T-type channel-mediated neurotransmitter release

Besides controlling a wide variety of cell functions, T-type channels have been shown to regulate neurotransmitter release in peripheral and central synapses and neuroendocrine cells. Growing evidence over the last 10 years suggests a key role of Cav3.2 and Cav3.1 channels in controlling basal neurosecretion near resting conditions and sustained release during mild stimulations. In some cases, the contribution of low-voltage-activated (LVA) channels is not directly evident but requires either the activation of coupled presynaptic receptors, block of ion channels, or chelation of metal ions. Concerning the coupling to the secretory machinery, T-type channels appear loosely coupled to neurotransmitter and hormone release. In neurons, Cav3.2 and Cav3.1 channels mainly control the asynchronous appearance of “minis” [miniature inhibitory postsynaptic currents (mIPSCs) and miniature excitatory postsynaptic currents (mEPSCs)]. The same loose coupling is evident from membrane capacity and amperometric recordings in chromaffin cells and melanotropes where the low-threshold-driven exocytosis possesses the same linear Ca²⁺ dependence of the other voltage-gated Ca²⁺ channels (Cav1 and Cav2) that is strongly attenuated by slow calcium buffers. The intriguing issue is that, despite not expressing a consensus “synprint” site, Cav3.2 channels do interact with syntaxin 1A and SNAP-25 and, thus, may form nanodomains with secretory vesicles that can be regulated at low voltages. In this review, we discuss all the past and recent issues related to T-type channel-secretion coupling in neurons and neuroendocrine cells.     

Calcium-channel number critically influences synaptic strength and plasticity at the active zone

How synaptic-vesicle release is controlled at the basic release structure, the active zone, is poorly understood. By performing cell-attached current and capacitance recordings predominantly at single active zones in rat calyces, we found that single active zones contained 5-218 (mean, 42) calcium channels and 1-10 (mean, 5) readily releasable vesicles (RRVs) and released 0-5 vesicles during a 2-ms depolarization. Large variation in the number of calcium channels caused wide variation in release strength (measured during a 2-ms depolarization) by regulating the RRV release probability (P(RRV)) and the RRV number. Consequently, an action potential opened ～1-35 (mean, ～7) channels, resulting in different release probabilities at different active zones. As the number of calcium-channels determined P(RRV), it critically influenced whether subsequent release would be facilitated or depressed. Regulating calcium channel density at active zones may thus be a major mechanism to yield synapses with different release properties and plasticity. These findings may explain large differences reported at synapses regarding release strength (release of 0, 1 or multiple vesicles), P(RRV), short-term plasticity, calcium transients and the requisite calcium-channel number for triggering release.     

Loose coupling between calcium channels and sites of exocytosis in chromaffin cells

Calcium microdomains generated by tight clusters of calcium channels regulate fusion of small vesicles at the synaptic terminal and have also been suggested to trigger exocytosis of large dense-core vesicles from neuroendocrine cells. To test this idea, we have compared sites of exocytosis and the spatial distribution of calcium channels in chromaffin cells. Fusion of individual vesicles was visualized using interference reflection microscopy and the submembranous calcium signal was assessed using total internal reflection fluorescence microscopy. Depolarization triggered a burst of exocytosis from up to seven sites in a membrane area of 11 μm², but these sites did not colocalize with calcium microdomains. Instead, calcium influx occurred in large patches (averaging 34 μm²) containing a mixture of P/Q- and N-type channels. About 20% of fusion events occurred outside calcium channel patches. Further, the delay between the onset of stimulation and a burst of exocytosis was prolonged for several seconds by increasing the concentration of the slow calcium chelator EGTA from 1.5 to 5 m m . These results demonstrate that while calcium channels and release sites tend to congregate in specialized regions of the surface membrane, these have dimensions of several micrometres. The dominant calcium signal regulating release in chromaffin cells is generated by the cooperative action of many channels operating over distances of many micrometres rather than discrete clusters of calcium channels generating localized microdomains.     

