Presynaptic α2δ-2 Calcium Channel Subunits Regulate Postsynaptic GABAA Receptor Abundance and Axonal Wiring

Abstract

Presynaptic α₂δ subunits of voltage-gated calcium channels regulate channel abundance and are involved in glutamatergic synapse formation. However, little is known about the specific functions of the individual α₂δ isoforms and their role in GABAergic synapses. Using primary neuronal cultures of embryonic mice of both sexes, we here report that presynaptic overexpression of α₂δ-2 in GABAergic synapses strongly increases clustering of postsynaptic GABA_ARs. Strikingly, presynaptic α₂δ-2 exerts the same effect in glutamatergic synapses, leading to a mismatched localization of GABA_ARs. This mismatching is caused by an aberrant wiring of glutamatergic presynaptic boutons with GABAergic postsynaptic positions. The trans-synaptic effect of α₂δ-2 is independent of the prototypical cell-adhesion molecules α-neurexins (α-Nrxns); however, α-Nrxns together with α₂δ-2 can modulate postsynaptic GABA_AR abundance. Finally, exclusion of the alternatively spliced exon 23 of α₂δ-2 is essential for the trans-synaptic mechanism. The novel function of α₂δ-2 identified here may explain how abnormal α₂δ subunit expression can cause excitatory-inhibitory imbalance often associated with neuropsychiatric disorders.SIGNIFICANCE STATEMENT Voltage-gated calcium channels regulate important neuronal functions such as synaptic transmission. α₂δ subunits modulate calcium channels and are emerging as regulators of brain connectivity. However, little is known about how individual α₂δ subunits contribute to synapse specificity. Here, we show that presynaptic expression of a single α₂δ variant can modulate synaptic connectivity and the localization of inhibitory postsynaptic receptors. Our findings provide basic insights into the development of specific synaptic connections between nerve cells and contribute to our understanding of normal nerve cell functions. Furthermore, the identified mechanism may explain how an altered expression of calcium channel subunits can result in aberrant neuronal wiring often associated with neuropsychiatric disorders such as autism or schizophrenia.

Keywords: auxiliary subunits; cacna2d; cultured hippocampal neurons; imaging; immunocytochemistry; voltage-gated calcium channels.

Figure 1.

Presynaptic expression of α₂δ-2 in cultured hippocampal neurons induces the formation of mismatched synapses. Representative immunofluorescence micrographs of cultured hippocampal neurons cotransfected with distinct α₂δ subunits and soluble eGFP. Putative presynaptic en passent boutons (arrowheads) were identified as eGFP-filled axonal varicosities along dendrites of untransfected neurons (axons are outlined with dashed lines). A, B, Double labeling of transfected neurons (20–30 DIV) for vGLUT1 and the GABA_AR _β2/3 subunit. Colocalization of fluorescence signals was analyzed using line scans. A, In control boutons (eGFP only), potential glutamatergic synapses are positive for presynaptic vGLUT1 but negative for postsynaptic GABA_AR (summarized in sketch). B, Presynaptic expression of α₂δ-2 (blue arrowheads) induces postsynaptic GABA_AR localization opposite transfected vGLUT1 positive nerve terminals. In contrast, postsynaptic GABA_ARs are not expressed opposite glutamatergic synapses expressing α₂δ-1, α₂δ-3, or eGFP only (control). C, D, Double labeling of transfected neurons (20–30 DIV) for vGAT and postsynaptic GABA_AR. Colocalization of fluorescence signals was analyzed using line scans. C, In control boutons (eGFP only), potential glutamatergic synapses are negative for presynaptic vGAT and postsynaptic GABA_AR (summarized in sketch). D, Transfected axonal varicosities were negative for vGAT in all conditions. Note the specific GABA_AR labeling opposite vGAT-negative nerve terminals expressing α₂δ-2 (blue arrowheads), which is in contrast to the colocalized fluorescence signals of vGAT and GABA_AR in untransfected GABAergic neighboring synapses (asterisks). Representative images of two independent culture preparations are shown. Scale bars, 10 μm (A,C) and 3 μm (B,D).

Figure 2.

