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. 2023 Sep 4;404(10):931–937. doi: 10.1515/hsz-2023-0235

Nanoscale organization of CaV2.1 splice isoforms at presynaptic terminals: implications for synaptic vesicle release and synaptic facilitation

Lorenzo A Cingolani 1,4,✉, Agnes Thalhammer 1,2,✉, Fanny Jaudon 1,3, Jessica Muià 1, Gabriele Baj 1,2
PMCID: PMC10695435  PMID: 37658578

Abstract

The distance between CaV2.1 voltage-gated Ca2+ channels and the Ca2+ sensor responsible for vesicle release at presynaptic terminals is critical for determining synaptic strength. Yet, the molecular mechanisms responsible for a loose coupling configuration of CaV2.1 in certain synapses or developmental periods and a tight one in others remain unknown. Here, we examine the nanoscale organization of two CaV2.1 splice isoforms (CaV2.1[EFa] and CaV2.1[EFb]) at presynaptic terminals by superresolution structured illumination microscopy. We find that CaV2.1[EFa] is more tightly co-localized with presynaptic markers than CaV2.1[EFb], suggesting that alternative splicing plays a crucial role in the synaptic organization of CaV2.1 channels.

Keywords: alternative splicing, CaV2.1, Munc13, presynaptic terminals, structural illumination microscopy, voltage-gated Ca2+ channels

The distance between voltage-gated Ca2+ channels (VGCCs) and the Ca2+ sensors responsible for releasing synaptic vesicles at the active zone of presynaptic terminals plays a crucial role in determining synaptic strength (Dittman and Ryan 2019). Because the diffusion time of Ca2+ is proportional to the square of distance, most synapses exhibiting high release probability and reliable transmission in response to single action potentials are characterized by a tight coupling between VGCCs and Ca2+ sensors. Conversely, synapses exhibiting low release probability and the ability to facilitate in response to repetitive action potentials generally have a loose coupling between VGCCs and Ca2+ sensors (Eggermann et al. 2012). During postnatal development, many glutamatergic excitatory synapses undergo a transition in the configuration of their release machinery. They switch from microdomains, where loosely coupled P/Q- (CaV2.1) and N-type (CaV2.2) VGCCs act cooperatively, to nanodomains, where tightly coupled P/Q-type VGCCs efficiently trigger synaptic vesicle exocytosis (Baur et al. 2015; Bornschein et al. 2019; Fedchyshyn and Wang 2005; Kusch et al. 2018; Nakamura et al. 2015).

However, the molecular mechanisms responsible for the transition of CaV2.1 from a loose to a tight configuration remain unknown. Notably, the pore-forming α1 subunit of CaV2.1 (α1A) undergoes extensive alternative splicing, potentially generating thousands of different splice isoforms (Soong et al. 2002). In particular, alternative splicing of two mutually exclusive exons (exons 37a and 37b) produces two isoforms of CaV2.1 (CaV2.1[EFa] and CaV2.1[EFb]), which differ in an EF-hand-like domain located in the proximal C terminus (Figure 1A; Bourinet et al. 1999; Chaudhuri et al. 2004; Jaudon et al. 2020; Soong et al. 2002; Thalhammer et al. 2020).

Figure 1:

Figure 1:

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Expression levels of CaV2.1 splice variants in primary cortical neurons. (A) Left, structural model of human CaV2.1[EFb] (UniProt ID: O00555; Martinez-Ortiz and Cardozo 2018), highlighting the full C-terminus (in green, cyan and blue colors), the part of the EF-hand-like domain shared between CaV2.1[EFa] and CaV2.1[EFb] (the E helix; in cyan) and the 33 residue-long sequence specific to CaV2.1[EFb] (the loop, the F helix and the downstream residues; in blue). Right, in scale drawing of exons 36, 37a, 37b and 38 of CACNA1A (CaV2.1) and mechanism of mutually exclusive splicing for exons 37a and 37b. Redrawn from (Thalhammer et al. 2020). (B) RT-qPCR quantification of total CaV2.1, CaV2.1[EFa] and CaV2.1[EFa] in 18 DIV primary cortical neurons expressing the indicated constructs (***p < 0.001, one-way ANOVA followed by Tukey’s post hoc test; n = 6 from 2 independent cultures). (C) Left, representative Western blot of membrane-enriched fractions from 18 DIV cortical primary neurons expressing the indicated constructs. Right, quantification of Western blots (**p < 0.009, one-way ANOVA followed by Tukey’s post hoc test; n = 3). (D) The relative abundance of CaV2.1[EFa] versus CaV2.1[EFb] splice variants was determined by absolute quantification using calibration curves with known concentrations of recombinant plasmids (***p < 0.001, Chi-square test; n = 6 from 2 independent cultures). Total CaV2.1, CaV2.1[EFa] and CaV2.1[EFb] in panels B and D were detected with primers m/h.CaV2.1_Fw/Rv (located in exons 21 and 22), m/h.CaV2.1[EFa]_Fw and m/h.CaV2.1[EFa/b]_Rv (located in exons 37a and 38), and m/h.CaV2.1[EFb]_Fw and m/h.CaV2.1[EFa/b]_Rv (located in exons 37b and 38), respectively (see Figure S3). For detailed methods, see supplementary materials and Ferrante et al. (2021).