Ultrastructural evidence of piecemeal degranulation in large dense-core vesicles of brain neurons

Large dense-core vesicles (LDCV) are a group of neuronal secretory organelles with different size and characteristically condensed morphology. LDCV release their specific cargo by regulated exocytosis, either in the form of “full fusion” or “kiss-and-run” exocytosis. In this paper, we provide ultrastructural evidence indicative of a slow and particulate mode of secretion from LDCV, called piecemeal degranulation (PMD). A number of LDCV in the nerve boutons of mouse brain presented marked increase in their size accompanied by reduction and also disappearance of content material. Residual secretory constituents in altered LDCV displayed eroded marginated patterns, leading to eccentric “haloed” morphologies. Remarkably, altered LDCV never appeared to be fused with each other or with the nerve plasma membrane. Very small vesicles, empty or apparently loaded with the same material making-up the LDCV content, could be seen near or attached to LDCV and the plasma membrane. First described in basophils, mast cells and eosinophils, PMD has recently been recognized in various neuro-endocrine cells, like adrenal chromaffin cells and endocrine cells of the gastro-intestinal epithelia. Here we suggest that PMD may be a hitherto unrecognized pathway of neuron secretion. It would represent the morphological correlate of a long-lasting and low-level process of neuro-transmitter release. It extends the patterns of neuron secretion and possibly opens new perspectives in understanding neuron plasticity.     

Expression of the P/Q (Cav2.1) calcium channel in nodose sensory neurons and arterial baroreceptors

The predominant calcium current in nodose sensory neurons, including the subpopulation of baroreceptor neurons, is the N-type channel, Cav2.2. It is also the primary calcium channel responsible for transmitter release at their presynaptic terminals in the nucleus of the solitary tract in the brainstem. The P/Q channel, Cav2.1, the other major calcium channel responsible for transmitter release at mammalian synapses, represents only 15–20% of total calcium current in the general population of sensory neurons and makes a minor contribution to transmitter release at the presynaptic terminal. In the present study we identified a subpopulation of the largest nodose neurons (capacitance > 50 pF) in which, surprisingly, Cav2.1 represents over 50% of the total calcium current, differing from the remainder of the population. Consistent with these electrophysiological data, anti-Cav2.1 antibody labeling was more membrane delimited in a subgroup of the large neurons in slices of nodose ganglia. Data reported in other synapses in the central nervous system assign different roles in synaptic information transfer to the P/Q-type versus N-type calcium channels. The study raises the possibility that the P/Q channel which has been associated with high fidelity transmission at other central synapses serves a similar function in this group of large myelinated sensory afferents, including arterial baroreceptors where a high frequency regular discharge pattern signals the pressure pulse. This contrasts to the irregular lower frequency discharge of the unmyelinated fibers that make up the majority of the sensory population and that utilize the N-type channel in synaptic transmission.     

T-type channels-secretion coupling: evidence for a fast low-threshold exocytosis

T-type channels are transient low-voltage-activated (LVA) Ca²⁺ channels that control Ca²⁺ entry in excitable cells during small depolarizations around resting potential. Studies in the past 20 years focused on the biophysical, physiological, and molecular characterization of T-type channels in most tissues. This led to a well-defined picture of the functional role of LVA channels in controlling low-threshold spikes, oscillatory cell activity, muscle contraction, hormone release, cell growth and differentiation. So far, little attention has been devoted to the role of T-type channels in transmitter release, which mainly involves channel types belonging to the high-voltage-activated (HVA) Ca²⁺ channel family. However, evidence is accumulating in favor of a unique participation of T-type channels in fast transmitter release. Clear data are now reported in reciprocal synapses of the retina and olfactory bulb, synaptic contacts between primary afferent and second order nociceptive neurons, rhythmic inhibitory interneurons of invertebrates and clonal cell lines transfected with recombinant α₁ channel subunits. T-type channels also regulate the large dense-core vesicle release of neuroendocrine cells where Ca²⁺ dependence, rate of vesicle release, and size of readily releasable pool appear comparable to those associated to HVA channels. This suggests that when sufficiently expressed and properly located near the release zones, T-type channels can trigger fast low-threshold secretion. In this study, we will review the main findings that assign a specific task to T-type channels in fast exocytosis, discussing their possible involvement in the control of the Ca²⁺-dependent processes regulating exocytosis like vesicle depletion and vesicle recycling.     