Presynaptic expression of α₂δ-2 alters the postsynaptic composition of glutamatergic synapses. A, B, Representative immunofluoresence micrographs of cultured hippocampal neurons (24–30 DIV) cotransfected with distinct α₂δ subunits and soluble eGFP (control condition with eGFP alone is summarized in sketch, C). Colocalization of fluorescence signals was analyzed using line scans. A, Colabeling of the AMPA receptor subtype GLUR1 and the GABA_AR. Presynaptic expression of α₂δ-2 (blue arrowheads) induces postsynaptic GABA_AR localization with a concomitant reduction of the GLUR1 fluorescence signal. In contrast, postsynaptic GABA_ARs are not expressed opposite putative glutamatergic synapses expressing α₂δ-1, α₂δ-3 or eGFP only (control). B, Colabeling of GLUR1 and gephyrin, the scaffolding protein of GABAergic synapses. Presynaptic expression of α₂δ-2 (blue arrowheads) induces postsynaptic gephyrin localization with a concomitant reduction of the GLUR1 fluorescence signal. In contrast, postsynaptic gephyrin immunoreactivity is absent opposite putative glutamatergic synapses expressing α₂δ-1, α₂δ-3, or eGFP only (control). D–F, Quantitative analysis of GABA_AR (D), GLUR1 (E), and gephyrin (F) fluorescence intensities. Values were normalized to α₂δ-2 (D, F) or eGFP only (control, E) fluorescence intensities within each culture preparation. Note the significant increase of postsynaptic GABA_AR (D) and gephyrin (F) clusters opposite presynaptic boutons expressing α₂δ-2. In contrast, integrated intensity of GLUR1 was significantly reduced opposite axonal varicosities transfected with α₂δ-2 or α₂δ-3. Values for individual cells (dots) and means (line) ± SEM are shown. Data are shown from three independent culture preparations; 11–25 (D), 22–30 (E), and 11–28 (F) cells were analyzed in each condition. Statistics: D, Kruskal–Wallis ANOVA with Dunn's post hoc analysis: H₍₄₎ = 51.6, p < 0.0001, post hoc: ****p < 0.0001 between α₂δ-2 and all other conditions; E, ANOVA on log10-transformed data with Holm–Sidak post hoc analysis: F_(3,100) = 18.6, p < 0.001, post hoc: ***p < 0.001 between control/α₂δ-1 and α₂δ-2/α₂δ-3; F, Kruskal–Wallis ANOVA with Dunn's post hoc analysis: H₍₄₎ = 64.6, p < 0.0001, post hoc: ****p < 0.0001 between α₂δ-2 and all other conditions. Asterisks in graphs indicate the significant difference compared with control. Scale bars, 3 μm.

Figure 3.

GABA_AR clusters are confined to the postsynaptic membrane. gSTED micrographs of cultured hippocampal (A) or MSNs (C) transfected with α₂δ-2 and mCherry or mCherry only (control; 20–30 DIV). Transfected neurons (red, detected in confocal mode) were immunolabeled for the GABA_AR (green, detected in gSTED mode). In all conditions, postsynaptic GABA_AR clusters are closely opposed to mCherry-positive presynaptic boutons. B, 3D model showing that the GABA_AR staining pattern depends on the orientation of the imaged synapse, which applies both for hippocampal as well as MSNs. Scale bar, 1 μm.

Figure 4.

Presynaptic expression of α₂δ-2 induces the recruitment of synaptic GABA_AR subtypes. Representative immunofluorescence micrographs of cultured hippocampal neurons cotransfected with distinct α₂δ subunits and soluble eGFP. Transfected permeabilized neurons (20–30 DIV) were double stained for vGLUT1 and different postsynaptic GABA_AR subtypes. Colocalization of fluorescence signals within eGFP-filled axonal varicosities (arrowheads, axons are outlined with dashed lines) was analyzed using line scans. A, B, Immunofluorescence analysis identified intensely fluorescent clusters of the GABA_AR subunits α₁, α₂, β₃, and γ₂ opposite α₂δ-2 expressing glutamatergic (vGLUT1-positive) nerve terminals (A). In contrast, these postsynaptic GABA_AR subtypes are absent opposite putative glutamatergic synapses expressing eGFP only (control), α₂δ-1, or α₂δ-3 (B, micrographs depict examples for α₁). C, D, Labeling of the GABA_AR subunits α₃, α₄, β₂, and δ displayed weak and mainly extrasynaptic immunoreactivity in all conditions. Note that all GABA_AR subtypes presented for α₂δ-2 were also analyzed in hippocampal neurons expressing eGFP only, α₂δ-1, and α₂δ-3. Representative images of two independent cultures are shown. Scale bars, 3 μm.

Figure 5.