Here, we use superresolution structured illumination microscopy (SIM) to investigate whether the two isoforms CaV2.1[EFa] and CaV2.1[EFb] exhibit distinct nanoscale organization at presynaptic excitatory terminals with varying coupling distances from the active zone. We focused on these isoforms as potential molecular correlates of the developmental switch in coupling distance between CaV2.1 and the release machinery for two main reasons. First, the two isoforms have opposite effects on neurotransmitter release and short-term synaptic plasticity. CaV2.1[EFa] enhances synchronous release and short-term synaptic depression, while CaV2.1[EFb] promotes asynchronous release and short-term synaptic facilitation (Thalhammer et al. 2017, 2018). Second, while CaV2.1[EFb] is expressed early in postnatal development, CaV2.1[EFa] expression increases during the postnatal period, in conjunction with the tightening of the coupling between CaV2.1 and the neurotransmitter release machinery (Bourinet et al. 1999; Chaudhuri et al. 2004; Soong et al. 2002; Thalhammer et al. 2020; Vigues et al. 2002).

Because of the well-known challenges associated with expressing exogenous CaV2.1 channels (Heck et al. 2019; Jaudon et al. 2022; Thalhammer et al. 2017), we infected primary cortical neurons at 7 DIV with lentiviruses expressing CaV2.1[EFa] or CaV2.1[EFb] under the control of a Synapsin promoter, and performed functional analyses at 18–20 DIV. The use of lentiviruses in conjunction with the Synapsin promoter allowed for a controlled and gradual increase in the levels of CaV2.1 splice isoforms. Specifically, exogenous expression of either CaV2.1[EFa] or CaV2.1[EFb] proved equally effective in augmenting total mRNA levels of CaV2.1 (approximately 4-fold increase for both CaV2.1[EFa] or CaV2.1[EFb]; Figure 1B, left panel) as well as protein levels of CaV2.1 (approximately 2-fold increase in membrane protein-enriched fractions for both CaV2.1[EFa] or CaV2.1[EFb]; Figure 1C). Furthermore, exogenous expression of CaV2.1[EFa] specifically enhanced the expression of CaV2.1[EFa], while leaving the levels of CaV2.1[EFb] unaltered (Figure 1B, middle panel). Conversely, exogenous expression of CaV2.1[EFb] selectively increased the expression of CaV2.1[EFb], while keeping the levels of CaV2.1[EFa] unchanged (Figure 1B, right panel). As a consequence, CaV2.1[EFa] overexpression led to an inversion in the relative distribution of CaV2.1[EFa]-CaV2.1[EFb] from 24–76 %, observed in naïve neurons, to 76–24 %, while CaV2.1[EFb] overexpression further decreased the relative distribution of CaV2.1[EFa]-CaV2.1[EFb] to 7.5–92.5 % (Figure 1D). Exogenous CaV2.1[EFa] and CaV2.1[EFb] did not impact total or isoform-specific expression of endogenous CaV2.1 (Figure S1).

We next assessed whether expression of the two CaV2.1 splice isoforms changed synaptic levels of CaV2.1. Consistent with the results from Western blotting analysis (Figure 1C), wide-field fluorescence imaging revealed that both CaV2.1[EFa] and CaV2.1[EFb] significantly increased CaV2.1 fluorescence intensity to a similar degree without altering expression of bassoon and vGlut1 (Figure S2A, B). However, when we employed SIM to obtain super-resolved fluorescence images, it became apparent that neither CaV2.1[EFa] nor CaV2.1[EFb] had any discernable impact on the number or size of CaV2.1 puncta (Figure 2A–C and S2C). Likewise, no changes were observed in the number or size of presynaptic boutons (identified by co-labeling for bassoon) or excitatory presynaptic boutons (identified by co-labeling for vGlut1; Figure 2A–C and S2C). Taken together, these results suggest that the exogenous CaV2.1 isoforms are expressed synaptically without causing major structural alterations to synaptic boutons.

