Receptor-mediated regulation of calcium channels and neurotransmitter release

Ca2+ influx into the nerve terminal is normally the trigger for the release of neurotransmitters. Many neurons possess presynaptic receptors whose activation results in changes in the quantity of neurotransmitter released by an action potential. This paper reviews studies that show that presynaptic receptors can regulate the activity of Ca2+ channels in the nerve terminal, resulting in changes in the influx of Ca2+ and in neurotransmitter release. Neurons possess several different types of voltage-sensitive Ca2+ channels. Ca2+ influx through N-type channels appears to trigger transmitter release in many instances. In other cases Ca2+ influx through L channels can influence transmitter release. Neurotransmitters can inhibit N channels through a G protein-mediated transduction mechanism. The G proteins are frequently pertussis toxin substrates. Inhibition of N channels appears to involve changes in their voltage dependence. Neurotransmitters can also regulate neuronal K+ channels. Activation of these K+ channels can lead to a reduction in Ca2+ influx and neurotransmitter release; these effects are also mediated by G proteins. Thus neurotransmitters may often regulate both presynaptic Ca2+ and K+ channels. These two effects may be synergistic mechanisms for the regulation of Ca2+ influx and neurotransmitter release.     

Measurement of intrasynaptosomal free calcium by using the fluorescent indicator quin-2.

The recently synthesized calcium indicator quin -2 was incorporated into synaptosomes from guinea-pig cerebral cortex following uptake and internal hydrolysis of quin -2 tetra-acetoxymethyl ester. Incubation in physiological media containing 1 mM- or 2 mM-CaCl2 led to equilibrium cytosolic ionized calcium concentrations of 85 +/- 10 nM and 205 +/- 5 nM respectively (mean +/- S.E.M. from eight and eighteen preparations respectively). Cytosolic Ca2+ was elevated following increases in external Ca2+ concentration, plasma membrane depolarization, mitochondrial inhibition, calcium ionophore addition or replacement of external sodium by lithium. Preliminary experiments were performed to assess changes in cytosolic Ca2+ accompanying the release of the neurotransmitter acetylcholine.     

Ricardo Miledi and the calcium hypothesis of neurotransmitter release

Ricardo Miledi has made significant contributions to our basic understanding of how synapses work. Here I discuss aspects of Miledi's research that helped to establish the requirement of presynaptic calcium for neurotransmitter release, from his earliest scientific studies to his classic experiments in the squid giant synapse.     

Exhaustion of calcium does not terminate evoked neurotransmitter release

Theories are considered which assume that termination of evoked release is caused by the exhaustion of intracellular Ca. It is shown that such theories predict, contrary to experiment, that total release is an unsaturated function of intracellular Ca whose duration depends strongly on extracellular Ca. These and other findings lead to the conclusion that termination must be due to the fast change of another parameter (not intracellular Ca).     

Functional interactions between presynaptic calcium channels and the neurotransmitter release machinery

In vertebrates, the physical coupling between presynaptic calcium channels and synaptic vesicle release proteins enhances the efficiency of neurotransmission. Recent evidence indicates that these synaptic proteins may feedback directly on synaptic release by negatively regulating calcium entry, and indirectly through pathways involving second messenger molecules. Studies of individual neurons from both vertebrates and invertebrates have provided novel insights into the roles of scaffolding proteins in calcium channel targeting and neurotransmitter release. These studies require us to expand current models of synaptic transmission.     

Cytosolic-free calcium and neurotransmitter release with decreased availability of glucose or oxygen

