E. A. Bukharaeva

Septin dynamics are essential for exocytosis

Background: Septins serve as scaffolds for membrane-associated protein complexes. Results: Knockdown of septin-2 or disruption of septin assembly/disassembly impairs interactions between exocytic proteins and inhibits late steps of exocytosis. Conclusion: Septins undergo dynamic reorganization to facilitate localized and timely interactions between exocytosis-essential proteins. Significance: Both the presence of septin-2 and active reorganization of septin oligomers are required for exocytosis. Septins are a family of 14 cytoskeletal proteins that dynamically form hetero-oligomers and organize membrane microdomains for protein complexes. The previously reported interactions with SNARE proteins suggested the involvement of septins in exocytosis. However, the contradictory results of up- or down-regulation of septin-5 in various cells and mouse models or septin-4 in mice suggested either an inhibitory or a stimulatory role for these septins in exocytosis. The involvement of the ubiquitously expressed septin-2 or general septin polymerization in exocytosis has not been explored to date. Here, by nano-LC with tandem MS and immunoblot analyses of the septin-2 interactome in mouse brain, we identified not only SNARE proteins but also Munc-18-1 (stabilizes assembled SNARE complexes), N-ethylmaleimide-sensitive factor (NSF) (disassembles SNARE complexes after each membrane fusion event), and the chaperones Hsc70 and synucleins (maintain functional conformation of SNARE proteins after complex disassembly). Importantly, α-soluble NSF attachment protein (SNAP), the adaptor protein that mediates NSF binding to the SNARE complex, did not interact with septin-2, indicating that septins undergo reorganization during each exocytosis cycle. Partial depletion of septin-2 by siRNA or impairment of septin dynamics by forchlorfenuron inhibited constitutive and stimulated exocytosis of secreted and transmembrane proteins in various cell types. Forchlorfenuron impaired the interaction between SNAP-25 and its chaperone Hsc70, decreasing SNAP-25 levels in cultured neuroendocrine cells, and inhibited both spontaneous and stimulated acetylcholine secretion in mouse motor neurons. The results demonstrate a stimulatory role of septin-2 and the dynamic reorganization of septin oligomers in exocytosis.

Modulation of the kinetics of evoked quantal release at mouse neuromuscular junctions by calcium and strontium

The effects of calcium and strontium on the quantal content of nerve‐evoked endplate currents and on the kinetic parameters of quantal release (minimal synaptic delay, value of main mode of synaptic delay histogram, and variability of synaptic delay) were studied at the mouse neuromuscular synapse. At low calcium ion concentrations (0.2–0.6 mmol/L), evoked signals with long synaptic delays (several times longer than the value of main mode) were observed. Their number decreased substantially when [Ca2+]o was increased (i.e. the release of transmitter became more synchronous). By contrast, the early phase of secretion, characterized by minimal synaptic delay and accounting for the main peak of the synaptic delay histogram, did not change significantly with increasing [Ca2+]o. Hence, extracellular calcium affected mainly the late, ‘asynchronous’, portion of phasic release. The average quantal content grew exponentially from 0.09 ± 0.01 to 1.04 ± 0.07 with the increase in [Ca2+]o without reaching saturation. Similar results were obtained when calcium was replaced by strontium, but the asynchronous portion of phasic release was more pronounced and higher strontium concentrations (to 1.2–1.4 mmol/L) were required to abolish responses with long delays. Treatment of preparations with 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid tetrakis acetoxymethyl ester (BAPTA‐AM) (25 µmol/L), but not with ethylene glycol‐bis(2‐aminoethylether)‐N,N,N′,N′‐tetraacetic acid acetoxymethyl ester (EGTA‐AM) (25 µmol/L), abolished the responses with long delays. The dependence of quantal content and synchrony of quantal release on calcium and strontium concentrations have quite different slopes, suggesting that they are governed by different mechanisms.

Modeling of quantal neurotransmitter release kinetics in the presence of fixed and mobile calcium buffers

The local calcium concentration in the active zone of secretion determines the number and kinetics of neurotransmitter quanta released after the arrival of a nerve action potential in chemical synapses. The small size of mammalian neuromuscular junctions does not allow direct measurement of the correlation between calcium influx, the state of endogenous calcium buffers determining the local concentration of calcium and the time course of quanta exocytosis. In this work, we used computer modeling of quanta release kinetics with various levels of calcium influx and in the presence of endogenous calcium buffers with varying mobilities. The results of this modeling revealed the desynchronization of quanta release under low calcium influx in the presence of an endogenous fixed calcium buffer, with a diffusion coefficient much smaller than that of free Ca2+, and synchronization occurred upon adding a mobile buffer. This corresponds to changes in secretion time course parameters found experimentally (Samigullin et al., Physiol Res 54:129–132, 2005; Bukharaeva et al., J Neurochem 100:939–949, 2007).

Opposite modulation of time course of quantal release in two parts of the same synapse by reactive oxygen species.

