Among the three families of Ca_V channels (Ca_V1, Ca_V2 and Ca_V3), the Ca_V2 family and more specifically Ca_V2.1 and Ca_V2.2 channels (generating P/Q and N-type currents, respectively) are particularly important for synaptic transmission in central and peripheral nervous systems (Dolphin, 2012). Ca_V2.1 and Ca_V2.2 are targeted to presynaptic terminals where they are responsible for triggering vesicular release (Catterall and Few, 2008; Zamponi et al., 2015). Ca_Vs are formed of several subunits: the α₁ subunit, that constitutes the Ca²⁺ selective pore and the voltage sensor, and auxiliary subunits β (cytoplasmic) and α₂δ (extracellular) (Flockerzi et al., 1986; Liu et al., 1996; Takahashi and Catterall, 1987; Witcher et al., 1993). Four genes coding for α₂δ subunits have been identified (Dolphin, 2012). They are translated into a single pre-protein α₂δ and post-translationally cleaved into α₂ and δ peptides, which remain attached by di-sulfide bonds (Dolphin, 2012). In α₂δ−1 and −2, α₂ contains a perfect metal ion adhesion site (MIDAS) motif essential for the interaction with α₁ subunit (Cantí et al., 2005; Hendrich et al., 2008) and δ which is glycophosphatidylinositol (GPI) anchored to the plasma membrane (Davies et al., 2010). The structure of the Ca_V1.1 channel complex has been recently determined using cryo-electron microscopy and has identified binding domains between Ca_V1.1 and α₂δ−1 including the interaction of the α₂δ MIDAS motif with loop I of the first repeated domain of Ca_V1.1 (Wu et al., 2016). Site-directed mutagenesis studies have confirmed a functional interaction between α₂δ−1 and the first extracellular loop of Ca_V1.2 (Bourdin et al., 2017) and Ca_V2.2 channels (unpublished results).

α₂δ subunits are important for the trafficking of α₁ subunits and their function, and they are also key proteins for synaptic function and synaptogenesis (Dickman et al., 2008; Eroglu et al., 2009; Hoppa et al., 2012; Saheki and Bargmann, 2009; Zamponi et al., 2015). We have recently shown that the proteolytic maturation of α₂δ−1 into disulfide-linked polypeptides α₂ and δ is an essential post-translational step enabling its modulatory effect on the activation and trafficking of N-type calcium channels in neurons (Kadurin et al., 2016). Indeed, we show that uncleaved α₂δ−1 inhibits presynaptic calcium transient-triggered action potential (AP) in hippocampal neurons and that this effect is reversed by the cleavage of α₂δ−1.

Here, we investigate the impact of the proteolytic maturation of α₂δ−1 on synaptic release. We used optical tools to measure vesicular release parameters (Ariel and Ryan, 2010; Hoppa et al., 2012). Our data show that an uncleaved α₂δ−1 reduces the probability of release in response to a single action potential, and also affects asynchronous release. These effects on presynaptic vesicular release are reversed when the cleavage of α₂δ−1 is restored. We provide additional evidence that cleaved α₂δ−1 interacts more than the uncleaved form with the Ca_V2.2 channel pore-forming subunit. Our data indicate that the proteolytic maturation of α₂δ−1 is important for its association with the Ca_V channel complex and its function on synaptic release.