Exposing brain slices to reduced oxygen tensions or impairing their ability to utilize oxygen with KCN decreases acetylcholine (ACh) but increases dopamine (DA) and glutamate in the medium at the end of a release incubation. To determine if these changes are due to alterations in the presynaptic terminals, release from isolated nerve endings (i.e. synaptosomes) was determined during histotoxic hypoxia (KCN). KCN reduced potassium-stimulated synaptosomal ACh release and increased dopamine and glutamate release. Since several lines of evidence suggest that altered calcium homeostasis underlies these changes in release, the effects of reducing medium calcium concentrations from 2.3 to 0.1-mM were determined. In low calcium medium, KCN still increased dopamine and glutamate release, but had no effect on ACh release. Hypoxia increased cytosolic-free calcium in both the normal and low calcium medium, although the elevation was less in the low calcium medium. Thus, the effects of histotoxic hypoxia on cytosolic free calcium concentration paralleled those on glutamate and dopamine release. Reducing the glucose concentration of the medium also increased cytosolic-free calcium. The data are consistent with the hypothesis that hypoxia and hypoglycemia increase cytosolic-free calcium, which stimulates the release of dopamine and glutamate, whose excessive release may lead to subsequent cellular damage postsynaptically.Abbreviations (cps) counts per second - (FAM) fura-2 acetoxymethylester - (ACh) acetylcholine - (Ca_i) cytosolic free calcium concentration - (DMSO) dimethylsulphoxide - (DA) dopamine - (TES) N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid - (R_min) the ratio of the fluorescence of fura at 510 nm after excitation at 340 nm to that after excitation at 380 nm in the absence of calcium - (R_max) or to that in the presence of saturating calcium - (SNK) Student-Newman-Keuls     

Synaptotagmin I functions as a calcium sensor to synchronize neurotransmitter release

To characterize Ca(2+)-mediated synaptic vesicle fusion, we analyzed Drosophila synaptotagmin I mutants deficient in specific interactions mediated by its two Ca(2+) binding C2 domains. In the absence of synaptotagmin I, synchronous release is abolished and a kinetically distinct delayed asynchronous release pathway is uncovered. Synapses containing only the C2A domain of synaptotagmin partially recover synchronous fusion, but have an abolished Ca(2+) cooperativity. Mutants that disrupt Ca(2+) sensing by the C2B domain have synchronous release with normal Ca(2+) cooperativity, but with reduced release probability. Our data suggest the Ca(2+) cooperativity of neurotransmitter release is likely mediated through synaptotagmin-SNARE interactions, while phospholipid binding and oligomerization trigger rapid fusion with increased release probability. These results indicate that synaptotagmin is the major Ca(2+) sensor for evoked release and functions to trigger synchronous fusion in response to Ca(2+), while suppressing asynchronous release.     

Multiple roles of calcium ions in the regulation of neurotransmitter release

The intracellular calcium concentration ([Ca(2+)]) has important roles in the triggering of neurotransmitter release and the regulation of short-term plasticity (STP). Transmitter release is initiated by quite high concentrations within microdomains, while short-term facilitation is strongly influenced by the global buildup of "residual calcium." A global rise in [Ca(2+)] also accelerates the recruitment of release-ready vesicles, thereby controlling the degree of short-term depression (STD) during sustained activity, as well as the recovery of the vesicle pool in periods of rest. We survey data that lead us to propose two distinct roles of [Ca(2+)] in vesicle recruitment: one accelerating "molecular priming" (vesicle docking and the buildup of a release machinery), the other promoting the tight coupling between releasable vesicles and Ca(2+) channels. Such coupling is essential for rendering vesicles sensitive to short [Ca(2+)] transients, generated during action potentials.     

Presynaptic calcium signals during neurotransmitter release: detection with fluorescent indicators and other calcium chelators.

Synthetic calcium buffers, including fluorescent calcium indicators, were microinjected into squid 'giant' presynaptic nerve terminals to investigate the calcium signal that triggers neurotransmitter secretion. Digital imaging methods, applied in conjunction with the fluorescent calcium indicator dye fura-2, reveal that transient rises in presynaptic calcium concentration are associated with action potentials. Transmitter release terminates within 1-2 ms after a train of action potentials, even though presynaptic calcium concentration remains at micromolar levels for many seconds longer. Microinjection of the calcium buffer, EGTA, into the presynaptic terminal has no effect on transmitter release evoked by single presynaptic action potentials. EGTA injection does, however, block the change in calcium concentration measured by fura-2. Therefore, the calcium signal measured by fura-2 is not responsible for triggering release. These results suggest that the rise in presynaptic calcium concentration that triggers release must be highly localized to escape detection with fura-2 imaging. Unlike EGTA, microinjection of BAPTA--a calcium buffer with an equilibrium affinity for calcium similar to that of EGTA--produces a potent, dose-dependent, and reversible block of action-potential evoked transmitter release. The superior ability of BAPTA to block transmitter release apparently is due to the more rapid calcium-binding kinetics of BAPTA compared to EGTA. Because EGTA should bind calcium within a few tens of microseconds under the conditions of our experiments, the inability of EGTA to block release indicates that transmitter release is triggered within a few tens of microseconds after the entry of calcium into the presynaptic terminal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neurotransmitter release from bradykinin-stimulated PC12 cells. Stimulation of cytosolic calcium and neurotransmitter release.