Potential mechanisms explaining the observed mismatched synapse formation of glutamatergic nerve terminals expressing α₂δ-2. A, Compensatory mechanism. Elevated expression of α₂δ subunits increases presynaptic calcium channel abundance and current densities and thus glutamate release. Therefore, GABA_ARs could be recruited to the dendritic spine in an attempt to compensate for excessive excitatory synaptic activity. If this is the case, then GABA_AR abundance at inhibitory synapses should not change upon α₂δ-2 overexpression in GABAergic presynaptic terminals. B, α₂δ-2 may be involved in trans-synaptically anchoring postsynaptic GABA_ARs. In this scenario, GABA_AR abundance should be increased when α₂δ-2 is overexpressed in both glutamatergic and GABAergic synapses. C, Trans-synaptic function of α₂δ-2 could also induce aberrant axonal wiring by guiding glutamatergic axons to GABAergic postsynaptic locations positioned along dendritic shafts. This is in contrast to the normal situation in which glutamatergic synapses are generally formed on dendritic spines.

Figure 6.

Striatum and cultured MSNs express three neuronal α₂δ subunits. A, Absolute qRT-PCR analysis revealed a stable expression of α₂δ-1, α₂δ-2, and α₂δ-3 in adult mouse striatum and monocultured MSNs (24–25 DIV). Although α₂δ-3 was the dominant isoform in striatal tissue, mRNA levels for α₂δ-2 and α₂δ-3 were similarly abundant in cultured MSNs. Error bars indicate mean ± SEM. Data from three independent culture/tissue preparations are shown. B, β-galactosidase staining of sagittal cryosections of α₂δ-3 knock-out mice carrying a LacZ cassette revealed intense labeling of striatum (Str), hippocampus (Hc), and olfactory tubercle (Ot). Lower expression was detected in the cortex (Cx), thalamus (Th), olfactory bulb (Ob), and parts of the cerebellum (Cb). C, Schematic representation of the epitope-tagged α₂δ subunits depicting the position of the extracellular 2HA tag inserted downstream of the signal peptide (SP), cache domains (C1–C4), and VWA (van Willebrand factor type A). Cultured MSNs were transfected with HA-tagged α₂δ subunits together with soluble eGFP and live labeled with an antibody against the HA epitope at 24 DIV. All α₂δ isoforms are expressed at the surface of presynaptic boutons, which is also shown by line scan analysis of α₂δ-1, α₂δ-2, and α₂δ-3 (red) in relation to synapsin (blue) and eGFP (green). The sketch summarizes the observed labeling patterns. Representative images of three (B) and one (C) independent preparation(s) are shown. Statistics: A, ANOVA on log10-transformed data with Holm–Sidak post hoc analysis: cultured MSNs: F_(3,8) = 460, p < 0.001; post hoc: p < 0.001 between all α₂δ subunits except α₂δ-2 vs α₂δ-3 (p = 0.34); striatum: F_(3,8) = 891, p < 0.001; post hoc: p < 0.001 between all α₂δ subunits except α₂δ-1 vs α₂δ-2 (p = 0.30). Scale bars, 2 mm (B) and 1 μm (C).

Figure 7.

Presynaptic and postsynaptic differentiation of GABAergic MSNs. A, Schematic illustration of the coculture procedure: Cerebral hemispheres were dissected and stripped of meninges (1). Parts of the prefrontal cortex and striatum were dissected as shown (2). Striatal and cortical tissue was collected separately and dissociated using trypsin-EDTA and trituration (3). Cortical neurons were plated on poly-l-lysine-coated coverslips while striatal neurons were transfected with soluble eGFP (4). Subsequently, striatal neurons were plated onto cortical neurons in a ratio of 3:2 (5) and maintained above a glial feeder layer (6). B, Lentiviral infection or lipofection with soluble eGFP allowed discriminating MSNs from cortical neurons. C–E, Double immunofluorescence of striatal–cortical cocultures (24–26 DIV) with presynaptic and postsynaptic markers for excitatory and inhibitory synapses. Neuronal morphology is outlined by eGFP. C, GABAergic synapses of transfected eGFP-positive MSNs showed immunoreactivity for vGAT, whereas vGLUT1 was absent. D, Axons of cortical neurons formed excitatory synapses on MSN spine heads (vGLUT1, white arrowheads), whereas GABAergic synapses were located on the dendritic shaft (vGAT, blue arrowhead). E, Labeling of PSD-95 in spine heads opposite synapsin-positive presynaptic terminals further indicated the presence of functional excitatory synapses on MSNs. F, Patch-clamp analysis of mIPSCs and mEPSCs in 14 DIV neurons confirmed the functionality of GABAergic and glutamatergic synapses. Representative micrographs of two independent cultures are shown. Scale bars, 50 μm (B), 3 μm (overview), and 1 μm (magnified selections; C–E).

