2. Presynaptic Ca²⁺ Channels

Ca²⁺ currents have diverse physiological roles and different pharmacological properties. Early investigations revealed distinct classes of Ca²⁺ currents which were identified with an alphabetical nomenclature [5]. P/Q-type, N-type, and R-type Ca²⁺ 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 Ca²⁺ currents initiate neurotransmitter release at most fast synapses [1,8,9]. The Ca²⁺ 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 loop between S5 and S6. The S1 through S4 segments serve as the voltage sensor module, whereas transmembrane segments S5 and S6 in each domain and the P loop between them form the pore module [11]. The intracellular segments serve as a signaling platform for Ca²⁺-dependent regulation of neurotransmission, as discussed below.

Ca²⁺ channel α1 subunits are encoded by ten distinct genes in mammals, which are divided into three subfamilies by sequence similarity [2,8,13]. The Ca_V2 subfamily members Ca_V2.1, Ca_V2.2, and Ca_V2.3 channels conduct P/Q-type, N-type, and R-type Ca²⁺ currents, respectively [2,8,9,13].

Ca_V channels are complexes of a pore-forming α1 subunit and auxiliary subunits. Skeletal muscle Ca_V channels have three distinct auxiliary protein subunits [8] (Figure 1a), the intracellular β subunit, the disulfide-linked α2δ subunit complex, and the γ subunit having four transmembrane segments. In contrast, brain neuron Ca_V2 channels are composed of the pore-forming α1 and the auxiliary β subunit [14]. The auxiliary subunits of Ca²⁺ channels have an important influence on their function [15,16]. The Ca_Vβ subunit shifts their kinetics and voltage dependence of activation and inactivation [15,16]. Cell surface expression of the α1 subunits is enhanced by the Ca_Vβ subunit [15,16]. The α2δ subunits are potent modulators of synaptic transmission. The α2δ subunits increase not only Ca_v1.2 but also Ca_v2.2, Ca_v2.1 currents, suggesting that the α2δ subunits enhance trafficking of the Ca_V channel complex [17]. Expression of α2δ subunits also appears to play a role in setting release probability [18]. Further details of these regulatory interactions are discussed below.

3. Intracellular Molecules Modulate Presynaptic Ca²⁺ Channels Activity

3.1. G Proteins

Presynaptic Ca²⁺ currents are reduced in magnitude by activation of G protein-coupled receptors for neurotransmitters at nerve terminals [19,20]. Gβγ subunits released from heterotrimeric G proteins of the Gi/Go class [19,20] bind directly to α1 subunits of the N-type Ca²⁺ channel [21,22] at the N terminus [23], the intracellular loop connecting domains I and II [21,24], and at the C terminus [25] (Figure 1b). Gβγ causes a positive shift in the voltage dependence of activation of the Ca²⁺ current [26,27,28]. The Gβγ-induced reduction of Ca²⁺ currents can be reversed by strong positive depolarization [26,27,28]. Reversal of this inhibition by depolarization provides a point of intersection between chemical and electrical signal transduction at the synapse and can potentially provide novel forms of short-term synaptic plasticity that do not rely on residual Ca²⁺.

The subtype of Ca_Vβ can influence the extent and kinetics of Gβγ mediated inhibition and this regulation also depends on the subtype of Gβ involved [29,30]. Gβγ interacts with multiple sites on the N-terminus, I–II linker, and the C-terminus of the α1 subunit. Binding of Gβγ causes a conformational shift that promotes interaction of the N-terminus “inhibitory module” with the initial one-third of the I–II-linker. Strong membrane depolarization leads to unbinding of Gβγ and loss of interaction between the N-terminus and the I–II linker. This depends upon binding of Ca_Vβ subunit to the α interaction domain (AID) on the I–II linker. In the absence of Ca_Vβ1 subunit binding with tryptophan mutation in the AID (W391) of the Ca_V2.2 α1 subunit, Ca²⁺ channel inhibition still occurs but cannot be reversed by strong depolarization. Ca_Vβ2a, that is palmitoylated at two N-terminal cysteine residues, can still bind to the α1 subunit and permit voltage-dependent relief of the inhibition [31]. It is possible that binding of Ca_Vβ1 to the AID induces a rigid α-helical link with domain IS6, and this transmits the movement of the voltage-sensor and activation gate to the I–II linker to alter the Gβγ binding pocket at depolarized potentials [32].

