Physiology of intracellular calcium buffering.

Calcium signaling underlies much of physiology. Almost all the Ca2+ in the cytoplasm is bound to buffers, with typically only ∼1% being freely ionized at resting levels in most cells. Physiological Ca2+ buffers include small molecules and proteins, and experimentally Ca2+ indicators will also buffer calcium. The chemistry of interactions between Ca2+ and buffers determines the extent and speed of Ca2+ binding. The physiological effects of Ca2+ buffers are determined by the kinetics with which they bind Ca2+ and their mobility within the cell. The degree of buffering depends on factors such as the affinity for Ca2+, the Ca2+ concentration, and whether Ca2+ ions bind cooperatively. Buffering affects both the amplitude and time course of cytoplasmic Ca2+ signals as well as changes of Ca2+ concentration in organelles. It can also facilitate Ca2+ diffusion inside the cell. Ca2+ buffering affects synaptic transmission, muscle contraction, Ca2+ transport across epithelia, and the killing of bacteria. Saturation of buffers leads to synaptic facilitation and tetanic contraction in skeletal muscle and may play a role in inotropy in the heart. This review focuses on the link between buffer chemistry and function and how Ca2+ buffering affects normal physiology and the consequences of changes in disease. As well as summarizing what is known, we point out the many areas where further work is required.

Different states of synaptic vesicle priming explain target cell type-dependent differences in neurotransmitter release.

Pronounced differences in neurotransmitter release from a given presynaptic neuron, depending on the synaptic target, are among the most intriguing features of cortical networks. Hippocampal pyramidal cells (PCs) release glutamate with low probability to somatostatin expressing oriens-lacunosum-moleculare (O-LM) interneurons (INs), and the postsynaptic responses show robust short-term facilitation, whereas the release from the same presynaptic axons onto fast-spiking INs (FSINs) is ~10-fold higher and the excitatory postsynaptic currents (EPSCs) display depression. The mechanisms underlying these vastly different synaptic behaviors have not been conclusively identified. Here, we applied a combined functional, pharmacological, and modeling approach to address whether the main difference lies in the action potential-evoked fusion or else in upstream priming processes of synaptic vesicles (SVs). A sequential two-step SV priming model was fitted to the peak amplitudes of unitary EPSCs recorded in response to complex trains of presynaptic stimuli in acute hippocampal slices of adult mice. At PC-FSIN connections, the fusion probability (Pfusion) of well-primed SVs is 0.6, and 44% of docked SVs are in a fusion-competent state. At PC-O-LM synapses, Pfusion is only 40% lower (0.36), whereas the fraction of well-primed SVs is 6.5-fold smaller. Pharmacological enhancement of fusion by 4-AP and priming by PDBU was recaptured by the model with a selective increase of Pfusion and the fraction of well-primed SVs, respectively. Our results demonstrate that the low fidelity of transmission at PC-O-LM synapses can be explained by a low occupancy of the release sites by well-primed SVs.

Complexin has a dual synaptic function as checkpoint protein in vesicle priming and as a promoter of vesicle fusion.

The presynaptic SNARE-complex regulator complexin (Cplx) enhances the fusogenicity of primed synaptic vesicles (SVs). Consequently, Cplx deletion impairs action potential-evoked transmitter release. Conversely, though, Cplx loss enhances spontaneous and delayed asynchronous release at certain synapse types. Using electrophysiology and kinetic modeling, we show that such seemingly contradictory transmitter release phenotypes seen upon Cplx deletion can be explained by an additional of Cplx in the control of SV priming, where its ablation facilitates the generation of a "faulty" SV fusion apparatus. Supporting this notion, a sequential two-step priming scheme, featuring reduced vesicle fusogenicity and increased transition rates into the faulty primed state, reproduces all aberrations of transmitter release modes and short-term synaptic plasticity seen upon Cplx loss. Accordingly, we propose a dual presynaptic function for the SNARE-complex interactor Cplx, one as a "checkpoint" protein that guarantees the proper assembly of the fusion machinery during vesicle priming, and one in boosting vesicle fusogenicity.

Fully-primed slowly-recovering vesicles mediate presynaptic LTP at neocortical neurons.

