The Nanophysiology of Fast Transmitter Release
Authors: Elise F. Stanley
Journal: Trends in Neurosciences (2016)

Single domain gating appears to predominate where transmission fidelity is paramount [73Bucurenciu I. et al.A small number of open Ca2+ channels trigger transmitter release at a central GABAergic synapse.Nat. Neurosci. 2010; 13: 19-21Crossref PubMed Scopus (85) Google Scholar]. Possible reasons include: minimal delay due to the virtually instantaneous access of Ca2+ to the SV sensor; protection against docked-SV depletion and transmission failure; a metabolic advantage by minimizing Ca2+ clearance; and avoiding detrimental effects of high cytoplasmic Ca2+ [8Stanley E.F. The calcium channel and the organization of the presynaptic transmitter release face.Trends Neurosci. 1997; 20: 404-409Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar]. Overlapping domain gating may predominate where the amplitude of transmitter release is more important than the frequency of impulses [76Goswami S.P. et al.Miniature IPSCs in hippocampal granule cells are triggered by voltage-gated Ca2+ channels via microdomain coupling.J. Neurosci. 2012; 32: 14294-14304Crossref PubMed Scopus (43) Google Scholar] or where release is subject to modulation of presynaptic CaVs. While rod synapses contradict this general idea, since they exhibit an exquisite dynamic range while relying on single domain gating [68Dittrich M. et al.An excess-calcium-binding-site model predicts neurotransmitter release at the neuromuscular junction.Biophys. J. 2013; 104: 2751-2763Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar], it is possible that this idea holds for neurons. Single domain-type synapses may pass through a pre-maturity overlapping domain stage, raising the possibility that amplitude coding is important during synapse formation [60Stanley E.F. Single calcium channel domain gating of synaptic vesicle fusion at fast synapses; analysis by graphic modeling.Channels (Austin). 2015; 9: 324-333Crossref PubMed Scopus (12) Google Scholar]. Action potentials invading the presynaptic terminal trigger discharge of docked synaptic vesicles (SVs) by opening voltage-dependent calcium channels (CaVs) and admitting calcium ions (Ca2+), which diffuse to, and activate, SV sensors. At most synapses, SV sensors and CaVs are sufficiently close that release is gated by individual CaV Ca2+ nanodomains centered on the channel mouth. Other synapses gate SV release with extensive Ca2+ microdomains summed from many, more distant CaVs. We review the experimental preparations, theories, and methods that provided principles of release nanophysiology and highlight expansion of the field into synaptic diversity and modifications of release gating for specific synaptic demands. Specializations in domain gating may adapt the terminal for roles in development, transmission of rapid impulse frequencies, and modulation of synaptic strength. Action potentials invading the presynaptic terminal trigger discharge of docked synaptic vesicles (SVs) by opening voltage-dependent calcium channels (CaVs) and admitting calcium ions (Ca2+), which diffuse to, and activate, SV sensors. At most synapses, SV sensors and CaVs are sufficiently close that release is gated by individual CaV Ca2+ nanodomains centered on the channel mouth. Other synapses gate SV release with extensive Ca2+ microdomains summed from many, more distant CaVs. We review the experimental preparations, theories, and methods that provided principles of release nanophysiology and highlight expansion of the field into synaptic diversity and modifications of release gating for specific synaptic demands. Specializations in domain gating may adapt the terminal for roles in development, transmission of rapid impulse frequencies, and modulation of synaptic strength. The seminal experiment demonstrating that transmitter release from presynaptic terminals requires both simultaneous nerve terminal depolarization and the presence of extracellular Ca2+ [1Katz B. Miledi R. The timing of calcium action during neuromuscular transmission.J. Physiol. 