Release probability of hippocampal glutamatergic terminals scales with the size of the active zone

Cortical synapses have structural, molecular and functional heterogeneity; our knowledge regarding the relationship between their ultrastructural and functional parameters is still fragmented. Here we asked how the neurotransmitter release probability and presynaptic [Ca(2+)] transients relate to the ultrastructure of rat hippocampal glutamatergic axon terminals. Two-photon Ca(2+) imaging-derived optical quantal analysis and correlated electron microscopic reconstructions revealed a tight correlation between the release probability and the active-zone area. Peak amplitude of [Ca(2+)] transients in single boutons also positively correlated with the active-zone area. Freeze-fracture immunogold labeling revealed that the voltage-gated calcium channel subunit Cav2.1 and the presynaptic protein Rim1/2 are confined to the active zone and their numbers scale linearly with the active-zone area. Gold particles labeling Cav2.1 were nonrandomly distributed in the active zones. Our results demonstrate that the numbers of several active-zone proteins, including presynaptic calcium channels, as well as the number of docked vesicles and the release probability, scale linearly with the active-zone area.     

Synapse-to-synapse variation of calcium channel subtype contributions in large mossy fiber terminals of mouse hippocampus

Both N- and P/Q-type voltage-dependent calcium channels are involved in fast transmitter release in the hippocampus, but are differentially regulated. Although variable contributions of voltage-dependent calcium channel subtypes to presynaptic Ca2+ influx have been suggested to give a neural network of great diversity, their presence has only been demonstrated in a culture system and has remained unclear in the brain. Here, the individual large mossy fiber presynaptic terminal was labeled with Ca2+/Sr2+-sensitive fluorescent dextrans in the hippocampal slice of the mouse. The fractional contribution of voltage-dependent calcium channel subtypes to presynaptic Ca2+/Sr2+ influx was directly measured by the sensitivity of Ca2+/Sr2+-dependent fluorescent increment to subtype-selective neurotoxins, omega-conotoxin GVIA (an N-type selective blocker), omega-agatoxin IVA (a P/Q-type selective blocker) and SNX-482 (an R-type selective blocker). Synapse-to-synapse comparison of large mossy fiber terminals revealed that the contributions of N- and R-type voltage-dependent calcium channels varied more widely than that of P/Q-type. Even two large mossy fiber presynaptic terminals neighboring on the same axon differed in the fractional contributions of N- and R-type voltage-dependent calcium channels. On the other hand, these terminals were similar in the fractional contributions of P/Q-type voltage-dependent calcium channels. These results provide direct evidence that individual large mossy fiber synapses are differential in the contribution of N- and R-type voltage-dependent calcium channel subtypes to presynaptic Ca2+/Sr2+ influx. We suggest that the synapse-to-synapse variation of presynaptic voltage-dependent calcium channel subtype contributions may be one of the mechanisms amplifying diversity of the hippocampal network.     

Splicing of alpha 1A subunit gene generates phenotypic variants of P- and Q-type calcium channels.

P-type and Q-type calcium channels mediate neurotransmitter release at many synapses in the mammalian nervous system. The alpha 1A calcium channel has been implicated in the etiologies of conditions such as episodic ataxia, epilepsy and familial migraine, and shares several properties with native P- and Q-type channels. However, the exact relationship between alpha 1A and P- and Q-type channels is unknown. Here we report that alternative splicing of the alpha 1A subunit gene results in channels with distinct kinetic, pharmacological and modulatory properties. Overall, the results indicate that alternative splicing of the alpha 1A gene generates P-type and Q-type channels as well as multiple phenotypic variants.     