α₂δ subunits play a crucial role in the trafficking of fully functional calcium channels to the plasma membrane and to presynaptic terminals (Dolphin, 2012). In order to determine the physiological impact of proteolytic maturation of α₂δ−1, we used the cleavage site mutant α₂(3C)δ−1 (Kadurin et al., 2016), which is resistant to endogenous proteolysis between α₂ and δ, to assess the effect of controlled cleavage by exogenous 3C-protease on vesicular release from presynaptic terminals, using the optical reporter vGlut-pHluorin. Transfected hippocampal neurons were identified by mCherry expression (Figure 1a). Neurons were subsequently stimulated (100 AP at 10 Hz), and fluorescence of vGlut-pHluorin was monitored to identify functional boutons (Figure 1a). We first examined the effect of expression of α₂(3C)δ−1 on synaptic release properties by measuring single AP-evoked exocytosis (Figure 1b). Single AP stimulations were repeated 10 to 12 times with a 45 s rest between each trial. Signals from each bouton were averaged and normalized to the fluorescence value obtained by rapid alkalinization of the entire labeled vesicle pool using NH₄Cl (Figure 1a and b). Overexpression of uncleaved α₂(3C)δ−1 induced a decrease of 29 ± 6% in exocytosis compared to the control empty vector condition (n = 28 and 41, respectively; p=0.04) (Figure 1c). Conversely, inducing controlled cleavage of α₂(3C)δ−1 by co-expressing 3C-protease resulted in an increase of 53 ± 18% in exocytosis compared to α₂(3C)δ−1 alone (n = 41 and 16, respectively; p=0.014), thus completely reversing the inhibitory effect of uncleaved α₂(3C)δ−1 (Figure 1b and c).

Figure 1

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Synaptic vesicle exocytosis properties are determined by the number of vesicles available for rapid release (the readily-releasable pool - RRP) and the probability (Pv) that a vesicle in the RRP will undergo fusion in response to a single AP stimulus (Schneggenburger et al., 2002). RRP can be determined using a high frequency stimulation (Ariel and Ryan, 2010; Ariel et al., 2012). During a 20 AP stimulus at 100 Hz, the fluorescence of vGlut-pHluorin in presynaptic terminals rapidly increases and reaches a plateau phase corresponding to the RRP (Figure 2a). The averaged response, obtained from 5 to 6 trials with a 5 min rest between each trial, were normalized to the size of the total presynaptic pool obtained with NH₄Cl application (Figure 2a). To examine whether proteolytic maturation of α₂δ−1 affects the size of the RRP, we imaged neurons transfected with either empty vector (Figure 2a) or α₂(3C)δ−1 (Figure 2b) or α₂(3C)δ−1 together with 3C-protease (Figure 2c) and compared the size of the RRP. As summarized in Figure 2d, no difference was recorded between the three conditions (empty vector, α₂(3C)δ−1 and α₂(3C)δ−1 with 3C-protease: 6.9 ± 0.4, 6.2 ± 0.3 and 6.4 ± 0.5% of total pool, n = 22, 16 and 19, respectively, p=0.78), indicating that proteolytic maturation of α₂δ−1 affects the Pv, rather than the size of the RRP.

Figure 2

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After the plateau phase corresponding to the RRP, an additional increase in fluorescence takes place during the stimulation, and continues for more than 500 ms after the end of the stimulus before reaching a stationary phase (Figure 3a). It was proposed that this secondary increase in fluorescence results from a combination of RRP refilling and slow decay of the elevated intracellular Ca²⁺ concentration (Ariel and Ryan, 2010). This late increase in fluorescence occurs at lower rate than the initial increase and represents post-stimulus exocytosis. Overexpression of uncleaved α₂(3C)δ−1 induced a decrease of about 30% in this phase of exocytosis compared to control empty vector condition (n = 22 and 31, respectively; p<0.001) (Figure 3b–c). This reduction of delayed exocytosis is completely prevented by the co-expression of α₂(3C)δ−1 with 3C-protease (Figure 3a–b).