Figure 2:

Figure 2:

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CaV2.1[EFa] is more efficiently targeted to excitatory synaptic boutons than CaV2.1[EFb]. (A) Reconstructed and Z-projected SIM images of primary cortical neurons for Bassoon (Bsn), vGlut1 and CaV2.1. Top, full field of view; bottom, magnification of indicated ROI. Arrowheads indicate CaV2.1 puncta co-localizing with Bsn; arrows indicate CaV2.1 puncta co-localizing with both Bsn and vGlut1. (B) Area of Bsn, vGlut1 and CaV2.1 puncta for the indicated experimental conditions. The size of Bsn, vGlut1 and CaV2.1 puncta is not altered by exogenous expression of either CaV2.1[EFa] or CaV2.1[EFb] (p = 0.610, p = 0.353 and p = 0.456, and n = 12, 12 and 24 fields of views for Bsn, vGlut1 and CaV2.1, respectively; one-way ANOVA). (C) Left, total number of Bsn particles per field of view; middle, number of vGlut1 particles normalized to the number of Bsn particles; right, number of CaV2.1 particles normalized to the number of Bsn particles. The number of Bsn, vGlut1 and CaV2.1 particles is not altered by exogenous expression of either CaV2.1[EFa] or CaV2.1[EFb] (p = 0.522, p = 0.542 and p = 0.851, and n = 12, 6 and 12 fields of views for Bsn, vGlut1 and CaV2.1, respectively; one-way ANOVA). (D) Reconstructed and Z-projected SIM images of Bsn and CaV2.1. White in merge highlights colocalization. (E) Percentage of Bsn puncta area co-localized with CaV2.1 puncta for images as in (D). Expression of CaV2.1[EFa] increases colocalization of CaV2.1 with Bsn (*p = 0.018, one-way ANOVA followed by Tukey’s post hoc test, n = 12 fields of views per condition). (F) As in (D) but for vGlut1 and CaV2.1. (G) Percentage of vGlut1 puncta area co-localized with CaV2.1 puncta for images as in (F). Expression of CaV2.1[EFa] increases colocalization of CaV2.1 with vGlut1 while expression of CaV2.1[EFb] decreases it (*p < 0.05, ***p < 0.001, one-way ANOVA followed by Tukey’s post hoc test, n = 12 fields of views per condition).

In line with previous findings (Thalhammer et al. 2017), the expression of CaV2.1[EFa] resulted in an increased level of colocalization between CaV2.1 and the presynaptic cyto-matrix protein bassoon. Under naïve condition, only 12 % of the area of bassoon positive puncta co-localized with CaV2.1; this percentage rose to 19 % in the presence of CaV2.1[EFa], while it remained unchanged at 13 % with CaV2.1[EFb] (Figure 2D, E and S2D, left). Interestingly, more pronounced effects were observed for excitatory synapses. Expression of CaV2.1[EFa] resulted in an increase in the level of colocalization between CaV2.1 and vGlut1 from 20 to 26 %, whereas CaV2.1[EFb] reduced it to 14 % (Figure 2F, G and S2D, right). These results collectively suggest that CaV2.1[EFa] is more tightly co-localized with presynaptic excitatory boutons than its mutually exclusive isoform CaV2.1[EFb].

Recent reports have highlighted the crucial role of Munc13-1 proteins in the vesicle release machinery. The number of Munc13-1 clusters at the active zone correlates closely with the number of docked vesicles and the estimated number of release complexes (Rebola et al. 2019; Sakamoto et al. 2018). In light of these findings, we co-stained for CaV2.1 and Munc13-1 to assess the nanoscale arrangement of presynaptic CaV2.1 in relation to Munc13-1.

Similar to what we observed for Bassoon and vGlut1, neither CaV2.1[EFa] nor CaV2.1[EFb] had any noticeable effect on fluorescence intensity or number and size of Munc13-1 puncta (Figure 3A, B). However, similar to the findings with Bassoon, expression of CaV2.1[EFa] led to an enhanced level of co-localization between CaV2.1 and Munc13-1. Under naïve condition, only 12 % of the area of Munc13-1 positive puncta co-localized with CaV2.1; this percentage increased to 19 % in the presence of CaV2.1[EFa], while it remained unchanged at 13 % with CaV2.1[EFb] (Figure 3C, D). To estimate the minimum distance between CaV2.1 and Munc13-1 at the active zone in the three experimental conditions, Gaussian bell curves were fit to the fluorescence intensity profiles of partially overlapping CaV2.1 and Munc13-1 puncta along the lines connecting their centers of mass (Figure 3E, F). Interestingly, the distance measured 32 nm for naïve neurons and the CaV2.1[EFa] condition, but increased to 64 nm in the CaV2.1[EFb] condition (Figure 3F, G).