Figure 8.

Presynaptic expression of α₂δ-2 induces upregulation of postsynaptic GABA_ARs in MSNs. A, B, Representative immunofluorescence micrographs of cultured MSNs cotransfected with distinct α₂δ subunits and soluble eGFP. Transfected neurons (21–28 DIV) were immunolabeled for vGAT and the GABA_AR. Colocalization of fluorescence signals within eGFP-filled axonal varicosities (arrowheads, axons are outlined with dashed lines) was analyzed using line scans. A, GABAergic synapses transfected with eGFP only (control) show matched presynaptic vGAT and postsynaptic GABA_AR immunoreactivity (summarized in sketch). B, Similar to control, postsynaptic GABA_ARs were localized opposite vGAT-positive presynaptic terminals expressing individual α₂δ isoforms (see also colocalization in line scans). Most importantly, GABA_AR clusters were larger and more intense opposite synaptic boutons expressing α₂δ-2 (blue arrowheads). C–F, Quantitative analysis of GABA_AR fluorescence intensity (C), cumulative frequency distribution of GABA_AR fluorescence intensity (D), bouton size (E), and vGAT fluorescence intensity (F). Values for individual cells (dots) and means (lines) ± SEM are shown. Values were normalized to the control within each culture preparation. Data from four independent culture preparations and 34–42 cells were analyzed in each condition. Statistics: ANOVA on log10-transformed data with Holm–Sidak post hoc analysis: C, F_(3,152) = 17.6, p < 0.001; post hoc: ***p < 0.001 between α₂δ-2 and control, ***p < 0.001 between α₂δ-2 and α₂δ-1/α₂δ-3, **p = 0.004 between control and α₂δ-3, p = 0.2 between control and α₂δ-1; E, F_(3,152) = 5.1, p < 0.01; post hoc: **p < 0.01 between α₂δ-3 and control/α₂δ-2, p = 0.08 between α₂δ-3 and α₂δ-1; F, F_(3,152) = 8.0, p < 0.001; post hoc: ***p < 0.001 between α₂δ-3 and control, **p < 0.01 between α₂δ-3 and α₂δ-1/α₂δ-2. Asterisks in graphs indicate the significant difference compared with control. Scale bars, 10 μm (A) and 3 μm (B).

Figure 9.

α-Nrxn modulates the effect of α₂δ-2 on GABA_ARs. Representative immunofluorescence micrographs of cultured hippocampal neurons (20–25 DIV) prepared from WT or α-Nrxn TKO mice. A, B, In control boutons (eGFP only), potential glutamatergic synapses are positive for presynaptic vGLUT1 but negative for postsynaptic GABA_AR staining (summarized in sketch, B). Consistent with our initial observation (see Fig. 1), presynaptic expression of α₂δ-2 (blue arrowheads) induces postsynaptic GABA_AR localization opposite vGLUT1-positive nerve terminals in WT neurons. Most importantly, in α-Nrxn TKO synapses, the expression of α₂δ-2 strongly induced postsynaptic GABA_AR localization (blue asterisks). C, Postsynaptic GABA_ARs clusters (integrated intensities) are significantly increased opposite glutamatergic nerve terminals expressing α₂δ-2 compared with control (eGFP only). This effect is much stronger in α-Nrxn TKO synapses compared with WT neurons (3-fold higher GABA_AR fluorescence intensity). D, Cumulative frequency distribution further reveals that the vast majority of glutamatergic boutons are positive for postsynaptic GABA_ARs in WT+α₂δ-2 (88%) and TKO+α₂δ-2 (97%) and that the population is shifted toward larger and more intense clusters. However, this analysis also demonstrates that mismatched GABA_ARs already form opposite α-Nrxn TKO synapses, although at a lower overall intensity. Values for individual cells (dots) and means (lines) ± SEM are shown. Values were normalized to the WT control within each culture preparation. Data from two independent culture preparations and 12–19 cells were analyzed within each condition. Statistics: C, ANOVA on log10-transformed data with Holm–Sidak post hoc analysis: genotype: F_(1,86) = 5.3, p = 0.023; transfection: F_(2,86) = 49.0, p < 0.001; genotype × transfection: F_(2,86) = 3.7, p = 0.03; post hoc: *p < 0.05, ***p < 0.001. Asterisks in graphs indicate the significant difference compared with control. Scale bar, 3 μm.

