleaved by calpains during membrane injury, with the cleavage motif encoded by alternately-spliced exon 40a. Herein we describe the cleavage of myoferlin, yielding a membrane-associated dual C2 domain ‘mini-myoferlin’. Myoferlin bears two enzymatic cleavage sites: a canonical cleavage site encoded by exon 38 within the C2DE domain; and a second cleavage site in the linker adjacent to C2DE, encoded by alternately-spliced exon 38a, homologous to dysferlin exon 40a. Both myoferlin cleavage sites, when introduced into dysferlin, can functionally substitute for exon 40a to confer Ca2+-triggered calpain cleavage in response to membrane injury. However, enzymatic cleavage of myoferlin is complex, showing both constitutive or Ca2+-enhanced cleavage in different cell lines, that is not solely dependent on calpains-1 or -2. The functional impact of myoferlin cleavage was explored through signalling protein phospho-protein arrays revealing specific activation of ERK1/2 by ectopic expression of cleavable myoferlin, but not an uncleavable isoform. In summary, we molecularly define two enzymatic cleavage sites within myoferlin and demonstrate ‘mini-myoferlin’ can be detected in human breast cancer tumour samples and cell lines. These data further illustrate that enzymatic cleavage of ferlins is an evolutionarily preserved mechanism to release functionally specialized mini-modules.

Preincubation of t-SNAREs with Complexin I Increases Content-Mixing Efficiency

Complexin (Cpx) is a major regulator for Ca(2+)-triggered fast neuroexocytosis which underlies neuronal communication. Many psychiatric and neurological disorders accompany changes in the Cpx expression level, suggesting that abnormal Cpx levels may elicit aberrant cognitive symptoms. To comprehend how the changes in the Cpx level might affect neuronal communication, we investigated Ca(2+)-triggered exocytosis at various Cpx concentrations. Ca(2+)-triggered content-mixing between a single proteoliposome of t-SNARE and another single proteoliposome of v-SNARE plus Ca(2+)-sensor synaptotagmin 1 was examined with total internal reflection microscopy. We find that Cpx enhances Ca(2+)-triggered vesicle fusion with the yield changing from approximately 10% to 70% upon increasing Cpx from 0 to 100 nM. Unexpectedly, however, the fusion efficiency becomes reduced when Cpx is increased further, dropping to 20% in the micromolar range, revealing a bell-shaped dose-response curve. Intriguingly, we find that the rate of vesicle fusion is nearly invariant through the entire range of Cpx concentrations studied, suggesting that a reevaluation of the current Cpx clamping mechanism is necessary. Thus, our results provide insights into how delicately Cpx fine-tunes neuronal communication.

Molecular Mechanism of the Synaptotagmin-SNARE Complex that is Essential for Synchronous Synaptic Neurotransmitter Release

Synaptotagmin-1 and neuronal SNARE proteins have key roles in evoked synchronous neurotransmitter release. However, it is still unknown how they cooperate to trigger synaptic vesicle fusion at the molecular level. We determined atomic-resolution crystal structures of calcium- and magnesium-bound complexes between synaptotagmin-1 and the neuronal SNARE complex. One of these structures was determined to atomic resolution with diffraction data from an X-ray free-electron laser (XFEL), leading to accurate rotamer assignments for many side chains, including those at the interfaces between molecules. This is one of the first new crystal structures determined using XFEL diffraction data. Moreover, in contrast to the tens of thousands to millions of crystals typically used in XFEL-based crystallography experiments, we obtained a reasonably complete data set from a few hundred images captured from 72 of the 148 crystals exposed to the XFEL beam. The electron density maps obtained from the XFEL diffraction data were notably superior to those of synchrotron data sets. The structures reveal several interfaces, including a large, specific, calcium independent and conserved interface. Tests of this interface by mutagenesis suggest that it is essential for calcium triggered neurotransmitter release in mouse hippocampal neuronal synapses and for calcium triggered vesicle fusion in a reconstituted single vesicle fusion system. We propose that this interface forms before calcium triggering and moves en bloc as calcium influx promotes the interactions between synaptotagmin-1 and the plasma membrane. The simultaneous membrane interactions of the calcium binding loops, of the polybasic region, and of membrane-proximal region of the SNARE complex would deform the plasma membrane. This morphological change of the plasma membrane juxtaposes the membranes closer than the critical distance (0.9 nm) to promote stalk formation, and subsequently leads to fusion pore opening.

Architecture of the synaptotagmin-SNARE machinery for neuronal exocytosis.