Pre- and postsynaptic forms of long-term potentiation (LTP) are candidate synaptic mechanisms underlying learning and memory. At layer 5 pyramidal neurons, LTP increases the initial synaptic strength but also short-term depression during high-frequency transmission. This classical form of presynaptic LTP has been referred to as redistribution of synaptic efficacy. However, the underlying mechanisms remain unclear. We therefore performed whole-cell recordings from layer 5 pyramidal neurons in acute cortical slices of rats and analyzed presynaptic function before and after LTP induction by paired pre- and postsynaptic neuronal activity. LTP was successfully induced in about half of the synaptic connections tested and resulted in increased synaptic short-term depression during high-frequency transmission and a decelerated recovery from short-term depression due to an increased fraction of a slow recovery component. Analysis with a recently established sequential two-step vesicle priming model indicates an increase in the abundance of fully-primed and slowly-recovering vesicles. A systematic analysis of short-term plasticity and synapse-to-synapse variability of synaptic strength at various types of synapses revealed that stronger synapses generally recover more slowly from synaptic short-term depression. Finally, pharmacological stimulation of the cyclic adenosine monophosphate and diacylglycerol signaling pathways, which are both known to promote synaptic vesicle priming, mimicked LTP and slowed the recovery from short-term depression. Our data thus demonstrate that LTP at layer 5 pyramidal neurons increases synaptic strength primarily by enlarging a subpool of fully-primed slowly-recovering vesicles.

A sequential two-step priming scheme reproduces diversity in synaptic strength and short-term plasticity.

Glutamatergic synapses display variable strength and diverse short-term plasticity (STP), even for a given type of connection. Using nonnegative tensor factorization and conventional state modeling, we demonstrate that a kinetic scheme consisting of two sequential and reversible steps of release-machinery assembly and a final step of synaptic vesicle (SV) fusion reproduces STP and its diversity among synapses. Analyzing transmission at the calyx of Held synapses reveals that differences in synaptic strength and STP are not primarily caused by variable fusion probability (pfusion) but are determined by the fraction of docked synaptic vesicles equipped with a mature release machinery. Our simulations show that traditional quantal analysis methods do not necessarily report pfusion of SVs with a mature release machinery but reflect both pfusion and the distribution between mature and immature priming states at rest. Thus, the approach holds promise for a better mechanistic dissection of the roles of presynaptic proteins in the sequence of SV docking, two-step priming, and fusion. It suggests a mechanism for activity-induced redistribution of synaptic efficacy.

Flowers for synaptic endocytosis.

Exocytosis and endocytosis of synaptic vesicles are tightly coordinated to maintain a steady supply of new vesicles during periods of extended neuronal stimulation. Yao et al. (2009) now report that a synaptic vesicle membrane protein named Flower promotes endocytosis at neuromuscular junctions in the fruit fly Drosophila.

Regulation of a subset of release-ready vesicles by the presynaptic protein Mover.

Neurotransmitter release occurs by regulated exocytosis from synaptic vesicles (SVs). Evolutionarily conserved proteins mediate the essential aspects of this process, including the membrane fusion step and priming steps that make SVs release-competent. Unlike the proteins constituting the core fusion machinery, the SV protein Mover does not occur in all species and all synapses. Its restricted expression suggests that Mover may modulate basic aspects of transmitter release and short-term plasticity. To test this hypothesis, we analyzed synaptic transmission electrophysiologically at the mouse calyx of Held synapse in slices obtained from wild-type mice and mice lacking Mover. Spontaneous transmission was unaffected, indicating that the basic release machinery works in the absence of Mover. Evoked release and vesicular release probability were slightly reduced, and the paired pulse ratio was increased in Mover knockout mice. To explore whether Mover's role is restricted to certain subpools of SVs, we analyzed our data in terms of two models of priming. A model assuming two SV pools in parallel showed a reduced release probability of so-called "superprimed vesicles" while "normally primed" ones were unaffected. For the second model, which holds that vesicles transit sequentially from a loosely docked state to a tightly docked state before exocytosis, we found that knocking out Mover selectively decreased the release probability of tight state vesicles. These results indicate that Mover regulates a subclass of primed SVs in the mouse calyx of Held.

Metabolomics and integrated network pharmacology analysis reveal Tricin as the active anti-cancer component of Weijing decoction by suppression of PRKCA and sphingolipid signaling.

