Presynaptic Calcium Channels

Presynaptic Ca2+ entry occurs through voltage-gated Ca2+ (CaV) channels which are activated by membrane depolarization. Depolarization accompanies neuronal firing and elevation of Ca2+ triggers neurotransmitter release from synaptic vesicles. For synchronization of efficient neurotransmitter release, synaptic vesicles are targeted by presynaptic Ca2+ channels forming a large signaling complex in the active zone. The presynaptic CaV2 channel gene family (comprising CaV2.1, CaV2.2, and CaV2.3 isoforms) encode the pore-forming α1 subunit. The cytoplasmic regions are responsible for channel modulation by interacting with regulatory proteins. This article overviews modulation of the activity of CaV2.1 and CaV2.2 channels in the control of synaptic strength and presynaptic plasticity.


Introduction
Presynaptic Ca 2+ entry into the active zone (AZ) occurs through voltage-gated Ca 2+ (Ca V ) channels which are activated membrane depolarization and triggers synchronous neurotransmitter release from synaptic vesicles (SVs). Multiple mechanisms regulate the function of presynaptic Ca 2+ channels [1][2][3][4]. The channel activity for opening, closing, or inactivation in response to membrane depolarization changes every few milliseconds during and after neuronal firing, resulting in control of synaptic strength [3,4]. Following a brief overview of Ca 2+ channel structure/function, this article reviews the molecular and cellular mechanisms that modulate the activity of presynaptic Ca 2+ channels in the regulation of neurotransmitter release and in the induction of short-term synaptic plasticity. To understand the physiological role of Ca 2+ channel modulation in the regulation of synaptic transmission, a model synapse formed between sympathetic, superior cervical ganglion (SCG) neurons in culture was employed for functional study of channel interaction with G proteins, SNARE proteins, and Ca 2+ -binding proteins which sense residual Ca 2+ in the AZ after the arrival of an action potential (AP).

Presynaptic Ca 2+ Channels
Ca 2+ currents have diverse physiological roles and different pharmacological properties. Early investigations revealed distinct classes of Ca 2+ currents which were identified with an alphabetical nomenclature [5]. P/Q-type, N-type, and R-type Ca 2+ currents are observed primarily in neurons, require strong depolarization for activation [6], and are blocked by specific polypeptide toxins from snail and spider venoms [7]. P/Q-type and N-type Ca 2+ currents initiate neurotransmitter release at most fast synapses [1,8,9]. The Ca 2+ channels are composed of four or five distinct subunits ( Figure 1a) [8,10]. The α1 subunit incorporates the conduction pore, the voltage sensors and gating apparatus, and target sites of toxins and intracellular regulators. The α1 subunit is composed of about 2000 amino acid residues and is organized in four homologous domains (I-IV) (Figure 1b). Each domain consists of six transmembrane α helices (S1 through S6) and a membrane-associated P

Active Zone Proteins
Rab-interacting molecule (RIM), an AZ protein required for SVs docking and priming [43][44][45][46][47][48], and synaptic plasticity [49], interacts with the C-terminal cytoplasmic tails of Ca V 2.1 and Ca V 2.2 channels [46,48,50,51] (Figure 1b). The interaction is essential for recruiting Ca 2+ channels to the presynaptic AZ [46] and determines channel density and SVs docking at the presynaptic AZ [48]. RIM-binding proteins, RIM-BPs, also interact with Ca V 2.1 and Ca V 2.2 channels [51], and are selectively required for high-fidelity coupling of AP-induced Ca 2+ influx to Ca 2+ -stimulated SVs exocytosis [52]. The tripartite complex of RIM, RIM-BPs, and C-terminal tails of the Ca V 2 channels regulate the recruitment of Ca V 2 channels to AZs. Interaction of RIM with Ca V β subunits shifts the voltage dependence of inactivation to more positive membrane potentials, increasing Ca 2+ channel activity [53]. In contrast, Ca V β subunits interaction with CAST/ERC2 shifts the voltage dependence of activation to more negative membrane potentials [54]. Positive regulation of presynaptic Ca 2+ channel activity by RIM and CAST/ERC2, in addition to their function in SVs docking, increase the release probability of SVs docked close to Ca V 2 channels. Furthermore, Munc13, required for SVs priming, controls Ca V 2 channels shortly after AP firing to guarantee transmitter release for continuous neural activity [55].