1967; 189: 535-544Crossref PubMed Scopus (378) Google Scholar] was the lifetime favorite (personal communication) of Sir Bernard Katz (Nobel Laureate in Physiology and Medicine, 1970). This key observation, together with the findings that depolarization opens presynaptic CaVs (see Glossary) [2Llinas R.R. et al.Presynaptic calcium currents and their relation to synaptic transmission: voltage clamp study in squid giant synapse and theoretical model for the calcium gate.Proc. Natl. Acad. Sci. U.S.A. 1976; 73: 2918-2922Crossref PubMed Scopus (212) Google Scholar] admitting Ca2+ into the nerve terminal cytoplasm [3Llinas R.R. et al.Calcium transients in presynaptic terminal of squid giant synapse: detection with aequorin.Science. 1972; 176: 1127-1129Crossref PubMed Scopus (116) Google Scholar] and that intracellular Ca2+ can gate transmitter release [4Miledi R. Transmitter release induced by injection of calcium ions into nerve terminals.Proc. Biol. Sci. 1969; 183: 421-425Crossref Scopus (324) Google Scholar] by the fusion of SVs [5Katz B. The Release of Neural Transmitter Substances. Liverpool University Press, 1969Google Scholar] with the surface membrane, formed the basis of the 'calcium hypothesis' of transmitter release gating. The essential link between influx of Ca2+ through the channel and its subsequent binding to the SV sensor was not discussed in Katz's monograph and is the subject of this review. Several previous reviews [6Eggermann E. et al.Nanodomain coupling between Ca2+ channels and sensors of exocytosis at fast mammalian synapses.Nat. Rev. Neurosci. 2011; 13: 7-21Crossref PubMed Scopus (312) Google Scholar, 7Meinrenken C.J. et al.Local routes revisited: the space and time dependence of the Ca2+ signal for phasic transmitter release at the rat calyx of Held.J. Physiol. 2003; 547: 665-689PubMed Google Scholar, 8Stanley E.F. The calcium channel and the organization of the presynaptic transmitter release face.Trends Neurosci. 1997; 20: 404-409Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 9Augustine G.J. How does calcium trigger neurotransmitter release?.Curr. Opin. Neurobiol. 2001; 11: 320-326Crossref PubMed Scopus (311) Google Scholar] provide additional perspectives on this subject. Because the vast majority of presynaptic terminals are small and inaccessible, progress in this field has relied heavily on a relatively few exemplar model synapses and the application of remarkably innovative experimental assay methods. The SGS (Figure 1A) was the first synapse at which it was possible to record directly from the presynaptic terminal [10Bullock T.H. Properties of a single synapse in the stellate ganglion of squid.J. Gen. Physiol. 1948; 11: 343-364Google Scholar]. Application of a two- or three-electrode voltage clamp (Figure 1A, lower panel) [2Llinas R.R. et al.Presynaptic calcium currents and their relation to synaptic transmission: voltage clamp study in squid giant synapse and theoretical model for the calcium gate.Proc. Natl. Acad. Sci. U.S.A. 1976; 73: 2918-2922Crossref PubMed Scopus (212) Google Scholar, 11Llinas R.R. et al.Presynaptic calcium currents in squid giant synapse.Biophys. J. 1981; 33: 289-321Abstract Full Text PDF PubMed Scopus (217) Google Scholar] heralded the modern era in synaptic research, relating presynaptic inward Ca2+ current to transmitter output. Three findings at the SGS were of particular significance with respect to release gating. First, a minimum latency of 0.2 ms between Ca2+ influx through the CaVs and transmitter release [12Llinas R.R. et al.Relationship between presynaptic calcium current and postsynaptic potential in squid giant synapse.Biophys. J. 1981; 33: 323-351Abstract Full Text PDF PubMed Scopus (496) Google Scholar] implied that at least some CaVs must lie within ∼100 nm from the SV sensors. Second, the demonstration that, typically, transmitter release was maximal during the repolarization phase of the action potential [13Llinas R.R. et al.Transmission by presynaptic spike-like depolarization in the squid giant synapse.Proc. Natl. Acad. Sci. U.S.A. 1982; 79: 2415-2419Crossref PubMed Scopus (148) Google Scholar] permitted calculation of realistic Ca2+ influx rates; and third, the finding by the Charlton group that fast- but not slow-binding intracellular Ca2+ scavengers block release [14Adler E.M. et al.Alien intracellular calcium chelators attenuate neurotransmitter release at the squid giant synapse.J. Neurosci. 