Figure 10.

Mismatched synapses show characteristics of GABAergic shaft synapses. A, D, Representative immunofluorescence micrographs of cultured hippocampal neurons cotransfected with α₂δ-2 and soluble eGFP. Colabeling of transfected neurons (24 DIV) for postsynaptic GLUR1 and gephyrin is shown. A, Presynaptic expression of α₂δ-2 induces a strong upregulation of postsynaptic gephyrin (asterisk) compared with neighboring endogenous GABAergic synapses situated on the same dendrite (arrowheads). Although GLUR1 expression was reduced in mismatched synapses (*; see Fig. 2), it was absent in endogenous GABAergic synapses (arrowheads, see sketch in B). C, Quantitative analysis of gephyrin fluorescence intensities in GABAergic neighboring and mismatched synapses. Dots represent values for individual boutons (mismatched synapses) and means of 10 endogenous clusters measured per image. Results were normalized to endogenous gephyrin intensities of neighboring synapses within each culture preparation. Data from three independent culture preparations are shown. Statistics: t test on log10-transformed data: t₍₅₀₎ = 9.5; ***p < 0.001. D, Left, Control boutons transfected with eGFP only form glutamatergic synapses on dendritic spines (gephyrin negative, GLUR1 positive; white arrowheads). Right, Expression of α₂δ-2 in putative glutamatergic boutons induces the formation of mismatched synapses (gephyrin-positive, GLUR1-negative) along the shaft of the dendrite (blue arrowheads). Dendritic morphology of untransfected neurons was outlined according to GLUR1 labeling. Scale bars, 5 μm (A) and 4 μm (B).

Figure 11.

Presynaptic α₂δ-2 induces the aberrant wiring of glutamatergic axons to dendritic shafts. A, D, To analyze the position of α₂δ-2-induced mismatched synapses on postsynaptic dendrites of excitatory (A, hippocampal neurons) and inhibitory neurons (D, MSNs), presynaptic (control or α₂δ-2) and postsynaptic neurons were labeled with mCherry (red) and eGFP (green), respectively (for details, see Materials and Methods). The magnified insets in A and D demonstrate the close association of presynaptic axonal varicosities (mCherry) with postsynaptic dendritic spines (eGFP), as expected for excitatory spine synapses. Using gSTED microscopy, the distance from the dendritic shaft to the contact zone of each synaptic bouton was measured (A, arrows and lines in the magnified inset). B, E, gSTED microscopy confirms the preferential location of excitatory synapses on dendritic spines of both glutamatergic or GABAergic neurons (white arrowheads and sketch in B) and, most importantly, suggests an aberrant wiring of putative glutamatergic axons expressing α₂δ-2 with postsynaptic sites along dendritic shafts (blue arrowheads and sketch in B), as typically observed for GABAergic synapses. C, Corresponding postsynaptic location of each contacting bouton was categorized as spine (green), shaft (gray), or as unclear (white; top) and the distance from the dendritic shaft to the contact point was measured (bottom). Although the vast majority of synaptic contact points in the control condition are located on dendritic spines (80%), presynaptic expression of α₂δ-2 shifted the preferential contact points to dendritic shafts (65%, top). This is further confirmed by a significantly decreased contact point to shaft distance of α₂δ-2-expressing compared with control synapses. Values for individual boutons (dots) and means (lines) ± SEM are shown. Dot colors (bottom) show the respective categorized synapse position (spine, green; shaft, gray; and unclear, white) presented in the top. Data from two independent culture preparations and 118 (control) and 75 (+α₂δ-2) boutons were analyzed. Statistics: C, top: χ² test: χ²₍₂₎ = 63.4, ***p < 0.001; bottom: Mann–Whitney U test: ***p < 0.001. E, F, gSTED microscopy (E) and high-resolution fluorescence microscopy (F) revealed that, similar to hippocampal neurons, presynaptic expression of α₂δ-2 in cortical neurons shifted the preferential contact points to dendritic shafts of GABAergic MSNs, as indicated by gephyrin labeling and the dendritic position (blue arrowheads and line scan in F). Representative micrographs of two independent cultures are shown. Scale bars, 50 μm (overview), 3 μm (insets, A, D), and 2 μm (B, E, F).

Figure 12.