SummarySynaptotagmin-1 and neuronal SNARE proteins play key roles in evoked synchronous neurotransmitter release. However, it is unknown how they cooperate to trigger synaptic vesicle fusion. Here we report atomic-resolution crystal structures of Ca2+- and Mg2+-bound complexes between synaptotagmin-1 and the neuronal SNARE complex, one of which was determined with diffraction data from an X-ray free electron laser, leading to an atomic-resolution structure with accurate rotamer assignments for many sidechains. The structures revealed several interfaces, including a large, specific, Ca2+-independent, and conserved interface. Tests of this interface by mutagenesis suggest that it is essential for Ca2+-triggered neurotransmitter release in neuronal synapses and for Ca2+-triggered vesicle fusion in a reconstituted system. We propose that this interface forms prior to Ca2+-triggering, and moves en bloc as Ca2+ influx promotes the interactions between synaptotagmin-1 and the plasma membrane, and consequently remodels the membrane to promote fusion, possibly in conjunction with other interfaces.

Complexins: small but capable.

Despite intensive research, it is still unclear how an immediate and profound acceleration of exocytosis is triggered by appropriate Ca2+-stimuli in presynaptic terminals. This is due to the fact that the molecular mechanisms of “docking” and “priming” reactions, which set up secretory vesicles to fuse at millisecond time scale, are extremely hard to study. Yet, driven by a fruitful combination of in vitro and in vivo analyses, our mechanistic understanding of Ca2+-triggered vesicle fusion has certainly advanced in the past few years. In this review, we aim to highlight recent progress and emerging views on the molecular mechanisms, by which constitutively forming SNAREpins are organized in functional, tightly regulated units for synchronized release. In particular, we will focus on the role of the small regulatory factor complexin whose function in Ca2+-dependent exocytosis has been controversially discussed for more than a decade. Special emphasis will also be laid on the functional relationship of complexin and synaptotagmin, as both proteins possibly act as allies and/or antagonists to govern SNARE-mediated exocytosis.

Calcium Binding Promotes Conformational Flexibility of the Neuronal Ca2+ Sensor Synaptotagmin

Synaptotagmin 1 (Syt1) is a synaptic vesicle protein that serves as a calcium sensor of neuronal secretion. It is established that calcium binding to Syt1 triggers vesicle fusion and release of neuronal transmitters, however, the dynamics of this process is not fully understood. To investigate how Ca2+ binding affects Syt1 conformational dynamics, we performed prolonged molecular dynamics (MD) simulations of Ca2+-unbound and Ca2+-bound forms of Syt1. MD simulations were performed at a microsecond scale and combined with Monte Carlo sampling. We found that in the absence of Ca2+ Syt1 structure in the solution is represented by an ensemble of conformational states with tightly coupled domains. To investigate the effect of Ca2+ binding, we used two different strategies to generate a molecular model of a Ca2+-bound form of Syt1. First, we employed subsequent replacements of monovalent cations transiently captured within Syt1 Ca2+-binding pockets by Ca2+ ions. Second, we performed MD simulations of Syt1 at elevated Ca2+ levels. All the simulations produced Syt1 structures bound to four Ca2+ ions, two ions chelated at the binding pocket of each domain. MD simulations of the Ca2+-bound form of Syt1 revealed that Syt1 conformational flexibility drastically increased upon Ca2+ binding. In the presence of Ca2+, the separation between domains increased, and interdomain rotations became more frequent. These findings suggest that Ca2+ binding to Syt1 may induce major changes in the Syt1 conformational state, which in turn may initiate the fusion process.

Transmitter release is evoked with low probability predominately by calcium flux through single channel openings at the frog neuromuscular junction.

The quantitative relationship between presynaptic calcium influx and transmitter release critically depends on the spatial coupling of presynaptic calcium channels to synaptic vesicles. When there is a close association between calcium channels and synaptic vesicles, the flux through a single open calcium channel may be sufficient to trigger transmitter release. With increasing spatial distance, however, a larger number of open calcium channels might be required to contribute sufficient calcium ions to trigger vesicle fusion. Here we used a combination of pharmacological calcium channel block, high-resolution calcium imaging, postsynaptic recording, and 3D Monte Carlo reaction-diffusion simulations in the adult frog neuromuscular junction, to show that release of individual synaptic vesicles is predominately triggered by calcium ions entering the nerve terminal through the nearest open calcium channel. Furthermore, calcium ion flux through this channel has a low probability of triggering synaptic vesicle fusion (∼6%), even when multiple channels open in a single active zone. These mechanisms work to control the rare triggering of vesicle fusion in the frog neuromuscular junction from each of the tens of thousands of individual release sites at this large model synapse.