The effect of bradykinin on intracellular free Ca2+ and neurotransmitter secretion was investigated in the rat pheochromocytoma cell line PC12. Bradykinin was shown to induce a rapid, but transient, increase in intracellular free Ca2+ which could be separated into an intracellular Ca2+ release component and an extracellular Ca2+ influx component. The bradykinin-induced stimulation of intracellular free Ca2+ displayed a similar time course, concentration dependencies and extracellular Ca2+ dependence as that found for neurotransmitter release, indicating an association between intracellular free Ca2+ levels and neurotransmitter secretion. The selective BK1-receptor antagonist des-Arg9,[Leu8]BK (where BK is bradykinin) did not significantly affect the stimulation of intracellular free Ca2+ or neurotransmitter release. In contrast, these effects of bradykinin were effectively blocked by the selective BK2-receptor antagonist [Thi5,8,D-Phe7]BK, and mimicked by the BK2 partial agonist [D-Phe7]BK in a concentration-dependent manner. The stimulation of intracellular free Ca2+ and neurotransmitter release induced by bradykinin was shown not to involve voltage-sensitive Ca2+ channels, since calcium antagonists had no effect on either response at concentrations which effectively inhibit depolarization-induced responses. These results indicate that bradykinin, acting through the interaction with the BK2 receptor, stimulates an increase in intracellular free Ca2+ leading to neurotransmitter secretion. Furthermore, bradykinin-induced responses involve the release of intracellular Ca2+ and the influx of extracellular Ca2+ that is not associated with the activation of voltage-sensitive Ca2+ channels.     

A theoretical study of calcium entry in nerve terminals, with application to neurotransmitter release

In his study of a kin selection model for the evolution of workers' behavior in an incipiently social insect, Craig (1979) found that the haplodiploid mode of sex determination, combined with a female-biased sex ratio, cannot accelerate an evolutionary trend toward eusociality. This seems to contradict to Hamilton's (1964) theory. It is my intention to prove that Craig's result is due to the dependence of relative reproductive success in each sex on the sex ratio of a population. The oviposition of unfertilized eggs by workers is indispensable, in primitive stages in the evolution of eusociality, for maintaining the relative reproductive success of a female. Assuming that workers control the sex ratios, we can distinguish the following three evolutionary phases in accordance with the ratio $\text{N}{}_2\text{N}{}_{10}$ (the ratio of the number of the queen's second brood to that of the progeny laid by workers derived from the same nest). During Phase 1 in which $\text{N}{}_2\text{N}{}_{10}$ < $\text{1}\text{2}$, the reproductive successes of a male and a female are equal, and Hamilton's rule holds. In Phase 2 in which $\text{1}\text{2 <}\text{N}{}_2\text{N}{}_{10}\text{<}\text{3}\text{2}$, the queen produces females, and workers oviposit males. As $\text{N}{}_2\text{N}{}_{10}$ increases, the sex ratio in the whole population becomes female-biased and relative reproductive success of a male increases. In Phase 3 in which N₂/N₁₀ >$\text{3}\text{2}$, the queen lays some male eggs in addition to female eggs. The sociality threshold ($\text{B}\text{C}$)_crit the minimum value of the benefit/cost ratio leading to the evolution of altruism, is $\text{2}\text{3}$on Phase 1. It rises threefold as N₂/N₁₀ increases in Phase 2, and, in Phase 3, it is twice as high as that in a diploid species. The anatomical and physiological characteristics of workers must have been so developed that they are efficient in helping activities after the beginning of eusociality. The evolutionary process before the beginning of helping behavior is also discussed. The egg laying by unmated females, which stay their mother's nest, seems to have been an important preadaptation for the evolution of eusociality in Hymenoptera.     