Presynaptic expression of α₂δ-2 affects synaptic transmission in aberrantly wired synapses. A–C, Experimental paradigm to study the functional consequences of presynaptic α₂δ-2 overexpression in mismatched glutamatergic synapses of cultured hippocampal neurons. A, Neurons were cultured in pairs of two synaptically connected cells in which one represents an untransfected control neuron (arrowhead) and the other an α₂δ-2-overexpressing neuron (eGFP labeled). B, Whole-cell patch-clamp recordings of excitatory synaptic transmission (13–18 DIV). An action potential in neuron 1 elicited by a given AP waveform recorded from WT hippocampal neurons caused eEPSCs in neuron 2. Synaptic transmission was analyzed in both directions of synaptically connected pairs. Therefore, putative glutamatergic spine synapses (control innervating α₂δ-2-overexpressing neuron) and putative mismatched synapses (α₂δ-2 overexpressing innervating control neuron) could be directly compared within the same pair. C, D, Quantitative analysis of mean eEPSC peak amplitudes (C) and paired-pulse response ratios (PPRs) as a measure for synaptic plasticity (D). C, eEPSCs were strongly reduced (48%) in mismatched synapses. D, Dashed line shows the boundary between paired-pulse depression (PPR < 1) and facilitation (PPR > 1). Note the slight significant increase in facilitation in mismatched synapses at 25 and 50 ms compared with the initial 10 ms interval. Values for individual pairs (dots) and means (lines) ± SEM are shown. Data from three independent culture preparations from seven (C) and six (D) pairs were analyzed. Statistics: C, paired t test: t₍₆₎ = 4.6; **p < 0.01; D, two-way repeated-measures ANOVA: condition: F_(1,45) = 0.02, p = 0.9; interval: F_(5,45) = 11.1, p < 0.001; condition × interval: F_(5,45) = 2.7, p = 0.03; Holm–Sidak post hoc analysis: **p = 0.004, ***p < 0.001 compared with 10 ms within α₂δ-2 to control. Scale bar, 20 μm.

Figure 13.

Lack of exon 23 in α₂δ-2 splice variants mediates the trans-synaptic effect on GABA_ARs. A, Schematic overview illustrating the approximate positions of three alternatively spliced regions of α₂δ-2. Sequence alignment between α₂δ-2-v1, the original construct used in this study, and two additional α₂δ-2 variants (v2, v3) reveals alternative splicing of exons 23, 30, and 38. B, Using homology modeling, we tested the potential consequences of alternative splicing on the structure prediction based on the high-resolution structure of α₂δ-1 (PDB code: 5GJV; Wu et al., 2016; see Fig. 14). Inclusion of exon 23 in α₂δ-2-v2 suggested the formation of an extra loop (arrowhead) leading to the disruption of an α-helix present in α₂δ-2-v1 and α₂δ-2-v3. C, Representative immunofluorescence micrographs of cultured hippocampal neurons (21–26 DIV) cotransfected with distinct α₂δ-2 splice variants and soluble eGFP. Presynaptic expression of either α₂δ-2-v1 (original construct) or α₂δ-2-v3, which both lack exon 23, robustly induces a mismatched GABA_AR localization opposite vGLUT1-positive nerve terminals (blue arrowheads). In contrast, presynaptic expression α₂δ-2-v2, which contains exon 23, failed to induce mismatched synapse formation (white arrowheads, line scans). Asterisks mark endogenous GABA_AR clusters from untransfected neighboring synapses not colocalizing with presynaptic vGLUT1. Representative images of two independent cultures are shown. Scale bar, 3 μm.

Figure 14.

Splicing sites of α₂δ-2 and implications on secondary structure. A, Using homology modeling, we tested the potential consequences of alternative splicing on the structure prediction of three distinct α₂δ-2 splice variants (v1–v3) based on the high-resolution structure of α₂δ-1 (PDB code: 5GJV; Wu et al., 2016). Although some of the longer loops generally seemed to be quite flexible and differently orientated in all three models (gray arrows), alternative splicing of exons 23, 30, and 38 resulted in differences between secondary structure elements in distinct variants (boxed regions in B–D). B, Higher magnification showing that the inclusion of exon 23 in α₂δ-2-v2 suggests the formation of an extra loop (arrowhead and green selection in overlay), leading to the disruption of an α-helix present in α₂δ-2-v1 and α₂δ-2-v3 (see also Fig. 13). C, Structure modeling further proposes that the insertion of three base pairs in exon 30 of α₂δ-2-v3 causes the formation of a short α-helix in the depicted loop, which is absent in α₂δ-2-v1 and α₂δ-2-v2. D, Moreover, models implicate that alternative splicing of exon 38 affects the length of a β sheet at this position, which is longer in α₂δ-2-v1 and α₂δ-2-v2.