t-SNAREs and Synaptotagmin-1
SV (v)-SNARE synaptobrevin 2 and presynaptic plasma membrane (t)-SNAREs syntaxin-1 and SNAP-25 are required for fusion of SVs with a plasma membrane to release neurotransmitters [56]. Both Ca V 2.1 and Ca V 2.2 channels at the presynaptic nerve terminals colocalize densely with syntaxin-1A [57][58][59], and also form a complex of with SNARE proteins [60][61][62] dependent on Ca 2+ with maximal binding at 20 µM and reduced binding at lower or higher concentrations of Ca 2+ [63]. The t-SNARE proteins syntaxin-1A and SNAP-25, but not the v-SNARE synaptobrevin, bind to the intracellular loop between domains II and III of the α 1 subunit of Ca V 2.2 (amino acid residues 718-963) named as the synprint site ( Figure 1b) [64,65]. Ca V 2.1 channels have an analogous synprint site, and different channel isoforms have distinct interactions with syntaxin and SNAP-25 [66,67], suggesting specialized regulatory properties for synaptic modulation.
t-SNAREs interacting with presynaptic Ca V 2.1 and Ca V 2.2 channels regulate channel activity ( Figure 3a). Syntaxin-1A or SNAP-25 shifts the voltage dependence of inactivation toward more negative membrane potentials and reduces the availability of the channels to open [68][69][70]. Coexpression of SNAP-25 can reverse the inhibitory effects of syntaxin-1A [69,71]. The transmembrane region of syntaxin-1A and only a short segment within the H3 helix are critical for channel modulation [72], whereas the synprint site binds to the entire H3 helix in the cytoplasmic domain of syntaxin-1A [63,64,72]. Deletion of the synprint site weakened the modulation of the channels by syntaxin-1A, but did not abolish it, arguing that the synprint site acts as an anchor in facilitating channel modulation but is not required absolutely for modulatory action.
Interaction of syntaxin-1A and SNAP-25 with the synprint site is controlled by phosphorylation of the synprint site with protein kinase C (PKC) (Figure 1b) [65] and Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) [77]. The negative shift of steady-state inactivation of Ca V 2.2 channels caused by syntaxin is blocked by PKC phosphorylation [65,71]. Thus, phosphorylation of the synprint site may serve as a biochemical switch controlling the SNARE-synprint interaction.