Calmodulin, synchronous and asynchronous release of neurotransmitter

Evidence collected from studies on a wide range of secretory cells suggests that calmodulin may play an important role in stimulus-secretion coupling. Work on synaptosomes, central synaptic preparations and chromaffin cell preparations indicates that calmodulin probably also acts as the intracellular Ca2+-receptor for secretion in neuronal cells, Ca2+-binding resulting in activation of protein kinases and phosphorylation of certain secretory vesicle proteins. Studies on the effects of calmodulin-binding drugs at peripheral synapses have given surprising results, particularly the finding that evoked (synchronous) transmitter release is not suppressed by calmodulin inhibition, though asynchronous release can be markedly inhibited. It is suggested that the insensitivity of synchronous release to drug treatment is due to the fact that only vesicle-bound calmodulin is involved in this form of transmitter secretion. Asynchronous release, however, involves recruitment of cytosolic calmodulin and can therefore be inhibited by calmodulin-binding drugs.     

Humanin derivative,HNG, enhances neurotransmitter release

BackgroundHumanin (HN) is an endogenous 24-residue peptide that was first identified as a protective factor against neuronal death in Alzheimer's disease (AD). We previously demonstrated that the highly potent HN derivative HNG (HN with substitution of Gly for Ser14) ameliorated cognitive impairment in AD mouse models. Despite the accumulating evidence on the antagonizing effects of HN against cognitive deficits, the mechanisms behind these effects remain to be elucidated.MethodsThe extracellular fluid in the hippocampus of wild-type young mice was collected by microdialysis and the amounts of neurotransmitters were measured. The kinetic analysis of exocytosis was performed by amperometry using neuroendocrine cells.ResultsThe hippocampal acetylcholine (ACh) levels were increased by intraperitoneal injection of HNG. HNG did not affect the physical activities of the mice but modestly improved their object memory. In a neuronal cell model, rat pheochromocytoma PC12 cells, HNG enhanced ACh-induced dopamine release. HNG increased ACh-induced secretory events and vesicular quantal size in primary neuroendocrine cells.ConclusionsThese findings suggest that HN directly enhances regulated exocytosis in neurons, which can contribute to the improvement of cognitive functions.General significanceThe regulator of exocytosis is a novel physiological role of HN, which provides a molecular clue for HN's effects on brain functions under health and disease.     

Rapid kinetics of depolarization-induced changes in intrasynaptosomal calcium concentrations.

The rapid kinetics of depolarization-evoked calcium influxes in isolated nerve terminals from rat cortex were monitored by stopped-flow spectrofluorimetry using specific indicators (Fluo-3, Indo-1). A very rapid increase in the intrasynaptosomal Ca(2+)-level was detected within the subsecond time range after depolarizing synaptosomes by mixing with physiological saline containing elevated K(+)-concentrations. About 15 mM [K+]o was determined as threshold concentration for inducing Ca(2+)-influx, which increased with higher concentration and saturated at [K+]o-concentrations of about 40 mM [K+]o.     

Leptinotarsin-D, a neurotoxic protein, evokes neurotransmitter release from, and calcium flux into, isolated electric organ nerve terminals

Previous work has demonstrated that the neurotoxin leptinotarsin elicits release of neurotransmitter from mammalian nerve terminals, and it has been suggested that the toxin may act either as a direct agonist of voltage-sensitive calcium channels in these terminals (Crosland et al., 1984) or as a calcium ionophore (Madeddu et al., 1985a,b). Preliminary studies (Yeager et al., 1987) demonstrated that leptinotarsin also evokes transmitter release from isolated elasmobranch electric organ nerve terminals. We now report further investigations of the effects of leptinotarsin in this system. The action of the toxin is saturable, releasing about the same small fraction of total transmitter as that released by depolarization. An upper limit for the concentration for half maximal release is estimated to be 4 nM. Leptinotarsin-evoked transmitter release exhibits behavior very similar to depolarization-evoked release with respect to dependence on Ca2+, Ba2+, and Sr2+ and blockade by Co2+, Cd2+, and trifluoperazine. Leptinotarsin also promotes the uptake of calcium into synaptosomes to a degree similar to that caused by depolarization by K+. The binding of leptinotarsin to nerve terminals is probably Ca2+ dependent and receptor mediated. Taken together with the behavior of leptinotarsin-evoked release in other preparations, these results are consistent with the hypothesis that this toxin acts by opening a presynaptic calcium channel. However, the possibility that leptinotarsin is a calcium ionophore cannot be excluded.     