1991; 11: 1496-1507Crossref PubMed Google Scholar] strengthened the argument that the SV sensors are located close to the CaV pores. Applying the patch clamp method to a calyx-type synapse isolated from the chick ciliary ganglion (Figure 1B, upper-left panel) provided the first opportunity to explore presynaptic Ca2+ currents [80Stanley E.F. Calcium currents in a vertebrate presynaptic nerve terminal: the chick ciliary ganglion calyx.Brain Res. 1989; 505: 341-345Crossref PubMed Scopus (43) Google Scholar, 81Stanley E.F. Goping G. Characterization of a calcium current in a vertebrate cholinergic presynaptic nerve terminal.J. Neurosci. 1991; 11: 985-993Crossref PubMed Google Scholar, 86Stanley E.F. Atrakchi A.H. Calcium currents recorded from a vertebrate presynaptic nerve terminal are resistant to the dihydropyridine nifedipine.Proc. Natl. Acad. Sci. U.S.A. 1990; 87: 9683-9687Crossref PubMed Scopus (49) Google Scholar] in vertebrates. Later these were correlated with transmitter release (Figure 1B, upper-right panel) [15Gentile L. Stanley E.F. A unified model of presynaptic release site gating by calcium channel domains.Eur. J. Neurosci. 2005; 21: 278-282Crossref PubMed Scopus (43) Google Scholar]. The chick CC came into its own as an experimental preparation with the application of the cell attached patch clamp recording method to the exposed presynaptic transmitter-release face (Figure 1B, lower-left panel) and the recording of single release site-associated CaVs [16Stanley E.F. Single calcium channels on a cholinergic presynaptic nerve terminal.Neuron. 1991; 7: 585-591Abstract Full Text PDF PubMed Scopus (76) Google Scholar]. The addition of a luminescent assay for transmitter release to this method permitted the direct correlation of single presynaptic CaV channel activity with SV fusion and discharge (Figure 1B, lower-right panel) [17Stanley E.F. Single calcium channels and acetylcholine release at a presynaptic nerve terminal.Neuron. 1993; 11: 1007-1011Abstract Full Text PDF PubMed Scopus (218) Google Scholar]. Nanometer-resolution observations on the active zone of the frog NMJ have provided key structural details of resting and actively secreting synapses. Belt-like active zones exhibit parallel pairs of transmembrane particles that are believed to include CaVs [18Pumplin D.W. Reese T.S. Membrane ultrastructure of the giant synapse of the squid.Neuroscience. 1978; 3: 685-696Crossref PubMed Scopus (24) Google Scholar, 19Heuser J.E. et al.Functional changes in frog neuromuscular junctions studied with freeze fracture.J. Neurocytol. 1974; 3: 109-131Crossref PubMed Scopus (360) Google Scholar, 20Robitaille R. et al.Strategic location of calcium channels at transmitter release sites of frog neuromuscular synapses.Neuron. 1990; 5: 773-779Abstract Full Text PDF PubMed Scopus (311) Google Scholar]; SV fusion profiles were observed abutting these particle rows [19Heuser J.E. et al.Functional changes in frog neuromuscular junctions studied with freeze fracture.J. Neurocytol. 1974; 3: 109-131Crossref PubMed Scopus (360) Google Scholar, 21Heuser J.E. et al.Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release.J. Cell Biol. 1979; 81: 275-300Crossref PubMed Scopus (1013) Google Scholar] (see [8Stanley E.F. The calcium channel and the organization of the presynaptic transmitter release face.Trends Neurosci. 1997; 20: 404-409Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar]) (Figure 1C, upper-left panel). Electron microscopy (EM) tomography revealed an associated submembrane scaffold (Figure 1C, upper-right panel) while a quantitative analysis of particle distributions in the freeze-fracture replicas argues that this scaffold contracts during fusion (Figure 1C, lower-left panel). With respect to release gating, the frog NMJ has had two important resurgences, the first associated with selective and irreversible toxin block of the CaVs [22Kerr L.M. Yoshikami D. A venom peptide with a novel presynaptic blocking action.Nature. 1984; 308: 282-284Crossref PubMed Scopus (287) Google Scholar, 23Yoshikami D. et al.The inhibitory effects of omega-conotoxins on Ca channel and synapses.Ann. N. Y. Acad. Sci. 1989; 560: 230-248Crossref PubMed Scopus (114) Google Scholar] and, more recently, the imaging of Ca2+ entry through individual channels (Figure 1C, lower-right panel) [24Luo F. et al.Transmitter release is evoked with low probability predominately by calcium flux through single channel openings at the frog neuromuscular junction.J. Neurophysiol. 