Negative Regulation of Neurotransmitter Release by Gβγ protein/Ca V 2 Channel Complex
Receptor-activated Gβγ modulation of presynaptic Ca V 2 channels is a potent negative regulation of neurotransmitter release. Electrophysiological recordings of Ca 2+ currents and synaptic transmission at the calyx of Held demonstrated this type of negative regulation by activation of GABA-B receptors or metabotropic glutamate receptors [91,92]. Optical measurements of Ca 2+ transients at the nerve terminals of the parallel fibers of cerebellar granule cells innervating Purkinje neurons has also demonstrated similar modulation by activation of CB1 receptors [93]. This Gβγ-mediated inhibition of Ca 2+ channels is relieved by depolarization. At autapses formed by single hippocampal pyramidal neurons, trains of AP-like stimuli relieve the inhibition of synaptic transmission caused by activation of GABA-B receptors, resulted in facilitation of synaptic transmission, which was blocked by inhibition of Ca V 2.1 channels with neurotoxins [94]. Thus, presynaptic firing could reverse the neurotransmitter-mediated G protein inhibition of synaptic transmission. Regulator of G protein signaling-2 (RGS-2), which speeds GTPase activity of the α subunit of the activated G protein α-GTP, determines short-term plasticity in hippocampal neurons by regulating Gi/o-mediated inhibition of presynaptic Ca 2+ channels. RGS-2 relieves the inhibition, resulting in a higher basal probability of release and synaptic facilitation [95]. However, at parallel fibers synapses onto Purkinje cells, this form of facilitation is not responsible for short-term synaptic plasticity [96].
In SCG neurons noradrenaline shortens AP duration by reducing Ca 2+ entry through Ca V 2.2 channels, resulting in a reduction of transmitter release [97]. Purified Gβγ microinjected into presynaptic SCG neurons in culture reduced synaptic transmission, and the Gβγ introduced neurons caused no further reduction of synaptic transmission with noradrenaline [97]. Thus, Gβγ is a potent negative regulator of neurotransmission inhibiting presynaptic Ca V 2.2 channels activity. The α1 subunit contains several Gβγ interaction sites, including the amino-terminal (NT) and I-II loop (Figure 1b). The "NT peptide" and an I-II loop α interaction domain "AID peptide" microinjected into presynaptic SCG neurons under long-term culture attenuated noradrenaline-induced G protein modulation ( Figure 2) and inhibited synaptic transmission [98]. In acutely dissociated SCG neurons, NT and AID peptides reduced whole-cell Ba 2+ current amplitude, modified voltage dependence of Ca 2+ channel activation, and attenuated noradrenaline-induced G protein modulation ( Figure 2) [98]. Co-application of NT and AID peptide negated inhibitory actions. Furthermore, a mutation within NT abolished inhibitory effects of the NT peptide [98]. Effects of Ca V 2.2 channel peptides demonstrate that the Ca V 2.2 amino-terminal and I-II loop serve as molecular determinants for Ca 2+ channel function to inhibit synaptic transmission and to attenuate G protein modulation. mediated G protein inhibition of synaptic transmission. Regulator of G protein signaling-2 (RGS-2), which speeds GTPase activity of the α subunit of the activated G protein α-GTP, determines short-term plasticity in hippocampal neurons by regulating Gi/o-mediated inhibition of presynaptic Ca 2+ channels. RGS-2 relieves the inhibition, resulting in a higher basal probability of release and synaptic facilitation [95]. However, at parallel fibers synapses onto Purkinje cells, this form of facilitation is not responsible for short-term synaptic plasticity [96].  Figure 1b). Ba 2+ currents were recorded from superior cervical ganglion (SCG) neurons acutely dissociated from 3-to 6-week-old Wistar rats, while the synaptic transmission was recorded from long-term cultured SCG neurons isolated from p7 rat. NA was bath-applied 30 min after injection of the peptide at 1 mM in the injection pipette. EPSPs were normalized to amplitude prior to NA application at time = 0 min. Bar graph summarizing NA effects, *p < 0.05 vs. NA effects in controls (Student's t-test). Adapted from [97].
In SCG neurons noradrenaline shortens AP duration by reducing Ca 2+ entry through CaV2.2 channels, resulting in a reduction of transmitter release [98]. Purified Gβγ microinjected into . Ba 2+ currents were recorded from superior cervical ganglion (SCG) neurons acutely dissociated from 3-to 6-week-old Wistar rats, while the synaptic transmission was recorded from long-term cultured SCG neurons isolated from p7 rat. NA was bath-applied 30 min after injection of the peptide at 1 mM in the injection pipette. EPSPs were normalized to amplitude prior to NA application at time = 0 min. Bar graph summarizing NA effects, *p < 0.05 vs. NA effects in controls (Student's t-test). Adapted from [98].

Synchronous Neurotransmitter Release Regulated by Ca 2+ Channel/SNARE Proteins Complex
Synprint peptides derived from Ca V 2.2 channels reduced transmitter release from the microinjected presynaptic SCG neurons in culture, due to competitive uncoupling of the endogenous Ca 2+ channel-SNARE proteins interaction in nerve terminals [99]. Synprint peptides selectively inhibited fast synchronous synaptic transmission, while they increased late asynchronous release ( Figure 3b). Similarly, synprint peptides reduced transmitter release from embryonic Xenopus spinal neurons [100]. Increasing the external Ca 2+ concentration effectively rescued this inhibition, implying that synprint peptides competitively displaces docked SVs away from Ca 2+ channels, and this effect can be overcome by increasing Ca 2+ influx into presynaptic terminals [100].
At the calyx of Held, presynaptic neurons express P/Q-, N-and R-type Ca 2+ currents in postnatal day 7 rats. P/Q-type Ca 2+ currents are more effective than N-type Ca 2+ currents and R-type Ca 2+ currents in eliciting neurotransmitter release [101][102][103]. The high efficiency of P/Q-type Ca 2+ currents to initiate neurotransmitter release is correlated with the close localization of Ca V 2.1 channels near docked SVs [104], as shown by immunocytochemistry [105], suggesting localization of Ca V 2 channels determines the efficiency of neurotransmitter release in response to neural activity.
Ca V 2 channels interaction with SNARE proteins, that is dependent on Ca 2+ concentration [63], have two opposing effects: at the pre-firing state synaptic transmission is blocked by enhancing Ca V 2 channels inactivation, whereas immediately after AP firing tethering SVs near the point of Ca 2+ entry enhances synaptic transmission. The overexpression of a syntaxin mutant that is unable to regulate Ca V 2.2 channels, but still binds to them [72], increased the efficiency of synaptic transmission at Xenopus neuromuscular junctions, as reflected in increased quantal content [106]. In contrast, injected synprint peptides reduced the basal efficiency of synaptic transmission, as reflected in reduced quantal content of synaptic transmission [106]. These results demonstrate a bidirectional regulation of synaptic transmission in vivo by interactions of Ca V 2.2 channels with SNARE proteins. day 7 rats. P/Q-type Ca 2+ currents are more effective than N-type Ca 2+ currents and R-type Ca 2+ currents in eliciting neurotransmitter release [101][102][103]. The high efficiency of P/Q-type Ca 2+ currents to initiate neurotransmitter release is correlated with the close localization of CaV2.1 channels near docked SVs [104], as shown by immunocytochemistry [105], suggesting localization of CaV2 channels determines the efficiency of neurotransmitter release in response to neural activity. CaV2 channels interaction with SNARE proteins, that is dependent on Ca 2+ concentration [63], have two opposing effects: at the pre-firing state synaptic transmission is blocked by enhancing CaV2 channels inactivation, whereas immediately after AP firing tethering SVs near the point of Ca 2+ entry