Reactive oxygen species (ROS) are potent regulators of transmitter release in chemical synapses, but the mechanism of this action remains almost unknown. Presynaptic modulation can change either the release probability or the time course of quantal release, which was recently recognized as an efficient mechanism determining synaptic efficiency. The nonuniform structure and a big size of the frog neuromuscular junction make it a useful model to study the action of ROS in compartments different in release probability and in time course of transmitter release. The time course (or kinetics) of quantal release could be estimated by measuring the dispersion of the synaptic delays for evoked uniquantal endplate currents (EPCs) under low release probability. Using two-electrode recording technique, the action of ROS on kinetics and release probabilities were studied at the proximal and distal parts within the same neuromuscular junction. The stable ROS hydrogen peroxide (H2O2) increased the dispersion of synaptic delays of EPCs (i.e. desynchronized quantal release) within the distal part but decreased delay dispersion (synchronized quantal release) within the proximal part of the same synapse. Unlike the opposite modulation of kinetics, H2O2 reduced release probability in both distal and proximal parts. Since ATP is released from motor nerve terminals together with acetylcholine and can be involved in ROS signaling, we tested the presynaptic action of ATP. In the presence of the pro-oxidant Fe2+, extracellular ATP, similarly to H2O2, induced significant desynchronization of release in the distal regions. The antioxidant N-acetyl-cysteine attenuated the inhibitory action of ATP on release probability and abolished the action of H2O2 and ATP in the presence of Fe2+, on release kinetics. Our data suggest that ROS induced during muscle activity could change the time course of transmitter release along the motor nerve terminal to provide fine tuning of synaptic efficacy.

Different sensitivities of rat skeletal muscles and brain to novel anti‐cholinesterase agents, alkylammonium derivatives of 6‐methyluracil (ADEMS)

The rat respiratory muscle diaphragm has markedly lower sensitivity than the locomotor muscle extensor digitorum longus (EDL) to the new acetylcholinesterase (AChE) inhibitors, alkylammonium derivatives of 6‐methyluracil (ADEMS). This study evaluated several possible reasons for differing sensitivity between the diaphragm and limb muscles and between the muscles and the brain.

Estimation of presynaptic calcium currents and endogenous calcium buffers at the frog neuromuscular junction with two different calcium fluorescent dyes

At the frog neuromuscular junction, under physiological conditions, the direct measurement of calcium currents and of the concentration of intracellular calcium buffers—which determine the kinetics of calcium concentration and neurotransmitter release from the nerve terminal—has hitherto been technically impossible. With the aim of quantifying both Ca2+ currents and the intracellular calcium buffers, we measured fluorescence signals from nerve terminals loaded with the low-affinity calcium dye Magnesium Green or the high-affinity dye Oregon Green BAPTA-1, simultaneously with microelectrode recordings of nerve-action potentials and end-plate currents. The action-potential-induced fluorescence signals in the nerve terminals developed much more slowly than the postsynaptic response. To clarify the reasons for this observation and to define a spatiotemporal profile of intracellular calcium and of the concentration of mobile and fixed calcium buffers, mathematical modeling was employed. The best approximations of the experimental calcium transients for both calcium dyes were obtained when the calcium current had an amplitude of 1.6 ± 0.08 pA and a half-decay time of 1.2 ± 0.06 ms, and when the concentrations of mobile and fixed calcium buffers were 250 ± 13 μM and 8 ± 0.4 mM, respectively. High concentrations of endogenous buffers define the time course of calcium transients after an action potential in the axoplasm, and may modify synaptic plasticity.

Temperature Effect on Proximal to Distal Gradient of Quantal Release of Acetylcholine at Frog Endplate

The conduction velocity of the nerve terminal, mean quantal content, and release latencies of uniquantal endplate currents (EPCs) were recorded in proximal, central, and distal parts of the terminal by extracellular pipettes located 5, 50, and 100 mm from the end of myelinated nerve trunk. The spike conduction velocity, minimal latency, modal value of the latency histograms, and time interval during which 90% of EPCs released (P90) at distal, central, and proximal part of the frog nerve terminal have different temperature dependency between 10° and 28°C. As shown by the size and time-course of reconstructed multiquantal EPCs, the secretion synchronization, which is greatest in distal parts, compensates at least partly for the progressive slowing of spike conduction velocity in the proximodistal direction, in particular at lower temperatures.

A method for assessing the kinetics of evoked secretion of transmitter quanta determining the generation of multiquantum endplate currents.