Currently, conventional methods of treating non-small cell lung cancer (NSCLC) have many disadvantages. An alternative effective therapy with minimal adverse reactions is urgently needed. Weijing decoction (WJD), which is a classic ancient Chinese herbal prescription, has been used successfully to treat pulmonary system diseases containing lung cancer in the clinic. However, the key active component and target of Weijing decoction are still unexplored. Therefore, for the first time, our study aims to investigate the pharmacological treatment mechanism of Weijing decoction in treating NSCLC via an integrated model of network pharmacology, metabolomics and biological methods. Network pharmacology results conjectured that Tricin is a main bioactive component in this formula which targets PRKCA to suppress cancer cell growth. Metabolomics analysis demonstrated that sphingosine-1-phosphate, which is regulated by sphingosine kinase 1 and sphingosine kinase 2, is a differential metabolite in plasma between the WJD-treated group and the control group, participating in the sphingolipid signaling. In vitro experiments demonstrated that Tricin had vital effects on the proliferation, pro-apoptosis, migration and colony formation of Lewis lung carcinoma cells. Through a series of validation assays, Tricin inhibited the tumor growth mainly by suppressing PRKCA/SPHK/S1P signaling and antiapoptotic signaling. On the other hand, Weijing formula could inhibit the tumor growth and prolong the survival time. A high dosage of Tricin was much more potent in animal experiments. In conclusion, we confirmed that Weijing formula and its primary active compound Tricin are promising alternative treatments for NSCLC patients.

Insect haptoelectrical stimulation of Venus flytrap triggers exocytosis in gland cells.

The Venus flytrap Dionaea muscipula captures insects and consumes their flesh. Prey contacting touch-sensitive hairs trigger traveling electrical waves. These action potentials (APs) cause rapid closure of the trap and activate secretory functions of glands, which cover its inner surface. Such prey-induced haptoelectric stimulation activates the touch hormone jasmonate (JA) signaling pathway, which initiates secretion of an acidic hydrolase mixture to decompose the victim and acquire the animal nutrients. Although postulated since Darwin's pioneering studies, these secretory events have not been recorded so far. Using advanced analytical and imaging techniques, such as vibrating ion-selective electrodes, carbon fiber amperometry, and magnetic resonance imaging, we monitored stimulus-coupled glandular secretion into the flytrap. Trigger-hair bending or direct application of JA caused a quantal release of oxidizable material from gland cells monitored as distinct amperometric spikes. Spikes reminiscent of exocytotic events in secretory animal cells progressively increased in frequency, reaching steady state 1 d after stimulation. Our data indicate that trigger-hair mechanical stimulation evokes APs. Gland cells translate APs into touch-inducible JA signaling that promotes the formation of secretory vesicles. Early vesicles loaded with H+ and Cl- fuse with the plasma membrane, hyperacidifying the "green stomach"-like digestive organ, whereas subsequent ones carry hydrolases and nutrient transporters, together with a glutathione redox moiety, which is likely to act as the major detected compound in amperometry. Hence, when glands perceive the haptoelectrical stimulation, secretory vesicles are tailored to be released in a sequence that optimizes digestion of the captured animal.

Superpriming of synaptic vesicles as a common basis for intersynapse variability and modulation of synaptic strength.

Glutamatergic synapses show large variations in strength and short-term plasticity (STP). We show here that synapses displaying an increased strength either after posttetanic potentiation (PTP) or through activation of the phospholipase-C-diacylglycerol pathway share characteristic properties with intrinsically strong synapses, such as (i) pronounced short-term depression (STD) during high-frequency stimulation; (ii) a conversion of that STD into a sequence of facilitation followed by STD after a few conditioning stimuli at low frequency; (iii) an equalizing effect of such conditioning stimulation, which reduces differences among synapses and abolishes potentiation; and (iv) a requirement of long periods of rest for reconstitution of the original STP pattern. These phenomena are quantitatively described by assuming that a small fraction of "superprimed" synaptic vesicles are in a state of elevated release probability (p ∼ 0.5). This fraction is variable in size among synapses (typically about 30%), but increases after application of phorbol ester or during PTP. The majority of vesicles, released during repetitive stimulation, have low release probability (p ∼ 0.1), are relatively uniform in number across synapses, and are rapidly recruited. In contrast, superprimed vesicles need several seconds to be regenerated. They mediate enhanced synaptic strength at the onset of burst-like activity, the impact of which is subject to modulation by slow modulatory transmitter systems.

Neurosecretion: what can we learn from chromaffin cells.