Figure 3

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We then wished to determine whether the results obtained on presynaptic release were due to differential interaction of cleaved and uncleaved α₂δ with the α1 subunit. We have previously shown that transient expression of α₂δ−1 in cell lines results in only a partial cleavage of wild type α₂δ−1, such that a mixture of cleaved and uncleaved α₂δ protein appears in the whole cell lysate (WCL) (Kadurin et al., 2012, 2016). We performed co-immunoprecipitation of wild type α₂δ−1 with Ca_V2.2 from tsA-201 cell WCL and found that the percentage of cleaved α₂δ−1 in the co-immunoprecipitated fractions is ~4 fold higher than the percentage of cleaved α₂δ−1 in the input WCL (from 10.0 ± 0.6% to 39.2 ± 1.6% in WCL and co-immunoprecipitated fractions, respectively, n = 3) (Figure 4), suggesting stronger association of mature cleaved α₂δ−1 with the Ca_V pore-forming subunit.

Figure 4

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Ca_V2 channels are important for synaptic transmission and their targeting to the active zone is tightly regulated (Catterall and Few, 2008; Simms and Zamponi, 2014). α₂δ subunits have been shown to control the trafficking of Ca_V2 to presynaptic terminals (Hoppa et al., 2012). α₂δ subunits are post-translationally proteolysed, and this process is key for their regulatory action on Ca_V2 channels (Kadurin et al., 2016). Here, we show that the post-translational proteolytic maturation of α₂δ−1 is also essential for these proteins to fulfil their regulatory function on vesicular release in presynaptic terminals of hippocampal neurons in culture. Interestingly, we show that both synchronous and asynchronous releases are affected, both release mechanisms being highly dependent on Ca²⁺ influx through Ca_V2 channels.

Vesicular release is characterized by two key presynaptic parameters: the RRP and Pv (Ariel and Ryan, 2012; Schneggenburger et al., 2002). A previous study has shown that over-expression of α₂δ subunits and knock-down of endogenous α₂δ increased and decreased Pv, respectively (Hoppa et al., 2012). In good agreement with this, our data show that uncleaved α₂δ−1 (α₂(3C)δ−1) reduces Pv, and co-expression of the 3C-protease restores the control Pv. Interestingly, Pv is modulated by the number of Ca_V2 channels in each active zone (Ermolyuk et al., 2012) and we have previously shown that uncleaved α₂δ subunits reduced the amplitude of calcium transients triggered by a single AP stimulation, by interfering with the trafficking of Ca_V2 channels (Kadurin et al., 2016). The active zone proteins Rab-3 interacting molecules (RIMs) and Munc-13, critical in the orchestration of synaptic vesicular release, have been shown to control the targeting of Ca_V2 channels within presynaptic terminals (de Jong et al., 2018; Südhof, 2012). These active zone proteins have also been shown to control the size of the RRP (Augustin et al., 1999; Calloway et al., 2015; Deng et al., 2011; Kaeser et al., 2011). The RRP is defined as a small fraction of vesicles in a presynaptic terminal that is available for immediate release with a brief stimulus train, and thus likely to equate to docked vesicles identified by electron microscopy (Ariel and Ryan, 2012; Rizzoli and Betz, 2005; Schneggenburger et al., 2002). Experimental methods used to estimate the size of the RRP have been recently reviewed and consist of two electrophysiological methods (post synaptic current recordings and presynaptic membrane capacitance measurements) and one optical method (Kaeser and Regehr, 2017). Here, we used the optical technique that was developed by Ariel and Ryan, (2010). This high-time resolution optical method measures exocytosis by detecting fluorescence from pHluorin tagged vGlut-1 (Voglmaier et al., 2006) associated with vesicle fusion. The high frequency stimulation protocol (20 APs at 100 Hz) induces a rapid rise in fluorescence followed by a plateau corresponding to a state during which all the vesicles in the RRP have fused with the membrane. The size of the RRP we describe here, which is determined by the amplitude of the fluorescence of the plateau (6–7% of the total pool of vesicles) is in good agreement with previously described values of RRP in neonatal rodent hippocampal neuron synapses (Ariel and Ryan, 2010; Fernández-Alfonso and Ryan, 2006; Rizzoli and Betz, 2005). A previous study has shown that wild-type α₂δ subunits have no effect on the size of the RRP (Hoppa et al., 2012). Consistent with that study, our data show that uncleaved α₂δ−1 does not affect the size of the RRP indicating that, unlike RIMs and Munc13, α₂δ−1 does not have the same dual function on synaptic vesicular release.