10] studies in recent years have demonstrated that changes in the time course of evoked secretion of transmitter quanta represent one of the most important presynaptic mechanisms in the modulation of synaptic transmission, leading to changes in the amplitude–time characteristics of multiquantum endplate currents (EPC) [1, 5, 6]. A general method for assessing the kinetics of evoked secretion of transmitter quanta is provided by measuring true synaptic delays of single-quantum EPC, which allows determination of the time at which each individual quantum is released in conditions of decreased EPC quantum composition [4, 7]. However, this direct approach cannot be used for identifying the kinetics of secretion in conditions of normal (multiquantum) release of transmitter quanta. Van der Kloot [11] suggested a deconvolution method for analyzing the time course of the secretion of multiquantum EPC, this being based on the numerical differentiation of averaged multiquantum EPC and single-quantum, or miniature, EPC (mEPC). This method has two significant disadvantages, as identified by the author himself. The first is the use of averaged mEPC, which are described by a smooth function with an instantaneous leading front, which does not correspond to the shape of real mEPC recorded in electrophysiological experiments. The second is that even insignificant noise distortion affecting the averaged mEPC results in significant distortions in the secretion time course as identified by numerical differentiation, and this prevents the analysis of the late phase. Thus, the question of developing methods for assessing the true kinetics of transmitter quantum release in conditions of multiquantum liberation remains relevant. We report here a new method of measuring the time course of secretion of the quanta making up a multiquantum EPC, based on the sequential subtraction, with a time shift, of averaged experimentally measured mEPC from the multiquantum EPC recorded in the same cell; this procedure yields a set of time intervals corresponding to the latent periods for the appearances of the quanta making up the multiquantum EPC, the number of time intervals corresponding to the quantum composition of the EPC. The subtraction procedure was based on recording multiquantum EPC and a set of single-quantum EPC, or mEPC, which were averaged because of the quite strong variability of the amplitude–time parameters of individual signals. This is illustrated in Fig. 1, A which shows an individual EPC recorded extracellularly, consisting of five quanta, as shown by the “steps” on the leading front of the signal (arrowed), along with the averaged mEPC. The subtraction procedure starts at the time point corresponding to 1 msec, before the start of the EPC. The curve corresponding to the time course of the averaged mEPC is sequentially and repeatedly subtracted, at defined time points (shifts along the time axis by 10 μsec each time, corresponding to the frequency at which the signal was digitized), from the curve describing the time course of a discrete EPC. This procedure leaves a residual curve. If the process of subtraction is started with a delay, i.e., at the moment at which the EPC appears, then the residual curve is negative, corresponding to the absence of a secreted quantum. As soon as the repeated subtraction with a time shift progresses to the Neuroscience and Behavioral Physiology, Vol. 32, No. 6, 2002

Kinetics of neurotransmitter release in neuromuscular synapses of newborn and adult rats.

The kinetics of the phasic synchronous and delayed asynchronous release of acetylcholine quanta was studied at the neuromuscular junctions of aging rats from infant to mature animals at various frequencies of rhythmic stimulation of the motor nerve. We found that in infants 6 (P6) and 10 (P10) days after birth a strongly asynchronous phase of quantal release was observed, along with a reduced number of quanta compared to the synapses of adults. The rise time and decay of uni‐quantal end‐plate currents were significantly longer in infant synapses. The presynaptic immunostaining revealed that the area of the synapses in infants was significantly (up to six times) smaller than in mature junctions. The intensity of delayed asynchronous release in infants increased with the frequency of stimulation more than in adults. A blockade of the ryanodine receptors, which can contribute to the formation of delayed asynchronous release, had no effect on the kinetics of delayed secretion in the infants unlike synapses of adults. Therefore, high degree of asynchrony of quantal release in infants is not associated with the activity of ryanodine receptors and with the liberation of calcium ions from intracellular calcium stores.

Characteristics of the time course of evoked secretion of transmitter quanta in different parts of the motor nerve ending in the frog.

Experiments were performed on neuromuscular preparations from frogs, in which three extracellular microelectrodes were used to record nerve ending currents and single-quantum endplate currents simultaneously from the proximal, central, and distal parts of single synaptic contacts. The rate of propagation of excitation across terminals was measured, along with the minimum synaptic delay, the intensity, and the degree of synchronicity of the secretion of transmitter quanta in different parts of the nerve ending, and the relationships between these factors and the calcium ion concentration in the medium. These studies showed that along with gradients in the rate of conduction of excitation and the intensity of secretion in different parts of the ending, there were also differences in the kinetics of the release of transmitter quanta. As the distance from the end of the myelinated part of the axon increased, the rate of conduction of the nerve impulse and the duration of the synaptic delay decreased, while the synchronicity of the release of quanta increased. Increases in the calcium concentration in the medium produced greater increases in the synchronicity of transmitter quantum release in the distal parts of the synapse than in the proximal parts. Mathematical modeling of multiple-quantum endplate currents showed that the characteristics of the kinetics of the secretion process observed here in different parts of the nerve ending represent a factor which partially compensates for the decrease in the amplitude and extending of the duration of the leading front of the multiple-quantum endplate current which are associated with the low rate of conduction of excitation across the nerve ending. The contribution of this compensation increases as the intensity of secretion of transmitter quanta increases in the distal parts of the synaptic contact.