Many of the molecular players in the stimulus-secretion chain are similarly active in neurosecretion and catecholamine release. Therefore, studying chromaffin cells uncovered many details of the processes of docking, priming, and exocytosis of vesicles. However, morphological specializations at synapses, called active zones (AZs), confer extra speed of response and another layer of control to the fast release of vesicles by action potentials. Work at the Calyx of Held, a glutamatergic nerve terminal, has shown that in addition to such rapidly released vesicles, there is a pool of "Slow Vesicles," which are held to be perfectly release-competent, but lack a final step of tight interaction with the AZ. It is argued here that such "Slow Vesicles" have many properties in common with chromaffin granules. The added complexity in the AZ-dependent regulation of "Fast Vesicles" can lead to misinterpretation of data on neurosecretion. Therefore, the study of Slow Vesicles and of chromaffin granules may provide a clearer picture of the early steps in the highly regulated process of neurosecretion.

Reduced endogenous Ca2+ buffering speeds active zone Ca2+ signaling.

Fast synchronous neurotransmitter release at the presynaptic active zone is triggered by local Ca(2+) signals, which are confined in their spatiotemporal extent by endogenous Ca(2+) buffers. However, it remains elusive how rapid and reliable Ca(2+) signaling can be sustained during repetitive release. Here, we established quantitative two-photon Ca(2+) imaging in cerebellar mossy fiber boutons, which fire at exceptionally high rates. We show that endogenous fixed buffers have a surprisingly low Ca(2+)-binding ratio (∼ 15) and low affinity, whereas mobile buffers have high affinity. Experimentally constrained modeling revealed that the low endogenous buffering promotes fast clearance of Ca(2+) from the active zone during repetitive firing. Measuring Ca(2+) signals at different distances from active zones with ultra-high-resolution confirmed our model predictions. Our results lead to the concept that reduced Ca(2+) buffering enables fast active zone Ca(2+) signaling, suggesting that the strength of endogenous Ca(2+) buffering limits the rate of synchronous synaptic transmission.

Calcium sensor kinase activates potassium uptake systems in gland cells of Venus flytraps.

The Darwin plant Dionaea muscipula is able to grow on mineral-poor soil, because it gains essential nutrients from captured animal prey. Given that no nutrients remain in the trap when it opens after the consumption of an animal meal, we here asked the question of how Dionaea sequesters prey-derived potassium. We show that prey capture triggers expression of a K(+) uptake system in the Venus flytrap. In search of K(+) transporters endowed with adequate properties for this role, we screened a Dionaea expressed sequence tag (EST) database and identified DmKT1 and DmHAK5 as candidates. On insect and touch hormone stimulation, the number of transcripts of these transporters increased in flytraps. After cRNA injection of K(+)-transporter genes into Xenopus oocytes, however, both putative K(+) transporters remained silent. Assuming that calcium sensor kinases are regulating Arabidopsis K(+) transporter 1 (AKT1), we coexpressed the putative K(+) transporters with a large set of kinases and identified the CBL9-CIPK23 pair as the major activating complex for both transporters in Dionaea K(+) uptake. DmKT1 was found to be a K(+)-selective channel of voltage-dependent high capacity and low affinity, whereas DmHAK5 was identified as the first, to our knowledge, proton-driven, high-affinity potassium transporter with weak selectivity. When the Venus flytrap is processing its prey, the gland cell membrane potential is maintained around -120 mV, and the apoplast is acidified to pH 3. These conditions in the green stomach formed by the closed flytrap allow DmKT1 and DmHAK5 to acquire prey-derived K(+), reducing its concentration from millimolar levels down to trace levels.

Some Subtle Lessons from the Calyx of Held Synapse.

The calyx of Held is a giant nerve terminal that forms a glutamatergic synapse in the auditory pathway. Due to its large size, it offers a number of advantages for biophysical studies, including voltage-clamp of both pre- and postsynaptic compartments and the loading with indicator dyes and caged compounds. Three aspects of recent findings on the calyx are reviewed here, each of which seems to have only subtle consequences for nerve-evoked excitatory postsynaptic currents: vesicle heterogeneity, refractoriness of release sites, and superpriming. Together, they determine short-term plasticity features that are superficially similar to those expected for a simple vesicle pool model. However, detailed consideration of these aspects may be required for the correct mechanistic interpretation of data from synapses with normal and perturbed function, as well as for modeling the dynamics of short-term plasticity.

Counting Vesicular Release Events Reveals Binomial Release Statistics at Single Glutamatergic Synapses.