Presynaptic Plasticity Induced by Ca 2+ -Sensors-Mediated Ca V 2.1 Channel Modulation
At most fast synapse in the central nervous system, Ca V 2 channels are expressed diversely. In contrast, synaptic transmission of long-term cultured sympathetic SCG neurons, forming a well-characterized cholinergic synapse [107,108], is mediated by Ca V 2.2 channels [109,110]. The physiological role of presynaptic Ca V 2.1 channel modulation by Ca 2+ -sensors was explored by exogenously expressed α1 subunit derived from the brain Ca V 2.1 channel that functionally generates P/Q type currents with other endogenous subunits in SCG neuron [111]. Section 6 describes presynaptic plasticity induced by modulation of the Ca V 2.1 channel that is mediated by CaM or expression of neuron-specific Ca 2+ -sensor proteins, monitoring excitatory postsynaptic potentials (EPSPs) evoked by various patterns of presynaptic APs firing in the presence of the blocker of endogenous Ca V 2.2 channels [109].

Ca 2+ /CaM Mediates Synaptic Depression and Facilitation
Modulation of presynaptic Ca 2+ channels has a powerful influence on synaptic transmission [90]. The cytoplasmic regions of the α1 subunit are the target of regulatory proteins for channel modulation ( Figure 1B). Brain-derived α1 subunit of the Ca V 2.1 channel mediates transmitter release from the transfected SCG neurons [111]. The transmitter release changes after AP firing due to modulation of Ca V 2.1 channel interacting with Ca 2+ bound CaM (Figure 4) [90]. CaM has two Ca 2+ binding sites, N and C robes. The N-robe sensing rapid and higher increase in Ca 2+ concentration [112] initiates synaptic depression, and following facilitation is mediated by the C-robe sensing lower Ca 2+ concentration. EPSPs recorded by pairs of APs with varied stimulation intervals show paired-pulse depression (PPD) and facilitation (PPF) (Figure 4a). PPD with a short interval (<50 ms) was blocked by deletion of the CBD, while PPF with intermediate interval (50-100 ms) was blocked by mutation of the IQ-like motif. Thus, the decline in Ca 2+ elevation after the first AP causes temporal regulation of the Ca V 2.1 channel interacting with CaM, resulting in a change in the transmitter release efficacy (Figure 4b). The time-dependent opposing modulation of the Ca V 2.1 channel activity may support a stable synaptic transmission.
Neural information in vivo is encoded in bursts of AP firing. Short-term presynaptic plasticity caused by APs bursts involves the CaM-dependent regulation of Ca V 2.1 channel. Mutation of the IQ-like motif potentiated reduction of the release efficacy, whereas the deletion of the CBD increased the release efficacy (Figure 4c, IM-AA/∆CBD). Thus, during APs bursts, CaM binding to the CBD controls negatively the release efficacy, whereas CaM binding to IQ-like motif controls it positively. At a higher frequency of APs burst over 20 Hz, the release efficacy of SCG neurons mediated by Ca V 2.1 channels reduced gradually (Figure 4c, WT), suggesting that the CaM-dependent inactivation of Ca V 2.1 channels shapes the time course of short-term synaptic plasticity by determining the timing of the peak of synaptic facilitation during APs bursts as well as the steady-state level of synaptic depression at the end of the APs bursts. dependent opposing modulation of the CaV2.1 channel activity may support a stable synaptic transmission.
Neural information in vivo is encoded in bursts of AP firing. Short-term presynaptic plasticity caused by APs bursts involves the CaM-dependent regulation of CaV2.1 channel. Mutation of the IQlike motif potentiated reduction of the release efficacy, whereas the deletion of the CBD increased the release efficacy (Figure 4c, IM-AA/ΔCBD). Thus, during APs bursts, CaM binding to the CBD controls negatively the release efficacy, whereas CaM binding to IQ-like motif controls it positively. At a higher frequency of APs burst over 20 Hz, the release efficacy of SCG neurons mediated by CaV2.1 channels reduced gradually (Figure 4c, WT), suggesting that the CaM-dependent inactivation of CaV2.1 channels shapes the time course of short-term synaptic plasticity by determining the timing of the peak of synaptic facilitation during APs bursts as well as the steady-state level of synaptic depression at the end of the APs bursts.
Synaptic transmission of SCG neurons transfected with CaBP1 and VILIP-2 changed by their modulation of CaV2.1 channels with binding residual Ca 2+ [113]. APs burst at 10 Hz induces synaptic facilitation followed by synaptic depression due to endogenous CaM. CaBP1 coexpressed with CaV2.1