2015; 113: 2480-2489Crossref PubMed Scopus (20) Google Scholar, 25Wachman E.S. et al.Spatial distribution of calcium entry evoked by single action potentials within the presynaptic active zone.J. Neurosci. 2004; 24: 2877-2885Crossref PubMed Scopus (56) Google Scholar]. The nCoH (Figure 1D, upper panels) in the auditory pathway of newborn rodents, another calyx-type synapse, stimulated a new wave of transmitter release research because of the relative ease of simultaneous pre- and postsynaptic patch clamp recording [26Borst J.G. et al.Pre- and postsynaptic whole-cell recordings in the medial nucleus of the trapezoid body of the rat.J. Physiol. 1995; 489: 825-840Crossref PubMed Scopus (359) Google Scholar, 27Takahashi T. et al.Presynaptic calcium current modulation by a metabotropic glutamate receptor.Science. 1996; 274: 594-597Crossref PubMed Scopus (342) Google Scholar]. Key discoveries included new perspectives on presynaptic input–output relations [7Meinrenken C.J. et al.Local routes revisited: the space and time dependence of the Ca2+ signal for phasic transmitter release at the rat calyx of Held.J. Physiol. 2003; 547: 665-689PubMed Google Scholar] and exploration of the SV sensor binding affinity (Figure 1D, upper-right panel) and kinetics [28Bollmann J.H. et al.Calcium sensitivity of glutamate release in a calyx-type terminal.Science. 2000; 289: 953-957Crossref PubMed Scopus (389) Google Scholar, 29Schneggenburger R. Neher E. Intracellular calcium dependence of transmitter release rates at a fast central synapse.Nature. 2000; 406: 889-893Crossref PubMed Scopus (568) Google Scholar]. The degree to which the findings at the nCoH could serve as a general model of synaptic transmission came into question when it became apparent that there is an abrupt change in release gating physiology during development (Figure 1D, lower-right panel) [30Fedchyshyn M.J. Wang L.Y. Developmental transformation of the release modality at the calyx of Held synapse.J. Neurosci. 2005; 25: 4131-4140Crossref PubMed Scopus (210) Google Scholar]. This functional switch coincided with the onset of hearing [31Hoffpauir B.K. et al.Synaptogenesis of the calyx of Held: rapid onset of function and one-to-one morphological innervation.J. Neurosci. 2006; 26: 5511-5523Crossref PubMed Scopus (95) Google Scholar], increased fenestration of the calyx (Figure 1D, lower-left panel), and high-frequency discharges [32Ford M.C. et al.Fenestration of the calyx of Held occurs sequentially along the tonotopic axis, is influenced by afferent activity, and facilitates glutamate clearance.J. Comp. Neurol. 2009; 514: 92-106Crossref PubMed Scopus (58) Google Scholar], suggesting that the nCoH was an intermediary stage in the development of the mature CoH. The evolution in our understanding of transmitter release physiology could serve as a textbook example of the cycle between conceptual models and their experimental scrutiny [33Popper K.R. Conjectures and Refutations: The Growth of Scientific Knowledge. Routledge, 1963Google Scholar]. We start here with a model in which the CaVs are distributed randomly across the presynaptic surface membrane and SV fusion is gated by the entering Ca2+ that fills the nerve terminal. This view, which we term the 'gross entry' model (Figure 2A), corresponds approximately to the state of the field at the time of Katz's monograph [5Katz B. The Release of Neural Transmitter Substances. Liverpool University Press, 1969Google Scholar]. However, attempts to create a mathematical model of synaptic transmission based on work at the SGS led to the realization that that not only must there be an abrupt entry of Ca2+ to initiate transmitter release; the ion must then be removed rapidly for release to also terminate abruptly [34Zucker R.S. Stockbridge N. Presynaptic calcium diffusion and the time courses of transmitter release and synaptic facilitation at the squid giant synapse.J. Neurosci. 