Figure 3:

Figure 3:

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CaV2.1[EFa] is more tightly co-localized with the active zone protein Munc13-1 than CaV2.1[EFb]. (A) Reconstructed and Z-projected SIM images of primary cortical neurons for Bsn, Munc13-1 and CaV2.1. Top, full field of view; bottom, magnification of indicated ROI. (B) Left, area of Munc13-1 puncta for the indicated experimental conditions (p = 0.141, n = 12 fields of view per condition, one-way ANOVA). Middle, number of Munc13-1 particles normalized to the number of Bsn particles. (p = 0.872, one-way ANOVA, n = 6 fields of view per condition). Right, quantification of Munc13-1 fluorescence intensity from wide-field fluorescence images before SIM reconstruction (p = 0.63, one-way ANOVA, n = 12 fields of view). Size and number of puncta as well as fluorescence intensity of Munc13-1 are not altered by exogenous expression of either CaV2.1[EFa] or CaV2.1[EFb]. (C) Reconstructed and Z-projected SIM images of Munc13-1 and CaV2.1. White in merge highlights colocalization. (D) Percentage of Munc13-1 puncta area co-localized with CaV2.1 puncta for images as in (C). Expression of CaV2.1[EFa] increases colocalization of CaV2.1 with Munc13-1 (*p < 0.04, one-way ANOVA followed by Tukey’s post hoc test, n = 12 fields of view). (E) Munc13-1 puncta (magenta) co-localized with CaV2.1 (green) for the indicated experimental conditions. Scanning line used for the intensity fluorescence profile is indicated in yellow. (F) Intensity fluorescence profile for images as in (E; n = 26 boutons). (G) Box plot of the distance between the peaks of the fluorescence intensity profiles for Munc13-1 and CaV2.1 puncta as for panels (E) and (F). Thick lines indicate median, boxes extend from the 25th to 75th percentiles. Expression of CaV2.1[EFb] increases the distance between Munc13-1 and CaV2.1 (*p = 0.012 and **p = 0.008, one-way Brown-Forsythe ANOVA followed by Dunnett T3 post hoc test, n = 26 boutons).

Collectively, these findings indicate significant and relevant differences in the spatial organization of two mutually exclusive isoforms of CaV2.1 at the active zone. CaV2.1[EFa] is positioned in close proximity to Munc13-1, indicative of its role in supporting synchronous release of synaptic vesicle through the generation of large (>10 μM) and brief (<1 ms) nanodomain calcium signals that activate fast low-affinity calcium sensors, such as synaptotagmin 1 (Eggermann et al. 2012; Geppert et al. 1994; Thalhammer et al. 2017). On the other hand, CaV2.1[EFb] is only loosely coupled to Munc13-1, indicative of its involvement in boosting the residual calcium signal (<1 μM) that persists for tens to hundreds of milliseconds in presynaptic boutons, thus contributing to short-term synaptic facilitation and asynchronous release, possibly via slow high-affinity calcium sensors, such as synaptotagmin 7 (Jackman et al. 2016; Kaeser and Regehr 2014; Thalhammer et al. 2017). Considering the limitations in spatial resolutions of the SIM technique, our estimations of the physical distance between Munc13-1 and the two CaV2.1 isoforms align well with electron microscopy studies that reported distances of 20 and 50 nm between Munc13-1 and CaV2.1 at strong and weak synapses, respectively (Rebola et al. 2019). We propose therefore that alternative splicing of CaV2.1 at exons 37a/b may account for many of the temporal and spatial variations in the nanoscale organization of CaV2.1 at the active zone that have recently been reported (Baur et al. 2015; Cingolani et al. 2019; Holderith et al. 2012; Kusch et al. 2018; Nakamura et al. 2015; Rebola et al. 2019).

Supplementary Material

Supplementary Material

j_hsz-2023-0235_suppl_001.pdf (1.9MB, pdf)

Acknowledgments

This work was supported by the Telethon foundation (proposal ID: GGP19181 to LAC).

Supplementary Material

This article contains supplementary material (https://doi.org/10.1515/hsz-2023-0235).

Footnotes

Research ethics: All animal procedures were in accordance with the guidelines for animal welfare used in scientific research of the Italian Government and the local governance.

Author contributions: AT and LAC concieved the project. AT, FJ, GB and LAC designed experiments. AT, FJ and JM performed experiments, AT and FJ analysed data, AT, FJ and LAC prepared figures and wrote the paper. The authors have accepted responsibility for the entire content of this manuscript and approved its submission.

Competing interests: The authors state no conflict of interest.

Research funding: Telethon foundation, proposal GGP19181.

Data availability: The raw data can be obtained on request from the corresponding author.

Contributor Information

Lorenzo A. Cingolani, Email: lcingolani@units.it.

Agnes Thalhammer, Email: agnes.thalhammer@units.it.

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Supplementary Materials

Supplementary Material

j_hsz-2023-0235_suppl_001.pdf (1.9MB, pdf)

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