Neuron-Specific Ca 2+ -Sensor Proteins Mediate Synaptic Depression and Facilitation
CaBP1, VILIP-2, and NCS-1 are members of a subfamily of neuron-specific Ca 2+ -sensor proteins (nCaS) that possess four EF-hand Ca 2+ -binding motifs. CaBP-1, VILIP-2, and NCS-1 bind to the same site as CaM, and modulate Ca V 2.1 channel activity. CaBP1, highly expressed in the brain and retina [114], causes rapid inactivation of Ca V 2.1 channels, binding to the CBD [84]. VILIP-2, highly expressed in the neocortex and hippocampus [115], increases Ca 2+ -dependent facilitation of Ca V 2.1 channels but inhibits Ca 2+ -dependent inactivation of Ca V 2.1 channels, binding to both IQ-like motif and CBD [83]. NCS-1, the classical example of facilitation of synaptic activity by nCaS, reduces Ca 2+ -dependent inactivation of P/Q-type Ca 2+ currents through interaction with the IQ-like motif and CBD without affecting peak current or activation kinetics [85].
Synaptic transmission of SCG neurons transfected with CaBP1 and VILIP-2 changed by their modulation of Ca V 2.1 channels with binding residual Ca 2+ [113]. APs burst at 10 Hz induces synaptic facilitation followed by synaptic depression due to endogenous CaM. CaBP1 coexpressed with Ca V 2.1 channels, significantly reduced the synaptic facilitation and enhanced the synaptic depression (Figure 4d) [113]. In contrast, VILIP-2 coexpressed with Ca V 2.1 reduced the synaptic depression and enhanced the synaptic facilitation (Figure 4d) [113]. CaBP1 and VILIP-2 have opposing effects on short-term synaptic plasticity, either favoring synaptic depression or facilitation, suggesting that nCaS via regulation of presynaptic Ca 2+ channels may play a critical role in determining the diversity of short-term synaptic plasticity at CNS synapses.
The expression of NCS-1 in presynaptic SCG neurons does not affect synaptic transmission, eliminating effects of this nCaS on endogenous N-type Ca 2+ currents [85]. However, in SCG neurons expressing Ca V 2.1 channels, coexpression of NCS-1 induces facilitation of synaptic transmission in response to paired APs and trains of APs, and this effect is lost in Ca V 2.1 channels with mutations in the IQ-like motif and CBD [85]. These results reveal that NCS-1 directly modulates Ca V 2.1 channels to induce short-term synaptic facilitation, and further demonstrate that nCaS are crucial in fine-tuning short-term synaptic plasticity.