1983; 3: 1263-1269Crossref PubMed Google Scholar]. In the ensuing 'radial diffusion' model (Figure 2B), Ca2+ removal from the release site was achieved by linear diffusion of the ion toward the nerve terminal interior combined with its sequestration by cytoplasmic scavengers [34Zucker R.S. Stockbridge N. Presynaptic calcium diffusion and the time courses of transmitter release and synaptic facilitation at the squid giant synapse.J. Neurosci. 1983; 3: 1263-1269Crossref PubMed Google Scholar]. An alternative to the radial diffusion model became necessary with the realization that although this model could account for Ca2+ dynamics during a single impulse, it could not do so for a high-frequency impulse train [35Parnas H. et al.Neurotransmitter release and it facilitation in the crayfish. VII. Another voltage dependent process besides Ca-entry controls time course of phasic release.Pflugers Arch. 1986; 406: 121-130Crossref PubMed Scopus (71) Google Scholar, 36Fogelson A.L. Zucker R.S. Presynaptic calcium diffusion from various arrays of single channels.Biophys. J. 1985; 48: 1003-1007Abstract Full Text PDF PubMed Scopus (180) Google Scholar]. The suggestion that Ca2+ gradients might occur not only radially into the terminal but also laterally along the membrane surface first appeared in a mathematical model of release [12Llinas R.R. et al.Relationship between presynaptic calcium current and postsynaptic potential in squid giant synapse.Biophys. J. 1981; 33: 323-351Abstract Full Text PDF PubMed Scopus (496) Google Scholar] and heralded the modern era of release site gating physiology. The concept of the microdomain has its roots in the realization that a single open CaV generates a discrete plume of Ca2+ centered on its pore [37Chad J.E. Eckert R. Calcium domains associated with individual channels can account for anomalous voltage relations of Ca-dependent responses.Biophys. J. 1984; 45: 993-999Abstract Full Text PDF PubMed Scopus (222) Google Scholar], now termed a nanodomain. This seminal concept served as the basis for the microdomain hypothesis (Figure 2C) in which the nanodomains from a tight cluster of individual CaVs overlapped and activated the SV sensors of nearby docked SVs (hence, overlapping domain gating) [36Fogelson A.L. Zucker R.S. Presynaptic calcium diffusion from various arrays of single channels.Biophys. J. 1985; 48: 1003-1007Abstract Full Text PDF PubMed Scopus (180) Google Scholar, 38Simon S.M. Llinas R.R. Compartmentalization of the submembrane calcium activity during calcium influx and its significance in transmitter release.Biophys. J. 1985; 48: 485-498Abstract Full Text PDF PubMed Scopus (443) Google Scholar, 39Zucker R.S. Fogelson A.L. Relationship between transmitter release and presynaptic calcium influx when calcium enters through discrete channels.Proc. Natl. Acad. Sci. U.S.A. 1986; 83: 3032-3036Crossref PubMed Scopus (144) Google Scholar, 40Llinas R. et al.Microdomains of high calcium concentration in a presynaptic terminal.Science. 1992; 256: 677-679Crossref PubMed Scopus (705) Google Scholar]. The microdomain concept in essence solved the issue of rapid Ca2+ clearance and survives as a model to explain release gating at least at some synapses [7Meinrenken C.J. et al.Local routes revisited: the space and time dependence of the Ca2+ signal for phasic transmitter release at the rat calyx of Held.J. Physiol. 2003; 547: 665-689PubMed Google Scholar, 41Zucker R.S. Calcium and transmitter release.J. Physiol. Paris. 1993; 87: 25-36Crossref PubMed Scopus (108) Google Scholar, 42Lisman J.E. et al.The sequence of events that underlie quantal transmission at central glutamatergic synapses.Nat. Rev. Neurosci. 2007; 8: 597-609Crossref PubMed Scopus (184) Google Scholar]. Since the microdomain model could account for release physiology, a study using the newly discovered irreversible CaV2.2 blocker ɷ-conotoxin GVIA that proposed a far more intimate release gating mechanism was met with some skepticism. Based on the principal of a parallel relationship between transmitter release and the number of active CaVs (Box 1), it was proposed that a single CaV could gate SV fusion if it was sufficiently close for its Ca2+ nanodomain to saturate all of the Ca2+-binding sites on the SV sensor (Figures 2D and 3C) [23Yoshikami D. et al.The inhibitory effects of omega-conotoxins on Ca channel and synapses.Ann. N. Y. Acad. Sci. 1989; 560: 230-248Crossref PubMed Scopus (114) Google Scholar]. Some support for this 'single domain' hypothesis was provided soon after at the SGS when the modeled Ca2+ current associated with action potential broadening during potassium channel block was related to transmitter output [43Augustine G.J. Regulation of transmitter release at the squid giant synapse by presynaptic delayed rectifier potassium current.J. Physiol. 