Many central glutamatergic synapses contain a single presynaptic active zone and a single postsynaptic density. However, the basic functional properties of such "simple synapses" remain unclear. One important step toward understanding simple synapse function is to analyze the number of synaptic vesicles released in such structures per action potential, but this goal has remained elusive until now. Here, we describe procedures that allow reliable vesicular release counting at simple synapses between parallel fibers and molecular layer interneurons of rat cerebellar slices. Our analysis involves local extracellular stimulation of single parallel fibers and deconvolution of resulting EPSCs using quantal signals as template. We observed a reduction of quantal amplitudes (amplitude occlusion) in pairs of consecutive EPSCs due to receptor saturation. This effect is larger (62%) than previously reported and primarily reflects receptor activation rather than desensitization. In addition to activation-driven amplitude occlusion, each EPSC reduces amplitudes of subsequent events by an estimated 3% due to cumulative desensitization. Vesicular release counts at simple synapses follow binomial statistics with a maximum that varies from 2 to 10 among experiments. This maximum presumably reflects the number of docking sites at a given synapse. These results show striking similarities, as well as significant quantitative differences, with respect to previous results at simple GABAergic synapses. SIGNIFICANCE STATEMENT: It is generally accepted that the output signal of individual central synapses saturates at high release probability, but it remains unclear whether the source of saturation is presynaptic, postsynaptic, or both presynaptic and postsynaptic. To clarify this and other issues concerning the function of synapses, we have developed new recording and analysis methods at single central glutamatergic synapses. We find that individual release events engage a high proportion of postsynaptic receptors (62%), revealing a larger component of postsynaptic saturation than anticipated. Conversely, we also find that the number of released synaptic vesicles is limited at each active zone. Altogether, our results argue for both presynaptic and postsynaptic contributions to signal saturation at single glutamatergic synapses.

Dynamics of volume-averaged intracellular Ca2+ in a rat CNS nerve terminal during single and repetitive voltage-clamp depolarizations.

KEY POINTS: The intracellular concentration of free calcium ions ([Ca2+ ]i ) in a nerve terminal controls both transmitter release and synaptic plasticity. The rapid triggering of transmitter release depends on the local micro- or nanodomain of highly elevated [Ca2+ ]i in the vicinity of open voltage-gated Ca2+ channels, whereas short-term synaptic plasticity is often controlled by global changes in residual [Ca2+ ]i , averaged over the whole nerve terminal volume. Here we describe dynamic changes of such global [Ca2+ ]i in the calyx of Held - a giant mammalian glutamatergic nerve terminal, which is particularly suited for biophysical studies. We provide quantitative data on Ca2+ inflow, Ca2+ buffering and Ca2+ clearance. These data allow us to predict changes in [Ca2+ ]i in the nerve terminal in response to a wide range of stimulus protocols at high temporal resolution and provide a basis for the modelling of short-term plasticity of glutamatergic synapses. ABSTRACT: Many aspects of short-term synaptic plasticity (STP) are controlled by relatively slow changes in the presynaptic intracellular concentration of free calcium ions ([Ca2+ ]i ) that occur in the time range of a few milliseconds to several seconds. In nerve terminals, [Ca2+ ]i equilibrates diffusionally during such slow changes, such that the globally measured, residual [Ca2+ ]i that persists after the collapse of local domains is often the appropriate parameter governing STP. Here, we study activity-dependent dynamic changes in global [Ca2+ ]i at the rat calyx of Held nerve terminal in acute brainstem slices using patch-clamp and microfluorimetry. We use low concentrations of a low-affinity Ca2+ indicator dye (100 µm Fura-6F) in order not to overwhelm endogenous Ca2+ buffers. We first study voltage-clamped terminals, dialysed with pipette solutions containing minimal amounts of Ca2+ buffers, to determine Ca2+ binding properties of endogenous fixed buffers as well as the mechanisms of Ca2+ clearance. Subsequently, we use pipette solutions including 500 µm EGTA to determine the Ca2+ binding kinetics of this chelator. We provide a formalism and parameters that allow us to predict [Ca2+ ]i changes in calyx nerve terminals in response to a wide range of stimulus protocols. Unexpectedly, the Ca2+ affinity of EGTA under the conditions of our measurements was substantially lower (KD  = 543 ± 51 nm) than measured in vitro, mainly as a consequence of a higher than previously assumed dissociation rate constant (2.38 ± 0.20 s-1 ), which we need to postulate in order to model the measured presynaptic [Ca2+ ]i transients.

The desert plant Phoenix dactylifera closes stomata via nitrate-regulated SLAC1 anion channel.