Timing and efficacy of transmitter release at mossy fiber synapses in the hippocampal network

It is widely accepted that the hippocampus plays a major role in learning and memory. The mossy fiber synapse between granule cells in the dentate gyrus and pyramidal neurons in the CA3 region is a key component of the hippocampal trisynaptic circuit. Recent work, partially based on direct presynaptic patch-clamp recordings from hippocampal mossy fiber boutons, sheds light on the mechanisms of synaptic transmission and plasticity at mossy fiber synapses. A high Na⁺ channel density in mossy fiber boutons leads to a large amplitude of the presynaptic action potential. Together with the fast gating of presynaptic Ca²⁺ channels, this generates a large and brief presynaptic Ca²⁺ influx, which can trigger transmitter release with high efficiency and temporal precision. The large number of release sites, the large size of the releasable pool of vesicles, and the huge extent of presynaptic plasticity confer unique strength to this synapse, suggesting a large impact onto the CA3 pyramidal cell network under specific behavioral conditions. The characteristic properties of the hippocampal mossy fiber synapse may be important for pattern separation and information storage in the dentate gyrus-CA3 cell network.     

CB1 modulation of temporally distinct synaptic facilitation among local circuit interneurons mediated by N-type calcium channels in CA1

One of the critical factors in determining network behavior of neurons is the influence of local circuit connections among interneurons. The short-term synaptic plasticity and the subtype of presynaptic calcium channels used at local circuit connections of inhibitory interneurons in CA1 were investigated using dual whole-cell recordings combined with biocytin and double immunofluorescence labeling in acute slices of P18- to 21-day-old rat stratum radiatum (SR) and stratum lacunosum molecular (SLM). Two forms of temporally distinct synaptic facilitation were observed among interneuron connections involving presynaptic cholecystokinin (CCK)-positive cells in SR, frequency-dependent facilitation, and a delayed onset of release (45-80 ms) with subsequent facilitation (DORF). Inhibition at both these synapses was under tonic cannabinoid-type 1 (CB1) receptor activity. DORF synapses did not display conventional release-dependent properties; however, blocking CB1 receptors with antagonist AM-251 (10 μM) altered the synaptic transmission to frequency-dependent depression with a fast onset of release (2-4 ms). Presynaptic CCK-negative interneurons in SLM elicited inhibitory postsynaptic potentials (IPSPs) insensitive to CB1 receptor pharmacology displayed frequency-dependent depression. Release of GABA at facilitating synapses was solely mediated via N-type presynaptic calcium channels, whereas depressing synapses utilized P/Q-type channels. These data reveal two distinct models of neurotransmitter release patterns among interneuron circuits that correlate with the subtype of presynaptic calcium channel. These data suggest that endocannabinoids act via CB1 receptors to selectively modulate N-type calcium channels to alter signal transmission.     

Vesicle pool partitioning influences presynaptic diversity and weighting in rat hippocampal synapses

Hippocampal synapses display a range of release probabilities. This is partially the result of scaling of release probability with the total number of releasable vesicles at each synapse. We have compared synaptic release and vesicle pool sizes across a large number of hippocampal synapses using FM 1-43 and confocal fluorescence microscopy. We found that the relationship between the number of recycling vesicles at a synapse and its release probability is dependent on firing frequency. During firing at 10 Hz, the release probability of each synapse is closely related to the number of recycling vesicles that it contains. In contrast, during firing at 1 Hz, different synapses turn over their recycling vesicle pools at different rates leading to an indirect relationship between recycling vesicle pool size and release probability. Hence two synapses may release vesicles at markedly different rates during low frequency firing, even if they contain similar numbers of vesicles. Both further kinetic analyses and manipulation of the number of vesicles in the readily releasable pool using phorbol ester treatment suggested that this imprecise scaling observed during firing at 1 Hz resulted from synapse-to-synapse differences in the proportion of recycling vesicles partitioned into the readily releasable pool. Hence differential partitioning between vesicle pools affects presynaptic weighting in a frequency-dependent manner. Since hippocampal single unit firing rates shift between 1 Hz and 10 Hz regimes with behavioural state, differential partitioning may be a mechanism for encoding information in hippocampal circuits.     