1990; 431: 343-364Crossref PubMed Scopus (177) Google Scholar, 44Augustine G.J. et al.The calcium signal for transmitter secretion from presynaptic nerve terminals.Ann. N. Y. Acad. Sci. 1991; 635: 365-381Crossref PubMed Scopus (220) Google Scholar, 45Zucker R.S. et al.Presynaptic calcium in transmitter release and posttetanic potentiation.Ann. N. Y. Acad. Sci. 1991; 635: 191-207Crossref PubMed Scopus (75) Google Scholar].Box 1The CaV Titration Domain Gating TestThe principle of this test is to vary the number of activated CaVs (hence, titrate) and correlate this with the number of released SVs [39Zucker R.S. Fogelson A.L. Relationship between transmitter release and presynaptic calcium influx when calcium enters through discrete channels.Proc. Natl. Acad. Sci. U.S.A. 1986; 83: 3032-3036Crossref PubMed Scopus (144) Google Scholar, 85Matveev V. et al.Ca2+ current versus Ca2+ channel cooperativity of exocytosis.J. Neurosci. 2009; 29: 12196-12209Crossref PubMed Scopus (20) Google Scholar]. Multiple Ca2+ ions (n) must bind to activate the SV sensor. For simplicity we use n = 4 in Box 1 and Box 2 but n = 5 in most recently published models [28Bollmann J.H. et al.Calcium sensitivity of glutamate release in a calyx-type terminal.Science. 2000; 289: 953-957Crossref PubMed Scopus (389) Google Scholar, 29Schneggenburger R. Neher E. Intracellular calcium dependence of transmitter release rates at a fast central synapse.Nature. 2000; 406: 889-893Crossref PubMed Scopus (568) Google Scholar, 36Fogelson A.L. Zucker R.S. Presynaptic calcium diffusion from various arrays of single channels.Biophys. J. 1985; 48: 1003-1007Abstract Full Text PDF PubMed Scopus (180) Google Scholar]. Where CaVs and SVs are remote from each other, the SV sensor can be activated only by a pooled microdomain from many channels (Figure IA). With recruitment of CaVs (top to bottom panels), the microdomain amplitude increases monotonically and the ion binds to a larger fraction of the SV sensor binding sites. However, the probability that all four sites on the same SV sensor will be occupied shows a curved relationship (Figure IA,C) and is described by [CaV]N. Hence, in our illustrated example N = 4. Thus, for pure microdomain gating the experimentally determined N value is equal to (but cannot be greater) than n. However, if each SV sensor is located within the nanodomain of an individual CaV (Figure IB), all four binding sites can be saturated when each channel opens. It follows, then, that as more channels are recruited there is a parallel, or linear, increase in the number of activated SV sensors (Figure IB,C) and N = 1. The simplest method to analyze CaV titration is to plot the quantity of transmitter released against the number of open channels using double-logarithmic coordinates (Figure ID). The slope of a fitted unbroken line is a direct measure of the N value.There are two main CaV titration assay variants. In the first, CaV recruitment is controlled by voltage clamp (CaV-TV). The protocol with the fewest ambiguities (avoiding confounding effects due to changes in membrane potential and hence Ca2+ driving force) is to trigger release using a family of Ca2+ tail currents generated by pulses of varying duration that depolarize the membrane to beyond the Ca2+ reversal potential (Figure 1B in main text, top-right panel). The amplitude of these tail currents is linearly related to the number of channels recruited and can then be plotted directly against the quantity of transmitter release [15Gentile L. Stanley E.F. A unified model of presynaptic release site gating by calcium channel domains.Eur. J. Neurosci. 