Influence of calcium ionophore A23187 on neurotransmitter release in rat brain synaptosomes

The effect of calcium ionophore A23187 on the release of nonmetabolizable glutamate analogues [3H]D-aspartate and the exocytosis registered by fluorescent dyes in synaptosomes was investigated. It was shown that A23187 is able to induce neurotransmitter release both in calcium-containing and calcium-free medium, the effect in the latter case being more pronounced. Calcium ionophore is able to induce exocytosis registered by acridine orange and FM 2-10. The influence of A23187 on the fluorescence of acridine orange was mainly calcium-independent, whereas the change in the fluorescence of FM 2-10 was calcium-dependent. It was suggested that the calcium-independent increase in acridine orange fluorescence is related to the dissipation of pH gradient in synaptic vesicles. Probably, the calcium-independent release of D-aspartate is also associated with the dissipation of pH gradient and subsequent leakage of neurotransmitters.     

Control of depolarization-evoked presynaptic neurotransmitter release by Cav2.1 calcium channel: old story, new insights

Depolarization-evoked synaptic transmission relies on the Ca²⁺-regulated release of quantal packets of neurotransmitters following the fusion of synaptic vesicles with the presynaptic plasma membrane. It is well known that neuronal voltage-gated Ca²⁺ channels (VGCC), mainly of the Ca_V2.1 and Ca_V2.2 subtypes, play a key role in the first steps of this process, by controlling extracellular Ca²⁺ influx into active zones of the synapse. These channels are in close association with the vesicle machinery and interact with several members of SNARE proteins (soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein receptor) including syntaxin 1A/1B and SNA P-25 (Q-SNARE s), and synaptotagmin 1 and synaptobrevin 2 (R-SNARE s) (reviewed in ref. ¹). All bind to the synprint (synaptic protein interaction) motif within the intracellular II -III linker of Ca_V2.1 and Ca_V2.2 channels and are responsible for a bidirectional coupling (i) linking the Ca²⁺ influx with the synaptic vesicle release machinery, which is essential for efficient, fast and spatially delimited neurotransmitter release² and (ii) providing regulation of Ca²⁺ channel activity and thus of Ca²⁺ influx.³Key words: calcium channel, Ca_V2.1 channel, P/Q channel, syntaxin, synaptotagmin, SNAP25, exocytosis, synaptic transmissionSeveral studies have proposed that synaptotagmin 1 is the Ca²⁺ sensor for release, linking Ca²⁺ influx to vesicle fusion (reviewed in ref. ⁴). Synaptotagmin 1 has two repeating domains that are rich in negative charges (C2A and C2B), each capable of binding Ca²⁺ ions. It is commonly thought that following Ca²⁺ entry through VGCCs, Ca²⁺ ions bind to C2A and C2B domains, allowing insertion of the Ca²⁺ binding loops of C2A domain in the target bilayer. This then pins the vesicle to the plasma membrane to trigger exocytotic fusion. This view was supported by a point mutation in the C2A domain of synaptotagmin 1 that caused a decrease in Ca²⁺ affinity with a concomitant decrease of neurotransmitter release.⁵ However, despite the fact that synaptotagmin 1 represents the most popular candidate for Ca²⁺ sensor, the initial Ca²⁺ binding event, which occurs during the dynamic process of release is at the EEEE locus within the Ca²⁺ channel itself. This makes the Ca²⁺ channel an excellent candidate for serving as a Ca²⁺ sensor of secretion.⁶Over the past few years, the group of Daphne Atlas has performed extensive studies to differentiate the role of Ca²⁺ binding at the pore of the channel from Ca²⁺ binding to intracellular proteins during evoked-neurotransmitter release. Substituting extracellular Ca²⁺ by lanthanum (La³⁺), a trivalent cation that effectively binds to the EEEE locus of VGCCs but is unable to permeate through the channel, is sufficient to support depolarization-evoked release of catecholamine in PC12 and primary chromaffin cells, as well as insulin release in pancreatic and insulinoma cells. These results led to the suggestion that evoked release may be dependent on ion channel pore occupancy as opposed to cation influx and elevation of intracellular Ca²⁺ concentration.^7–9 This model was further supported by experiments in which depolarization-evoked secretion of catecholamine in chromaffin cells was supported by Ca²⁺ bound at the selectivity filter of a non-conducting Ca_V1.2 channel.¹⁰ These studies are consistent with the proposal that conformational changes subsequent to Ca²⁺ binding at the selectivity filter of the channel are the primary trigger of secretion, whereas synaptotagmin 1 is associated with the channel and acts as a vesicle docking protein (reviewed in ref. ¹¹).In a recent issue of Channels, Cohen-Kutner et al. extended this concept to the neuronal Ca_V2.1 channel.¹² Using the two-electrode voltage-clamp technique on BAPTA-injected Xenopus oocyte expressing the human Ca_V2.1 channel (in combination with β₃ and α₂δ auxiliary subunits), the authors show that overexpression of syntaxin 1A (Stx1A) depresses whole-cell inward barium (Ba²⁺) current in a dose-dependent manner (Fig. 1, reviewed in ref. ¹²). As previously reported by Bezprozvanny et al.