Direct interactions between C. elegans RAB-3 and Rim provide a mechanism to target vesicles to the presynaptic density

Rim is a multi-domain, active zone protein that regulates exocytosis and is implicated in vesicle priming and presynaptic plasticity. We recently demonstrated that synaptic defects associated with loss of Caenorhabditis elegans Rim (termed UNC-10) are accompanied by a reduction in docked vesicles adjacent to the presynaptic density. Since Rim is known to interact with the vesicle-associated GTPase Rab3A, here we asked whether UNC-10-dependent recruitment of synaptic vesicles to the presynaptic density was through an UNC-10/Rab-3 interaction. We first established that C. elegans Rab3 (termed RAB-3) in its GTP but not GDP-bound state interacts with UNC-10. We then demonstrated by EM analysis that rab-3 mutant synapses exhibit the same vesicle-targeting defect as unc-10 mutants. Furthermore, unc-10;rab-3 double mutants phenocopy the targeting defects of the single mutants, suggesting UNC-10 and RAB-3 act in the same pathway to target vesicles at the presynaptic density. Endogenous release of unc-10;rab-3 double mutants was similar to that of unc-10 single mutants, but more severe than rab-3 mutants, suggesting the common targeting defects are reflected by the milder rab-3 release defect. Rim has recently been shown to positively regulate calcium influx through direct interactions with calcium channels. Consistent with this notion we found UNC-10 colocalized with the calcium channel, UNC-2 at C. elegans presynaptic densities and synaptic release in unc-10 and rab-3 mutants exhibit reduced calcium-sensitivity. Together these results suggest that vesicles targeted to the presynaptic density by RAB-3/UNC-10 interactions are ideally positioned for efficient calcium-dependent release.     

NMDA receptors induce somatodendritic secretion in hypothalamic neurones of lactating female rats

Many neurones in the mammalian brain are known to release the content of their vesicles from somatodendritic locations. These vesicles usually contain retrograde messengers that modulate network properties. The back-propagating action potential is thought to be the principal physiological stimulus that evokes somatodendritic release. In contrast, here we show that calcium influx through NMDA receptor (NMDAR) channels, in the absence of postsynaptic cell firing, is also able to induce vesicle fusion from non-synaptic sites in nucleated outside-out patches of dorsomedial supraoptic nucleus (SON) neurones of adult female rats, in particular during their reproductive stages. The physiological significance of this mechanism was characterized in intact brain slices, where NMDAR-mediated release of oxytocin was shown to retrogradely inhibit presynaptic GABA release, in the absence of postsynaptic cell firing. This implies that glutamatergic synaptic input in itself is sufficient to elicit the release of oxytocin, which in turn acts as a retrograde messenger leading to the depression of nearby GABA synapses. In addition, we found that during lactation, when oxytocin demand is high, NMDA-induced oxytocin release is up-regulated compared to that in non-reproductive rats. Thus, in the hypothalamus, local signalling back and forth between pre- and postsynaptic compartments and between different synapses may occur independently of the firing activity of the postsynaptic neurone.     