Specific Gβ subunits have been shown to be responsible for the Ca_V2 channel modulation in different neurons. In rat SCG neurons Ca_V2.2 channels are differentially modulated by different types of Gβ subunits, with Gβ₁ and Gβ₂ being most effective, Gβ₅ showing weaker modulation, and Gβ₃ and Gβ₄ being ineffective [33,34,35]. In contrast, in rat stellate ganglion neurons, Gβ₂ and Gβ₄ but not Gβ₁ subunit are responsible for the coupling of Ca_V2.2 channels with noradrenaline receptors [36]. In the transfected human embryonic kidney tsA-201 cell line, Ca_V2.2 channel inhibition, with Gβ₁ and Gβ₃ being more effective than Gβ₄ and Gβ₂, and no significant modulation being induced by Gβ₅ [37]. Gβ subunit-induced inhibition of Ca_V2.1 channel differed from those observed with the Ca_V2.2 channel. Ca_V2.1 channels exhibited more rapid rates of recovery from inhibition than those observed with Ca_V2.2 channels, on average, twice as rapidly for the Ca_V2.1 channels, indicating that Gβ binding to this channel subtype is less stable [37].

Regulation of the Ca_V2.2 channels also involves the interplay between Ca²⁺ channels and G protein interaction. Syntaxin-1A, a presynaptic plasma membrane protein, is required for G protein inhibition of presynaptic Ca²⁺ channels [38]. Physical interaction between syntaxin-1A and Ca²⁺ channels is a prerequisite for tonic Gβγ modulation of Ca_V2.2 channels, suggesting that syntaxin-1A mediates a colocalization of Gβγ subunits and Ca_V2.2 channels, thus resulting in a more effective G protein coupling to, and regulation of, the channel. The interactions between syntaxin, G proteins, and Ca_V2.2 channels are part of the structural specialization of the presynaptic terminal [39].

G proteins also induce voltage-independent inhibition of Ca_V2 channels through intracellular signaling pathways [1,19,40]. This often involves the Gq family of G proteins, which regulate the levels of phosphatidylinositide lipids by inducing hydrolysis of phosphatidylinositol bisphosphate via activation of phospholipase C enzymes [41]. Acetylcholine release from rat sympathetic neurons is reduced through this pathway via presynaptic muscarinic receptors activation [42].

3.2. 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_V2.1 and Ca_V2.2 channels [46,48,50,51] (Figure 1b). The interaction is essential for recruiting Ca²⁺ 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_V2.1 and Ca_V2.2 channels [51], and are selectively required for high-fidelity coupling of AP-induced Ca²⁺ influx to Ca²⁺-stimulated SVs exocytosis [52]. The tripartite complex of RIM, RIM-BPs, and C-terminal tails of the Ca_V2 channels regulate the recruitment of Ca_V2 channels to AZs. Interaction of RIM with Ca_Vβ subunits shifts the voltage dependence of inactivation to more positive membrane potentials, increasing Ca²⁺ channel activity [53]. In contrast, Ca_Vβ subunits interaction with CAST/ERC2 shifts the voltage dependence of activation to more negative membrane potentials [54]. Positive regulation of presynaptic Ca²⁺ 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_V2 channels. Furthermore, Munc13, required for SVs priming, controls Ca_V2 channels shortly after AP firing to guarantee transmitter release for continuous neural activity [55].

3.3. 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_V2.1 and Ca_V2.2 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 Ca²⁺ with maximal binding at 20 μM and reduced binding at lower or higher concentrations of Ca²⁺ [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 α₁ subunit of Ca_V2.2 (amino acid residues 718-963) named as the synprint site (Figure 1b) [64,65]. Ca_V2.1 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_V2.1 and Ca_V2.2 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.