³ this effect is mainly due to a hyperpolarized shift of the steady-state inactivation curve, which decreases the number of available channels at typical resting membrane potentials. A recovery of channel activity is observed following co-expression of botulinium neurotoxin C1 (BoNT/C1) (Fig. 3, reviewed in ref. ¹²). In contrast, expression of the other Q-SNARE protein SNAP-25 drastically increases inward Ba²⁺ current (Fig. 2, reviewed in ref. ¹²). However, when both Q-SNARE proteins are co-expressed, Ca_V2.1 channel recovers wild-type P/Q kinetics and current amplitude (Fig. 2, reviewed in ref. ¹²). Similarly, increases in P/Q currents by expressing the R-SNARE synaptobrevin (VAMP-2) are reversed by the Q-SNARE proteins (Fig. 4, reviewed in ref. ¹²). Taken together these results suggest that: (i) when expressed in BAPTA injected Xenopus oocyte, each of the SNARE proteins is able to modulate the kinetic properties of Ca_V2.1 channel and (ii) when co-expressed, SNARE proteins no longer affect channel activity but rather form a Ca²⁺-independent excitosome complex with a fully functional channel. These data fit nicely with previous work from the Catterall laboratory on P/Q-type channels,¹³ and with previous work on N-type channels.¹⁴To investigate the relevance of Ca_V2.1 channel interaction with SNARE proteins for depolarization-evoked secretion, membrane capacitance changes induced in Xenopus oocytes were monitored in the presence of extracellular Ca²⁺, as previously shown for Ca_V1.2 and Ca_V2.2.¹⁵ While expression of Ca_V2.1 alone in this reconstituted release assay produced only a small change in capacitance, coexpression with the SNARE proteins efficiently induced a BoNT/C- and BoNT/A-sensitive membrane fusion, particularly when all SNARE proteins were co-expressed, i.e., when all members of the excitosome complex are present (Fig. 5, reviewed in ref. ¹²). Hence, increasing the amount of excitosome promotes the capability of Ca_V2.1 channels to produce evoked-secretion, probably by increasing the number of functional excitosome complexes (Fig. 6, reviewed in ref. ¹²).In summary, Cohen-Kutner et al. provide evidence that when expressed in Xenopus oocyte (and possibly in other cellular systems), Ca_V2.1 channels could associate with SNARE proteins at resting intracellular Ca²⁺ concentrations, resulting in tethering the vesicle to the channel and thereby generating docked but non-releasable vesicles. Calcium entry following membrane depolarization would switch the vesicle from the non-releasable to a releasable state by Ca²⁺-binding to Syt1 C2 domains. The fusion of releasable vesicles requires a conformational change of the complex that occurs within the channel itself, during an incoming action potential (Fig. 1).Open in a separate windowFigure 1A putative model of functional coupling between Ca_V2.1 channel and vesicle release machinery. At resting membrane potential, Ca_V2.1 channel associates with SNARE proteins to form an excitosome complex, in turn generating docked but non-releasable vesicle (A). Calcium entry following membrane depolarization would switch the vesicle from the non-releasable to a releasable state by Ca²⁺-binding to Synaptotagmin 1 C2 domains (B). The fusion of the releasable vesicle requires a conformational change of the excitosome complex that occurs within the channel itself, during an incoming action potential (C).The concept that Ca_V2.1 channels, besides sustaining Ca²⁺ influx, could also work as a molecular on/off-switch of secretion by controlling the ultimate stage of the process (i.e., the conformational change of the releasing complex) is intriguing and is worthy of further investigation. To better dissociate secretion events linked to Ca²⁺ entry through Ca_V2.1 channel from those induced by conformational changes of the channel, it would be necessary to measure secretion in the presence of a non-permeant cation such as La³⁺. Furthermore, one would also need to evaluate mediation of secretion by a non-conducting Ca_V2.1 channel, as already done for L-type channels (Ca_V1.2).^7,9,10 Moreover, the possibility that Ca_V2.1 channels could control secretion via a conformational change of the releasing complex raises questions concerning the preferential channel-gating mode controlling this process. It was recently shown that application of the gating modifier BayK 8644 to non-conducting Ca_V1.2 channels modifies secretion kinetics of catecholamine in chromaffin cells.¹⁶ It is also well known that the auxiliary β-subunit of VGCCs modulates Ca_V2.1 gating modes.¹⁷ Therefore, comparing secretion mediated by a non-conducting Ca_V2.1 channel in the presence of different types of β-subunits would provide important information on the molecular mechanisms through which Ca_V2.1 channels control evoked-secretion, both at the fundamental and physiopathological levels.In conclusion, since the pioneering work by Katz and Miledi in 1967 on the importance of the extracellular Ca²⁺ in the “electro-secretory” process,¹⁸ the identification of the calcium channel as the Ca²⁺ sensor of secretion is one of the most recent and exciting steps that have been made in the understanding of the molecular aspects of the mechanisms involved in the control of depolarization-evoked neurotransmitter release.     