Temporal Regulation of Release Efficacy by Ca 2+ -Sensor Proteins
The opening of Ca 2+ channel creates a steep gradient of Ca 2+ elevation in the AZ, where each nCaS has a different affinity and binding speed to Ca 2+ [112]. The affinity is CaM (5-10 µM) > CaBP1 (2.5 µM) >VILIP-2 (~1 µM) [116]. CaM has a lower affinity and a higher binding speed to Ca 2+ than nCaS, suggesting a temporal regulation of Ca V 2.1 channel activity by CaM versus nCaS. Their affinity and binding speed to Ca 2+ determinate timing of the Ca V 2.1 channel modulation. Thus differential effects of CaM and nCaS on facilitation and inactivation of the presynaptic Ca V 2.1 channels would substantially change the encoding of the synaptic properties in response to bursts of APs firing [117].
Time window of the CaM-and nCaS-induced Ca V 2.1 channel modulation after AP firing can be estimated by the paired-pulse protocol applying to SCG neurons transfected with Ca V 2.1 channels. CaM mediated PPD with a short interval (<100 ms), and PPF with intermediate interval (20-100 ms) (Figure 2a). In contrast, NCS-1 induced PPF with a shorter interval (30-50 ms) [85]. CaBP1 induced PPD with interval <150 ms, while VILIP-2 induced PPF with an interval of 50-250 ms [113]. These data suggest that CaM modulates Ca V 2.1 channels shortly after Ca 2+ entry and lasts 100 ms, while NCS-1 acts much shorter and CaBP1 and VILIP-2 actions last longer than CaM effects. The time-dependent action of CaM and nCaS reflects the decline rate of Ca 2+ concentration at the Ca V 2.1 channels after an AP firing. The divergent actions of CaM and nCaS on Ca V 2.1 channels fine-tune the function and regulatory properties of presynaptic P/Q-type Ca 2+ currents, allowing a greater range of input-output relationships and causing various short-term plasticity at different synapses [4].

CaMKII Saves as Effector Checkpoint for Ca 2+ Entry
CaMKII is the most prominent Ca 2+ /CaM-dependent regulator of postsynaptic response [118][119][120][121] and presynaptic function [122][123][124][125]. The autophosphorylated form of CaMKII [7], which does not require the catalytic activity of the enzyme [126], binds to the α1 subunit of Ca V 2.1 channels upstream of the IQ-like motif, and enhances the activity by slowing inactivation and positively shifting the voltage dependence of inactivation [126]. The dephosphorylation of CaMKII does not reverse the binding [127]. The presence of a competing peptide that blocks the interaction of CaMKII with presynaptic Ca V 2.1 channels of SCG neurons prevented both PPD and PPF, suggesting that binding of CaMKII to Ca V 2.1 channels is required for the expression of this regulatory effect. Similarly, the expression of the brain-specific CaMKII inhibitor CaMKIIN [128], which prevents CaMKII binding to Ca V 2.1 channels [126], also prevented PPD and PPF. Thus, the noncatalytic regulation of Ca V 2.1 channels by bound CaMKII controls the activity of those channels that have the effector of the Ca 2+ signal (i.e., CaMKII) in position to bind the entering Ca 2+ and respond to it [126]. SNARE proteins and RIM similarly serve as effectors of the Ca 2+ signal for initiation of SVs exocytosis increasing the activity of the Ca V 2.1 channels by the formation of a complete SNAREs complex with synaptotagmin and RIM bound [53,70]. This "effector checkpoint" mechanism serves to focus Ca 2+ entry through those Ca 2+ channels whose effectors are bound and ready to respond.
Furthermore, autophosphorylated CaMKII bound to Ca V 2.1 channels also binds to synapsin-1, a phosphoprotein of the SVs, increases its phosphorylation and induces oligomers of synapsin-1 [127]. Synapsin-1 is a major presynaptic phosphoprotein that is a prominent substrate for CaMKII, and phosphorylation by CaMKII regulates the effects of synapsin-1 on the trafficking of SVs [129]. The phosphorylation of synapsin-1 by CaMKII increases synaptic transmission at the squid giant synapse [122,123]. Formation of the ternary complex of Ca V 2.1 and synapsin-1 bound to CaMKII would modulate the dynamics of SV function in AZs containing these proteins [127].

Neuronal Firing and Presynaptic Short-Term Plasticity
Neuronal firing regulates presynaptic Ca 2+ channels by Ca 2+ bound CaM and nCaS and causes facilitation and inactivation of neurotransmitter release. The differential expression of these Ca 2+ -dependent regulatory proteins may provide a means of cell-type-specific regulation of presynaptic Ca 2+ channels and short-term synaptic plasticity. The short-term plasticity of neurotransmitter release shapes the postsynaptic response to bursts of impulses and is crucial for the fine-grained encoding of information in the nervous system [117,130].