2005; 21: 278-282Crossref PubMed Scopus (43) Google Scholar].The alternative method (CaV-TB) is to trigger release with an action potential while gradually blocking the channels [23Yoshikami D. et al.The inhibitory effects of omega-conotoxins on Ca channel and synapses.Ann. N. Y. Acad. Sci. 1989; 560: 230-248Crossref PubMed Scopus (114) Google Scholar] using either an irreversible blocker such as ɷ-conotoxin GVIA (CaV2.2) or ɷ-agatoxin GIVA (CaV2.1) or a pore-blocking cation with a relatively slow binding off rate. This method serves as a useful Ca titration method for synapses with small or dispersed terminals that are inaccessible to direct voltage clamp analysis. The principle of this test is to vary the number of activated CaVs (hence, titrate) and correlate this with the number of released SVs [39Zucker R.S. Fogelson A.L. Relationship between transmitter release and presynaptic calcium influx when calcium enters through discrete channels.Proc. Natl. Acad. Sci. U.S.A. 1986; 83: 3032-3036Crossref PubMed Scopus (144) Google Scholar, 85Matveev V. et al.Ca2+ current versus Ca2+ channel cooperativity of exocytosis.J. Neurosci. 2009; 29: 12196-12209Crossref PubMed Scopus (20) Google Scholar]. Multiple Ca2+ ions (n) must bind to activate the SV sensor. For simplicity we use n = 4 in Box 1 and Box 2 but n = 5 in most recently published models [28Bollmann J.H. et al.Calcium sensitivity of glutamate release in a calyx-type terminal.Science. 2000; 289: 953-957Crossref PubMed Scopus (389) Google Scholar, 29Schneggenburger R. Neher E. Intracellular calcium dependence of transmitter release rates at a fast central synapse.Nature. 2000; 406: 889-893Crossref PubMed Scopus (568) Google Scholar, 36Fogelson A.L. Zucker R.S. Presynaptic calcium diffusion from various arrays of single channels.Biophys. J. 1985; 48: 1003-1007Abstract Full Text PDF PubMed Scopus (180) Google Scholar]. Where CaVs and SVs are remote from each other, the SV sensor can be activated only by a pooled microdomain from many channels (Figure IA). With recruitment of CaVs (top to bottom panels), the microdomain amplitude increases monotonically and the ion binds to a larger fraction of the SV sensor binding sites. However, the probability that all four sites on the same SV sensor will be occupied shows a curved relationship (Figure IA,C) and is described by [CaV]N. Hence, in our illustrated example N = 4. Thus, for pure microdomain gating the experimentally determined N value is equal to (but cannot be greater) than n. However, if each SV sensor is located within the nanodomain of an individual CaV (Figure IB), all four binding sites can be saturated when each channel opens. It follows, then, that as more channels are recruited there is a parallel, or linear, increase in the number of activated SV sensors (Figure IB,C) and N = 1. The simplest method to analyze CaV titration is to plot the quantity of transmitter released against the number of open channels using double-logarithmic coordinates (Figure ID). The slope of a fitted unbroken line is a direct measure of the N value. There are two main CaV titration assay variants. In the first, CaV recruitment is controlled by voltage clamp (CaV-TV). The protocol with the fewest ambiguities (avoiding confounding effects due to changes in membrane potential and hence Ca2+ driving force) is to trigger release using a family of Ca2+ tail currents generated by pulses of varying duration that depolarize the membrane to beyond the Ca2+ reversal potential (Figure 1B in main text, top-right panel). The amplitude of these tail currents is linearly related to the number of channels recruited and can then be plotted directly against the quantity of transmitter release [15Gentile L. Stanley E.F. A unified model of presynaptic release site gating by calcium channel domains.Eur. J. Neurosci. 2005; 21: 278-282Crossref PubMed Scopus (43) Google Scholar]. The alternative method (CaV-TB) is to trigger release with an action potential while gradually blocking the channels [23Yoshikami D. et al.The inhibitory effects of omega-conotoxins on Ca channel and synapses.Ann. N. Y. Acad. Sci. 1989; 560: 230-248Crossref PubMed Scopus (114) Google Scholar] using either an irreversible blocker such as ɷ-conotoxin GVIA (CaV2.2) or ɷ-agatoxin GIVA (CaV2.1) or a pore-blocking cation with a relatively slow binding off rate. This method serves as a useful Ca titration method for synapses with small or dispersed terminals that are inaccessible to di