Modeling study of the effects of membrane surface charge on calcium microdomains and neurotransmitter release

Synchronous neurotransmitter release is mediated by the opening of voltage-gated Ca²⁺ channels and the build-up of submembrane Ca²⁺ microdomains. Previous models of Ca²⁺ microdomains have neglected possible electrostatic interactions between Ca²⁺ ions and negative surface charges on the inner leaflet of the plasma membrane. To address the effects of these interactions, we built a computational model of ion electrodiffusion described by the Nernst-Planck and Poisson equations. We found that inclusion of a negative surface charge significantly alters the spatial characteristics of Ca²⁺ microdomains. Specifically, close to the membrane, Ca²⁺ ions accumulate, as expected from the strong electrostatic attraction exerted on positively charged Ca²⁺ ions. Farther away from the membrane, increasing the surface charge density results in a reduction of the Ca²⁺ concentration because of the preferential spread of Ca²⁺ ions along lateral directions. The model also predicts that the negative surface charge will decrease the spatial gradient of the Ca²⁺ microdomain in the lateral direction, resulting in increased overlap of microdomains originating from different Ca²⁺ channels. Finally, we found that surface charge increases the probability of vesicle release if the Ca²⁺ sensor is located within the electrical double layer, whereas this probability is decreased if the Ca²⁺ sensor lies at greater distances from the membrane. Our data suggest that membrane surface charges exert a significant influence on the profile of Ca²⁺ microdomains, and should be taken into account in models of neurotransmitter release.