Presynaptic Short-Term Facilitation
The Calyx of Held, the large presynaptic terminal enabling to record directly presynaptic Ca 2+ current by voltage-clamp methods, suggests that neuronal firing controls P/Q-and N-type currents to modulate differentially synaptic transmission. Presynaptic Ca 2+ current consists of a combination of P/Q-and N-type currents in young mice and shows activity-dependent facilitation that predicts the amount of synaptic facilitation according to the power law [131,132]. Ca V 2.1 knockout lost both facilitation of the presynaptic Ca 2+ current and synaptic facilitation [101,131,132]. The remaining N-type Ca 2+ currents are less efficient in mediating synaptic transmission and do not support facilitation of synaptic transmission, but they are more sensitive to modulation by G protein-coupled receptors [101]. These results suggest that Ca V 2.1 channels are responsible for neuronal activity-dependent synaptic facilitation, while Ca V 2.2 channels have strong G protein regulation.
Presynaptic short APs bursts generate augmentation and longer APs bursts generate post-tetanic potentiation (PTP) relying on residual Ca 2+ . The optical measurement of presynaptic Ca 2+ transients with the induction of PTP in the calyx of Held showed an increase in the Ca 2+ influx to the extent that predicted PTP when the power law of neurotransmission was applied, and the Ca 2+ transient decayed with a time course of the decay of PTP [133]. In Ca V 2.1-transfected SCG neurons, PTP was not significantly affected by mutations at the IQ-like motif [90]. In contrast, PPF and augmentation share a common mechanism involving an increase in instantaneous Ca 2+ entry through Ca V 2.1 channels by CaM-and nCaS-binding in an activity-dependent manner, which in turn facilitates neurotransmitter release. It is likely that facilitation of presynaptic Ca 2+ currents may contribute to short-term facilitation [90,132], and the augmentation and the PTP represent overlapping processes caused by differential combinations of mechanisms at different synapses [130].
The expression of Ca V β subunits has a strong influence on synaptic facilitation in hippocampal synapses through their effects on Ca 2+ channel function [134]. Cavβ2 and Cavβ4 subunits distribute in clusters and localize to synapses. Caβ2 induces depression, whereas Cavβ4 induces PPF followed by synaptic depression during longer stimuli trains. The induction of PPF by Cavβ4 correlates with a reduction in the release probability and cooperativity of the transmitter release. These results suggest that Cavβ subunits determine the gating properties of the presynaptic Ca 2+ channels within the presynaptic terminal in a subunit-specific manner and may be involved in the organization of the Ca 2+ channel relative to the release machinery [134].
The mutation of Ca V 2.1 channels at the IQ-like motif in hippocampal neurons confirmed the mechanism of short-term synaptic facilitation dependent nCaS regulation of Ca V 2.1 channels with brief and local Ca 2+ elevation [135]. In addition, long-term potentiation of synaptic transmission at the Schaffer collateral-CA1 synapse, that is thought to be primarily generated postsynaptically, is substantially weakened by the mutation. Furthermore, the impairments in short-term and long-term plasticity due to Ca V 2.1 channel mutation at the IQ-like motif are associated with pronounced deficits in spatial learning and memory in context-dependent fear conditioning and in the Barnes circular maze. Thus, regulation of Ca V 2.1 channels by CaM and nCaS is required for not only presynaptic facilitation but also induction of postsynaptic long-term potentiation, and spatial learning and memory [136].

Presynaptic Short-Term Depression
At the calyx of Held, presynaptic stimulation at 100 Hz induces robust synaptic depression [88]. Synaptic depression during high-frequency APs bursts in presynaptic neurons is generally thought to be a result of SVs depletion [130]. In a prominent feature of synaptic transmission, the depression is caused by a decrease in release probability [103]. The release probability is determined by docked SVs and Ca 2+ current in the AZ. Presynaptic loading of peptides that disrupt CaM interactions reduced both Ca 2+ -dependent inactivation of the P/Q-type Ca 2+ current and PPD [88]. The Ca 2+ -dependent inactivation of the presynaptic Ca 2+ current, rather than SVs depletion, causes rapid synaptic depression for stimuli ranging from 2 to 30 Hz [87,88].
The transfection of SCG neurons with Ca V 2.1 channels lacking the CBD, a mutation reducing Ca 2+ -dependent inactivation in heterologous expression systems [80,81], blocked PPD, and reduced synaptic depression during APs burst up to 40 Hz [90]. CaBP1 expression, which blocks Ca 2+ -dependent facilitation of P/Q-type Ca 2+ current, induced PPD, and synaptic depression during APs burst. However, the synaptic depression was absent in the presynaptic neuron coexpressed with CaBP1 and Ca V 2.1 channels lacking the CBD [113]. These results further demonstrate that rapid synaptic depression is caused by inactivation of presynaptic Ca V 2.1 channel bound with CaM or CaBP1. During APs burst at 30 Hz and 40 Hz, a slower phase of synaptic depression is likely caused by SVs depletion.
Data from the calyx of Held and Ca V 2.1-transfected SCG neurons suggest a conserved mechanism for generating rapid synaptic depression evoked by physiological rate and duration (at 40 Hz for 1 s) of APs bursts in multiple synapses where neuronal activity elevates presynaptic Ca 2+ transient, and such a Ca 2+ rise dependent binding of nCaS to Ca V 2 channels inactivates presynaptic Ca 2+ channels. Studies of β subunits within cultured hippocampal neurons also support an important role for Ca V 2 channels modulation in synaptic plasticity: the overexpression of Ca V β4 favors facilitation whereas the overexpression of Ca V β2 favors depression [134].