Date palm Phoenix dactylifera is a desert crop well adapted to survive and produce fruits under extreme drought and heat. How are palms under such harsh environmental conditions able to limit transpirational water loss? Here, we analysed the cuticular waxes, stomata structure and function, and molecular biology of guard cells from P. dactylifera. To understand the stomatal response to the water stress phytohormone of the desert plant, we cloned the major elements necessary for guard cell fast abscisic acid (ABA) signalling and reconstituted this ABA signalosome in Xenopus oocytes. The PhoenixSLAC1-type anion channel is regulated by ABA kinase PdOST1. Energy-dispersive X-ray analysis (EDXA) demonstrated that date palm guard cells release chloride during stomatal closure. However, in Cl- medium, PdOST1 did not activate the desert plant anion channel PdSLAC1 per se. Only when nitrate was present at the extracellular face of the anion channel did the OST1-gated PdSLAC1 open, thus enabling chloride release. In the presence of nitrate, ABA enhanced and accelerated stomatal closure. Our findings indicate that, in date palm, the guard cell osmotic motor driving stomatal closure uses nitrate as the signal to open the major anion channel SLAC1. This initiates guard cell depolarization and the release of anions together with potassium.

Complexin stabilizes newly primed synaptic vesicles and prevents their premature fusion at the mouse calyx of held synapse.

Complexins (Cplxs) are small synaptic proteins that cooperate with SNARE-complexes in the control of synaptic vesicle (SV) fusion. Studies involving genetic mutation, knock-down, or knock-out indicated two key functions of Cplx that are not mutually exclusive but cannot easily be reconciled, one in facilitating SV fusion, and one in "clamping" SVs to prevent premature fusion. Most studies on the role of Cplxs in mammalian synapse function have relied on cultured neurons, heterologous expression systems, or membrane fusion assays in vitro, whereas little is known about the function of Cplxs in native synapses. We therefore studied consequences of genetic ablation of Cplx1 in the mouse calyx of Held synapse, and discovered a developmentally exacerbating phenotype of reduced spontaneous and evoked transmission but excessive asynchronous release after stimulation, compatible with combined facilitating and clamping functions of Cplx1. Because action potential waveforms, Ca(2+) influx, readily releasable SV pool size, and quantal size were unaltered, the reduced synaptic strength in the absence of Cplx1 is most likely a consequence of a decreased release probability, which is caused, in part, by less tight coupling between Ca(2+) channels and docked SV. We found further that the excessive asynchronous release in Cplx1-deficient calyces triggered aberrant action potentials in their target neurons, and slowed-down the recovery of EPSCs after depleting stimuli. The augmented asynchronous release had a delayed onset and lasted hundreds of milliseconds, indicating that it predominantly represents fusion of newly recruited SVs, which remain unstable and prone to premature fusion in the absence of Cplx1.

Protein scaffolds in the coupling of synaptic exocytosis and endocytosis.

Mechanisms that ensure robust long-term performance of synaptic transmission over a wide range of activity are crucial for the integrity of neuronal networks, for processing sensory information and for the ability to learn and store memories. Recent experiments have revealed that such robust performance requires a tight coupling between exocytic vesicle fusion at defined release sites and endocytic retrieval of synaptic vesicle membranes. Distinct presynaptic scaffolding proteins are essential for fulfilling this requirement, providing either ultrastructural coordination or acting as signalling hubs.

Superpriming of synaptic vesicles after their recruitment to the readily releasable pool.

Recruitment of release-competent vesicles during sustained synaptic activity is one of the major factors governing short-term plasticity. During bursts of synaptic activity, vesicles are recruited to a fast-releasing pool from a reluctant vesicle pool through an actin-dependent mechanism. We now show that newly recruited vesicles in the fast-releasing pool do not respond at full speed to a strong Ca(2+) stimulus, but require approximately 4 s to mature to a "superprimed" state. Superpriming was found to be altered by agents that modulate the function of unc13 homolog proteins (Munc13s), but not by calmodulin inhibitors or actin-disrupting agents. These findings indicate that recruitment and superpriming of vesicles are regulated by separate mechanisms, which require integrity of the cytoskeleton and activation of Munc13s, respectively. We propose that refilling of the fast-releasing vesicle pool proceeds in two steps, rapid actin-dependent "positional priming," which brings vesicles closer to Ca(2+) sources, followed by slower superpriming, which enhances the Ca(2+) sensitivity of primed vesicles.