There are two potential mechanisms to account for the reduction in Pv by α₂(3C)δ−1. It is likely that α₂(3C)δ−1 reduces the trafficking of endogenous Ca_V2 channels into active zones, as we showed for exogenously expressed Ca_V2.2 (Kadurin et al., 2016). However, α₂(3C)δ−1 can also traffic alone into presynaptic terminals (Kadurin et al., 2016), where it could then displace the endogenous α₂δ interacting with channels in active zones, thus forming non-functional channels. The finding here that uncleaved α₂δ interacts less than cleaved α₂δ with Ca_V2.2 may indicate that the former mechanism is more likely.

Several reports have also described a role for α₂δ subunits in synaptogenesis, independently from their role as a Ca_V auxiliary subunit (Dickman et al., 2008; Eroglu et al., 2009; Kurshan et al., 2009). α₂δ subunits are extracellular proteins anchored to the plasma membrane via a GPI moiety (Davies et al., 2010) which makes them potentially good candidates to interact with extracellular ligands such as thrombospondins, low density lipoprotein receptor-related protein and α-neurexin (Eroglu et al., 2009; Kadurin et al., 2017; Tong et al., 2017). Although a direct interaction between α₂δ and thrombospondin and its role in the mediation of synaptogenesis remains controversial (Lana et al., 2016; Xu et al., 2010), altogether these reports suggest that α₂δ subunits could play a role as an extracellular coordinator of synaptic function. Furthermore, the modulation of presynaptic Ca_V channels by proteolytic cleavage of α₂δ subunits could serve as an additional regulatory mechanism for their complex synaptic functions at the post-translational level.

Ca_V2 channels and BK potassium channels are known to be part of multi-molecular complexes in neurons (Berkefeld et al., 2006; Müller et al., 2010). α₂δ−1 has very recently been shown to interact with BK channels, and this interaction was found to reduce the stability of Ca_V2.2 channels at the plasma membrane by preventing α₂δ−1 interacting with Ca_V2.2 channels (Zhang et al., 2018). Functionally, BK channels were shown to control neurotransmitter release by shortening the AP duration and reducing Ca²⁺ influx into presynaptic elements at neuro-muscular junctions (Protti and Uchitel, 1997; Yazejian et al., 1997). Although their presence in presynaptic boutons has been disputed (Hoppa et al., 2014), BK channels are also expressed in axons from central neurons (Debanne et al., 2011; Johnston et al., 2010). Furthermore, α₂δ−1 has also recently been shown to interact with NMDA glutamate receptors (NMDARs) (Chen et al., 2018), albeit via a C-terminal domain of α₂δ−1 that is beyond the GPI-anchor attachment site and would therefore not be present in a mature GPI-anchored form (Davies et al., 2010; Kadurin et al., 2012; Wu et al., 2016). This interaction was found to promote the trafficking of the NMDARs to synaptic sites between peripheral dorsal root ganglion neurons and dorsal horn neurons in the spinal cord and is involved in the development of neuropathic pain (Chen et al., 2018). Therefore, it will be of great interest to determine whether fully mature α₂δ−1 is required for the interaction with BK potassium channels and with NMDARs.