Calcium signaling and exocytosis in adrenal chromaffin cells

At a given cytosolic domain of a chromaffin cell, the rate and amplitude of the Ca2+ concentration ([Ca2+]c) depends on at least four efficient regulatory systems: 1) plasmalemmal calcium channels, 2) endoplasmic reticulum, 3) mitochondria, and 4) chromaffin vesicles. Different mammalian species express different levels of the L, N, P/Q, and R subtypes of high-voltage-activated calcium channels; in bovine and humans, P/Q channels predominate, whereas in felines and murine species, L-type channels predominate. The calcium channels in chromaffin cells are regulated by G proteins coupled to purinergic and opiate receptors, as well as by voltage and the local changes of [Ca2+]c. Chromaffin cells have been particularly useful in studying calcium channel current autoregulation by materials coreleased with catecholamines, such as ATP and opiates. Depending on the preparation (cultured cells, adrenal slices) and the stimulation pattern (action potentials, depolarizing pulses, high K+, acetylcholine), the role of each calcium channel in controlling catecholamine release can change drastically. Targeted aequorin and confocal microscopy shows that Ca2+ entry through calcium channels can refill the endoplasmic reticulum (ER) to nearly millimolar concentrations, and causes the release of Ca2+ (CICR). Depending on its degree of filling, the ER may act as a sink or source of Ca2+ that modulates catecholamine release. Targeted aequorins with different Ca2+ affinities show that mitochondria undergo surprisingly rapid millimolar Ca2+ transients, upon stimulation of chromaffin cells with ACh, high K+, or caffeine. Physiological stimuli generate [Ca2+]c microdomains in which the local subplasmalemmal [Ca2+]c rises abruptly from 0.1 to approximately 50 microM, triggering CICR, mitochondrial Ca2+ uptake, and exocytosis at nearby secretory active sites. The fact that protonophores abolish mitochondrial Ca2+ uptake, and increase catecholamine release three- to fivefold, support the earlier observation. This increase is probably due to acceleration of vesicle transport from a reserve pool to a ready-release vesicle pool; this transport might be controlled by Ca2+ redistribution to the cytoskeleton, through CICR, and/or mitochondrial Ca2+ release. We propose that chromaffin cells have developed functional triads that are formed by calcium channels, the ER, and the mitochondria and locally control the [Ca2+]c that regulate the early and late steps of exocytosis.     

Purinergic synapses formed between rat sensory neurons in primary culture

Though there is some evidence to the contrary, dogma claims that primary sensory neurons in the dorsal root ganglion do not interact, that the ganglion serves as a through-station in which no signal processing occurs. Here we use patch clamp and immunocytochemistry to show that sensory neurons in primary culture can form chemical synapses on each other. The resulting neurotransmitter release is calcium dependent and uses synaptotagmin-containing vesicles. On many cells studied, the postsynaptic receptor for the neurotransmitter is a P2X receptor, an ion channel activated by extracellular ATP. This shows that sensory neurons have the machinery to form purinergic synapses on each other and that they do so when placed in short-term tissue culture.     

Reciprocal intraglomerular excitation and intra- and interglomerular lateral inhibition between mouse olfactory bulb mitral cells

How patterns of odour-evoked glomerular activity are transformed into patterns of mitral cell action potentials (APs) in the olfactory bulb is determined by the functional connectivity of the cell populations in the bulb. We have used paired whole-cell voltage recordings from olfactory bulb slices to compare the functional connectivity of mitral cells to the known anatomy of the mitral cell network. Both inhibitory and excitatory coupling were observed between pairs of mitral cells. Inhibitory coupling was seen as an increased frequency of small, asynchronous GABAergic IPSPs following APs in the presynaptic cell. Excitatory coupling was short in latency, beginning about 1.3 ms after the presynaptic AP and was mediated by both NMDA and AMPA receptors. Mitral cell pairs were coupled by excitation if and only if their apical dendrites terminated in the same glomerulus. The excitatory coupling between mitral cells resembles conventional fast synaptic transmission in its time course, amplitude and latency, despite the absence of evidence for anatomically defined synapses between mitral cells.     