Single-Molecule Diffusion Measurements Indicate Independent Membrane Insertion by the Tandem C2 Domains of Synaptotagmin 7

The synaptotagmin (Syt) family of proteins is characterized by the presence of tandem C2 domains, C2A and C2B, which sense Ca2+ to trigger vesicle fusion during exocytosis. The widely studied Syt1 is central to rapid neurotransmitter release, while Syt7 is involved in slower secretion of large dense-core vesicles and has C2 domains that dock much more tightly to target membranes. Despite recent progress in understanding mechanisms of vesicle fusion, the molecular mechanisms of synaptotagmin C2AB membrane docking remain incompletely characterized. For example, the two C2 domains of Syt1 are reported to cooperatively insert into target membranes, but specific interdomain contacts have not been identified. To test whether the two C2 domains from Syt7 interact on a planar lipid bilayer, lateral diffusion constants of fluorescent-tagged C2A, C2B, and C2AB domains from human Syt7 were measured on PC:PS (3:1) bilayers using total internal reflection fluorescence microscopy with single-particle tracking. The Syt7 C2AB tandem exhibits a lateral diffusion constant half the value of the isolated single domains, and does not change when additional residues are engineered into the C2A-C2B linker. This is the expected result if C2A and C2B are separated when membrane-bound; theory predicts that C2AB diffusion would be faster if the two domains interact. Furthermore, ensemble stopped-flow measurements of membrane dissociation kinetics also support an absence of interdomain interactions, as EDTA-induced dissociation kinetics of the C2AB tandem are similar to the isolated C2A domain and remain unchanged when rigid or flexible linker extensions are included. Together, the results suggest that the two C2 domains of Syt7 bind independently to membranes. Ongoing efforts seek to perform analogous measurements with Syt1, whose C2 domains have much shorter membrane-bound lifetimes.

Lateral diffusion of proteins on supported lipid bilayers: additive friction of synaptotagmin 7 C2A-C2B tandem domains.

The synaptotagmin (Syt) family of proteins contains tandem C2 domains, C2A and C2B, which bind membranes in the presence of Ca2+ to trigger vesicle fusion during exocytosis. Despite recent progress, the role and extent of interdomain interactions between C2A and C2B in membrane binding remain unclear. To test whether the two domains interact on a planar lipid bilayer (i.e., experience thermodynamic interdomain contacts), diffusion of fluorescent-tagged C2A, C2B, and C2AB domains from human Syt7 was measured using total internal reflection fluorescence microscopy with single-particle tracking. The C2AB tandem exhibits a lateral diffusion constant approximately half the value of the isolated single domains and does not change when additional residues are engineered into the C2A–C2B linker. This is the expected result if C2A and C2B are separated when membrane-bound; theory predicts that C2AB diffusion would be faster if the two domains were close enough together to have interdomain contact. Stopped-flow measurements of membrane dissociation kinetics further support an absence of interdomain interactions, as dissociation kinetics of the C2AB tandem remain unchanged when rigid or flexible linker extensions are included. Together, the results suggest that the two C2 domains of Syt7 bind independently to planar membranes, in contrast to reported interdomain cooperativity in Syt1.

Calcium influx activates adenylyl cyclase 8 for sustained insulin secretion in rat pancreatic beta cells.

Insulin is a key metabolic regulator in health and diabetes. In pancreatic beta cells, insulin release is regulated by the major second messengers Ca(2+) and cAMP: exocytosis is triggered by Ca(2+) and mediated by the cAMP/protein kinase A (PKA) signalling pathway. However, the causal link between these two processes in primary beta cells remains undefined. Time-resolved confocal imaging of fluorescence resonance energy transfer signals was performed to visualise PKA activity, and combined membrane capacitance recordings were used to monitor insulin secretion from patch-clamped rat beta cells. Membrane depolarisation-induced Ca(2+) influx caused an increase in cytosolic PKA activity via activating a Ca(2+)-sensitive adenylyl cyclase 8 (ADCY8) subpool. Glucose stimulation triggered coupled Ca(2+) oscillations and PKA activation. ADCY8 knockdown significantly reduced the level of depolarisation-evoked PKA activation and impaired replenishment of the readily releasable vesicle pool. Pharmacological inhibition of PKA by two inhibitors reduced depolarisation-induced PKA activation to a similar extent and reduced the capacity for sustained vesicle exocytosis and insulin release. Our findings suggest that depolarisation-induced Ca(2+) influx plays dual roles in regulating exocytosis in rat pancreatic beta cells by triggering vesicle fusion and replenishing the vesicle pool to support sustained insulin release. Therefore, Ca(2+) influx may be important for glucose-stimulated insulin secretion.