CaMKII Regulates Short-Term Synaptic Plasticity
The binding of CaMKII to Ca V 2.1 channels enhances their functional activity by inhibiting their inactivation [126] and enhances the activity of CaMKII by increasing its autophosphorylation [127]. SCG neurons introduced a competing peptide that blocks the interaction of CaMKII with Ca V 2.1 channels or SCG neurons transfected the brain-specific CaMKII inhibitor CaMKIIN [128] which prevents CaMKII binding to Ca V 2.1 channels [126] prevented not only PPF and PPD but also synaptic depression during APs burst and augmentation after a conditioning APs burst. It is unlikely that the basal release probability is affected by competing for peptide injection or CaMKIIN expression because the mean amplitudes of the first EPSPs are unchanged. Binding of CaMKII to the Ca V 2.1 channel is required for both up-regulation of channel activity in presynaptic facilitation and for Ca 2+ -independent activation of CaMKII by Ca V 2.1, and one or both of these effects is necessary for normal short-term synaptic plasticity.

Ca 2+ -Binding Molecules Regulate Short-Term Synaptic Plasticity
Synaptotagmin-1, 2, and 9 serve as Ca 2+ sensors to mediate the fast synchronous transmitter release as discussed above [56,73,74]. In contrast, synaptotagmin-7 that binds slowly to Ca 2+ via its C 2 A domain [137] is not required for the synchronous synaptic transmission but mediates asynchronous transmitter release [111]. Synaptotagmin-7 is also required for the short-term facilitation, such as PPF and synaptic facilitation during APs burst, at several synapses [138]. Synaptotagmin-7 has a stronger contribution to membrane binding, and perhaps to bridging the vesicle and plasma membranes [111] that may enhance the fast transmitter release in response to repetitive APs firing.
Short-term plasticity may be a combination of the three molecular mechanisms, Ca 2+ channel modulation, synaptotagmin-7 action and Ca 2+ buffering, activated by Ca 2+ elevation with neuronal firing. Ca 2+ channel modulation with CaM and nCaS is a response to millisecond Ca 2+ dynamics. The slower synaptotagmin-7 action integrates local and global Ca 2+ entry, and Ca 2+ buffering may control the spread Ca 2+ accumulation [146].

Conclusions
In response to presynaptic AP firing, Ca 2+ binding proteins triggers SVs exocytosis and regulate the probability. Thus, modulation of presynaptic Ca 2+ channels has a powerful influence on synaptic transmission. At the pre-firing state, Ca 2+ channels activity is inhibited by interaction with AZ proteins. AP firing relieves the inhibition by switching to interact with SNAREs and synaptotagmin, the effectors for Ca 2+ -dependent exocytosis. During and post firing, the activity of the Ca V 2.1 channel is regulated by interaction with CaM and nCaS dependent on individual speed and affinity of binding to residual Ca 2+ . Interacting with CaMKII, the Ca V 2.1 channel increases the binding to CaM and nCaS and their interaction causes short-term facilitation and depression of synaptic transmission. Fine-tuning the function and regulatory properties of presynaptic P/Q-type Ca 2+ currents allow a greater range of input-output relationships and short-term plasticity. In contrast, tonic inhibition of N-type Ca 2+ currents is activated by G-protein coupled-autoreceptors and retrograde signaling receptors.
Acknowledgments: The author expresses her sincere thanks to all the collaborators concerned with the studies described in this review.

Conflicts of Interest:
The authors declare no conflict of interest.