RIM1 confers sustained activity and neurotransmitter vesicle anchoring to presynaptic Ca2+ channels

The molecular organization of presynaptic active zones is important for the neurotransmitter release that is triggered by depolarization-induced Ca2+ influx. Here, we demonstrate a previously unknown interaction between two components of the presynaptic active zone, RIM1 and voltage-dependent Ca2+ channels (VDCCs), that controls neurotransmitter release in mammalian neurons. RIM1 associated with VDCC beta-subunits via its C terminus to markedly suppress voltage-dependent inactivation among different neuronal VDCCs. Consistently, in pheochromocytoma neuroendocrine PC12 cells, acetylcholine release was significantly potentiated by the full-length and C-terminal RIM1 constructs, but membrane docking of vesicles was enhanced only by the full-length RIM1. The beta construct beta-AID dominant negative, which disrupts the RIM1-beta association, accelerated the inactivation of native VDCC currents, suppressed vesicle docking and acetylcholine release in PC12 cells, and inhibited glutamate release in cultured cerebellar neurons. Thus, RIM1 association with beta in the presynaptic active zone supports release via two distinct mechanisms: sustaining Ca2+ influx through inhibition of channel inactivation, and anchoring neurotransmitter-containing vesicles in the vicinity of VDCCs.     

Testosterone modulates Ca(v2.2) calcium channels' functional expression at rat levator ani neuromuscular junction

Spinal nucleus of bulbocavernosus and its target musculature, the bulbocavernosus and levator ani muscles, are sexually dimorphic, and their sexual differentiation depends on plasmatic levels of testosterone. Electrophysiological and immunocytochemical studies have demonstrated that at mammalian adult neuromuscular junctions only P/Q-type Ca2+ channels (Ca(v2.1)), mediate evoked transmitter release. Here we report that N-type Ca2+ channel (Ca(v2.2)) blocker omega-Conotoxin GVIA, as well as Ca(v2.1) blocker omega-Agatoxin IVA, significantly reduced quantal content of transmitter release by approximately 80% and approximately 70% respectively at levator ani muscle of the adult rats, indicating that neuromuscular transmission is jointly mediated by both types of channels. In these synapses, we also observed that castration and restitution of plasmatic testosterone in rats resulted in changes in the sensitivity to omega-Conotoxin GVIA. Castration induced, whereas testosterone treatment avoided, functional loss of Ca(v2.2), as mediators of transmitter release in these synapses. Strikingly, the expression and localization of alpha1B subunits, which form the pore of the Ca(v2.2) channel, were similar at control, gonadectomized and gonadectomized testosterone-treated rats, suggesting that testosterone may regulate the coupling mechanisms between Ca(v2.2) and transmitter release at the neuromuscular junctions of these sexually dimorphic motoneurons.     

Brevity of the Ca2+ microdomain and active zone geometry prevent Ca2+-sensor saturation for neurotransmitter release

The brief time course of the calcium (Ca2+) channel opening combined with the molecular-level colocalization of Ca2+ channels and synaptic vesicles in presynaptic terminals predict sub-millisecond calcium concentration ([Ca2+]) transients of > or = 100 microM in the immediate vicinity of the vesicle. This [Ca2+] is much higher than some of the recent estimates for the equilibrium dissociation constant of the Ca2+ sensor(s) that control neurotransmitter release, suggesting release should be close to saturation, yet it is well known that release is highly sensitive to changes in Ca2+ influx. We show that due to the brevity of the Ca2+ influx the binding kinetics of the Ca2+ sensor rather than its equilibrium affinity determine receptor occupancy. For physiologically relevant Ca2+ currents and forward Ca2+ binding rates, the effective affinity of the Ca2+ sensor can be several-fold lower than the equilibrium affinity. Using simple models, we show redundant copies of the binding sites increase effective affinity of the Ca2+ sensor for release. Our results predict that different levels of expression of Ca2+ binding sites could account for apparent differences in Ca2+ sensor affinities between synapses. Using Monte Carlo simulations of Ca2+ dynamics with nanometer resolution, we demonstrate that these kinetic constraints combined with vesicles acting as diffusion barriers can prevent saturation of the Ca2+-sensor(s) for neurotransmitter release. We further show the random positioning of the Ca2+-sensor molecules around the vesicle can result in the emergence of two distinct populations of the vesicles with low and high release probability. These considerations allow experimental evidence for the Ca2+ channel-vesicle colocalization to be reconciled with a high equilibrium affinity for the Ca2+ sensor of the release machinery.