Dependent on Ca²⁺ concentration, syntaxin-1 interacts with either the synprint site or synaptotagmin-1; at low Ca²⁺ concentrations, syntaxin-1 binds synprint, while at higher concentrations (>30 μM) it associates with synaptotagmin-1 [63]. Synaptotagmin-1, -2, and -9 serve as the Ca²⁺ sensors for the fast, synchronous neurotransmitter release [56,73,74]. The Ca²⁺ binding site C2B domain of synaptotagmin-1 interacts with the synprint sites of both Ca_V2.1 and Ca_V2.2 channels (Figure 1b) [75]. Synaptotagmin-1 can relieve the inhibitory effects of SNAP-25 on Ca_V2.1 channels [70,76]. Relief of Ca²⁺ channel inhibition by the formation of the synaptotagmin/SNARE complex favors Ca²⁺ influx. This is a potential mechanism to increase the release probability of SVs docked close to Ca_V2 channels [4].

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 Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) [77]. The negative shift of steady-state inactivation of Ca_V2.2 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.

3.4. Ca²⁺-Sensor Proteins

Ca²⁺ elevation regulates Ca_V2.1 channels activity by its binding to CaM [8,78,79,80,81] and related neuron-specific Ca²⁺-binding proteins, CaBP1, VILIP-2 [82,83,84], and NCS-1 (frequenin) [85]. The presynaptic Ca_V2.1 channel proteins consist of a pore-forming α₁ subunit associated with β, and possibly α₂δ subunits (Figure 1a) [86]. The intracellular C terminus of the α1 subunit [81] called the IQ-like motif, which begins with the sequence isoleucine-methionine (IM) instead of isoleucine-glutamine (IQ), and the nearby downstream CaM-binding domain (CBD) are the interacting sites with these Ca²⁺-binding proteins (Figure 1b). Displacement with alanine in the IQ-like domain inhibited Ca²⁺-dependent Ca_V2.1 channels facilitation [78,81], whereas deletion of CBD inhibited Ca²⁺-dependent Ca_V2.1 channels inactivation [79,80,81,83,84]. Ca²⁺/CaM-dependent inactivation of Ca_V2.1 channels, dependent on global elevations of Ca²⁺, is observed in transfected cells overexpressing Ca_V2.1 channels [78,79,80] and in the nerve terminals of the calyx of Held [87,88] where Ca_V2.1 channels are densely localized. In contrast, the large neuronal cell bodies of Purkinje neurons [89] or SCG neurons [90] rarely show Ca²⁺-dependent Ca_V2.1 channels inactivation.

4. Synchronous Neurotransmitter Release Regulated by Ca²⁺ Channel/SNARE Proteins Complex

Synprint peptides derived from Ca_V2.2 channels reduced transmitter release from the microinjected presynaptic SCG neurons in culture, due to competitive uncoupling of the endogenous Ca²⁺ 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 Ca²⁺ concentration effectively rescued this inhibition, implying that synprint peptides competitively displaces docked SVs away from Ca²⁺ channels, and this effect can be overcome by increasing Ca²⁺ influx into presynaptic terminals [100].

Figure 3. Spatial regulation of transmitter release by the I-II loop interaction with SNAREs. (a) The I-II loop interacts with t-SNAREs, resulting in inhibition of Ca2.2 channels opening. Once AP opens the channels, an increase in Ca²⁺ mediates interaction with SNAREs complex and induces transmitter release. Adapted from [4]. (b) Triple APs induces a large synchronous transmitter release from the first AP. In contrast, asynchronous transmitter release was observed in the presence of 130 μM synprint peptide (see Figure 1b). Adapted from [99].

At the calyx of Held, presynaptic neurons express P/Q-, N- and R-type Ca²⁺ currents in postnatal day 7 rats. P/Q-type Ca²⁺ currents are more effective than N-type Ca²⁺ currents and R-type Ca²⁺ currents in eliciting neurotransmitter release [101,102,103]. The high efficiency of P/Q-type Ca²⁺ currents to initiate neurotransmitter release is correlated with the close localization of Ca_V2.1 channels near docked SVs [104], as shown by immunocytochemistry [105], suggesting localization of Ca_V2 channels determines the efficiency of neurotransmitter release in response to neural activity.

Ca_V2 channels interaction with SNARE proteins, that is dependent on Ca²⁺ concentration [63], have two opposing effects: at the pre-firing state synaptic transmission is blocked by enhancing Ca_V2 channels inactivation, whereas immediately after AP firing tethering SVs near the point of Ca²⁺ entry enhances synaptic transmission. The overexpression of a syntaxin mutant that is unable to regulate Ca_V2.2 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_V2.2 channels with SNARE proteins.