Synchronous stimulated release is often followed by a delayed release occurring after the end of the stimulus, also called asynchronous release (Atluri and Regehr, 1998; Goda and Stevens, 1994; Kaeser and Regehr, 2014). Asynchronous release is thought to be activated by residual Ca²⁺ remaining in the presynaptic terminal after the stimulation (Atluri and Regehr, 1998; Cummings et al., 1996). Although the source of Ca²⁺ responsible for the initiation of synchronous release is indisputably identified from many studies as voltage-gated calcium channels within the active zone (Catterall and Few, 2008; Dolphin, 2012; Nakamura et al., 2015; Zamponi et al., 2015), the source of Ca²⁺ involved in asynchronous release remains uncertain. To study asynchronous release in this work, we took advantage of the optical method developed previously (Ariel and Ryan, 2010) to monitor the slow increase of fluorescence of pHluorin tagged to vGlut-1 after the end of the high frequency stimulation (20 AP at 100 Hz). We show that asynchronous release is reduced in hippocampal presynaptic terminals when uncleaved α₂δ−1 (α₂(3C)δ−1) is expressed, and this inhibitory effect is abolished when 3C-protease is co-expressed. Together with our previous report showing that the proteolytic cleavage of α₂δ is critical for the functional trafficking of Ca_V2.2 channels to the presynaptic terminals (Kadurin et al., 2016), our data demonstrate that asynchronous release is mediated by Ca²⁺ influx generated by Ca_V localized at the presynaptic terminals. Relevant to our data, a study has characterized an asynchronous Ca²⁺ current, recorded after the end of the stimulation pulse, conducted by both Ca_V2.1 and Ca_V2.2 channels and activated by the increase of intracellular Ca²⁺ generated by the activity of these channels (Few et al., 2012). This asynchronous current was also identified in mouse hippocampal neurons and this led the authors to suggest that the asynchronous current could contribute to asynchronous release (Few et al., 2012). Other Ca²⁺ sources for asynchronous release have been proposed (Kaeser and Regehr, 2014). Ca²⁺-permeable P2X2 ATP receptors have been involved in asynchronous release in excitatory synapses between CA3 neurons and interneurons in the CA1 region in the hippocampus (Khakh, 2009). At these synapses, P2X2 receptors would be activated by ATP released from vesicles in presynaptic terminals. Further pharmacological characterization would be needed to ascertain the involvement of P2X2 receptors in the asynchronous release we are monitoring in our experimental model. Additionally, in the nucleus of the solitary tract, TRPV1 channels had been suggested to be a source of Ca²⁺ for asynchronous release at excitatory synapses from unmyelinated cranial visceral primary afferent neurons (Peters et al., 2010). However, recent data from the same group have suggested instead that the Ca²⁺ source for asynchronous release would originate from spill-over of intracellular Ca²⁺ from Ca²⁺ nanodomains created by Ca_V2 channels (Fawley et al., 2016). This latter hypothesis would fit well with our data showing that mature α₂δ−1 is needed to traffic Ca_V to the presynaptic terminals to modulate asynchronous release.

Building on our previous report, we show here that the maturation of α₂δ is crucial for Ca_V channels to fulfil their functional role on synaptic transmission. As α₂δ−1 expression is upregulated during chronic pain and increases presynaptic Ca_V2 trafficking (Bauer et al., 2009; Kadurin et al., 2016; Patel et al., 2013; Zamponi et al., 2015), α₂δ−1 represents a therapeutic target (Zamponi, 2016), and an important question to address for future studies will be to identify endogenous protease(s) involved in the proteolytic maturation of α₂δ proteins.

All data generated or analysed during this study are included in the manuscript.

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Author details

1. Laurent Ferron

   Department of Neuroscience, Physiology and Pharmacology, University College London, London, United Kingdom

   Contribution

   Conceptualization, Formal analysis, Investigation, Writing—original draft, Writing—review and editing

   For correspondence

   l.ferron@ucl.ac.uk

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2547-7417

2. Ivan Kadurin

   Department of Neuroscience, Physiology and Pharmacology, University College London, London, United Kingdom

   Contribution

   Conceptualization, Formal analysis, Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

3. Annette C Dolphin

   Department of Neuroscience, Physiology and Pharmacology, University College London, London, United Kingdom

   Contribution

   Conceptualization, Funding acquisition, Project administration, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4626-4856

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