Introduction

Autism spectrum disorder (ASD) is a complex neuro-developmental disorder and affects about 1% of the population worldwide (Won et al. 2013). Even though ASD is mainly seen as a genetic disease, the exact etiology is only known in about 20% of the cases, mostly for monogenetic forms of ASD like Timothy syndrome (Splawski et al. 2004; Miles 2011). In ASD, the wide range of genetic findings points to various signaling pathways involved in the manifestation of this disorder (O’Roak et al. 2011; Ebert and Greenberg 2013). A gene pathway analysis with over 2600 patients recognized the “calcium signaling pathway” among the top three pathways involved in ASD (Skafidas et al. 2014). Since the discovery that a single point mutation in the α1-subunit of L-type calcium channels (CaV1.2) causes Timothy syndrome, many other genes encoding voltage-gated calcium channel (VGCC) subunits have been associated with ASD like the genes CACNA1B-F encoding pore-forming subunits, CACNB1 and CACNB2 encoding auxiliary β-subunits and, CACNA2D3 and CACNA2D4 for auxiliary α2-δ-subunits (for a comprehensive overview, see Breitenkamp et al. 2015).

Voltage-gated calcium channels (VGCC) are heteromultimeric protein complexes whose composition of pore-forming and auxiliary subunits determines their biophysical properties. High-voltage activated VGCCs are heteromers composed of up to four subunits (CaVα1, CaVβ, CaVα2-δ, CaVγ) (Buraei and Yang 2010; Campiglio and Flucher 2015). The subunit of particular importance for the regulation of channel function is the CaVβ-subunit, with four different isoforms (CaVβ1-4) involved in the regulation of voltage dependence, channel activity, current kinetics, and also the membrane targeting of functional channels (Meir et al. 2000; Dolphin 2012). Structurally, CaVβ-subunits consist of five domains: a variable N-terminus, a conserved Src homology 3 domain (SH3), a conserved but alternatively spliced HOOK domain, a guanylate kinase domain (GK), and a variable C-terminus (Buraei and Yang 2010). With the exception of some rare splice variants, all CaVβ-subunits have the same functional structure and show a significant scale of homology and conservation indicating their functional importance (Hibino et al. 2003; Hofmann et al. 2014).

In a previous study, we found three rare missense mutations in highly conserved regions within the susceptibility gene CACNB2 in ASD-affected patients, all of them within the HOOK-domain (Breitenkamp et al. 2014). In whole-cell recordings, two of the resulting CaVβ2 mutants (p.G167S, p.S197F) led to a slowed and the third (p.F240L) to an accelerated current inactivation. In a detailed subsequent study, we could show that these mutations and an N-terminal mutation at position 2 (p.V2D) led to a significantly higher single-channel activity compared to channels containing a wild-type (WT) CaVβ2 (Despang et al. 2020). Mutated and WT CaVβ2 subunits interacted with the inhibitory RGK-protein Gem but differed in the extent and characteristics of being modulated by Gem. More recently, we described a homozygous N-terminal CaVβ2 mutant at position 70 (p.R70C) that also showed an increased activity in terms of a reduced inactivation of L-type calcium currents at the whole-cell level (Graziano et al. 2021).

We report here the identification and first electrophysiological characterization of CaVβ1b_R296C, a novel variant of the β1-isoform of VGCC subunits found in an ASD patient. Different genetic studies suggested that similar to CaVβ2, CaVβ1 may contribute to ASD. A strong evidence of linkage was found for the chromosomal region 17q11-21 that contains CACNB1, the gene encoding CaVβ1 (Cantor et al. 2005). In a meta-analysis combining affected sib pairs of five genome-wide linkage scans for ASD, no significant chromosomal region for ASD was found (Trikalinos et al. 2006). However, the authors found two chromosomal regions with a suggestive significance, namely, 17p11.2–q12 and 10p12– q11.1, which comprise the genes encoding CaVβ1 and CaVβ2, respectively.

Given our previous studies indicating Ca2+-current enhancement as a common feature of CaVβ2 variants found in ASD patients, we characterized the electrophysiological properties of the new CaVβ1 variant introduced here. Since the different CaVβ2 variants have shown a kind of “electrophysiological” fingerprint including differential interaction with the RGK-protein Gem, we analyzed CaVβ1-Gem interaction, too. We chose CaV1.2 as a pore-forming subunit for several reasons: (1) CaV1.2 is expressed in neuronal tissue and has been associated with several neuro-psychiatric diseases (Catterall et al. 2021). (2) A mutant CaV1.2 protein characterized by impaired current inactivation has been shown to underly the Timothy syndrome, a monogenetic “model disease” for ASD (Splawski et al. 2004; Breitenkamp et al. 2015). (3) In our previous studies, we identified functional effects of CaVβ2 variants when co-expressed with CaV1.2 (Breitenkamp et al. 2014; Despang et al. 2020; Graziano et al. 2021). For the following reasons, CaV2.2 was chosen as a second VGCC pore subunit for our analysis of the CaVβ1b_R296C variant: CaV2.2 is expressed predominantly in neurons where most of the central nervous system’s synapses rely on CaV2.2 and CaV2.1 for fast synaptic transmission (Wheeler et al. 1994; Catterall et al. 2021). Three different CaVβ subunits associate with the pore-forming subunit CaV2.2, of which CaVβ1b is an isoform (Scott et al. 1996; Müller et al. 2010).

Material and methods

DNA constructs and site-directed mutagenesis

For functional analysis, the mutation p.R296C was introduced in human CaVβ1b (NM_000723) by site-directed mutagenesis (Stratagene QuikChange Kit) and verified by sequencing. EGFP was used as reporter gene, which was co-expressed together with the CaVβ-subunit by the bicistronic pIRES2-EGFP vector (Clontech). R296C forward primer 5'-cattgagcgctccaacacatgctccagcct-3'; R296C reverse primer 5'-aggctggagcatgtgttggagcgctcaatg-3'. For co-immunoprecipitation experiments, a hemagglutinin (HA) tag was introduced in the C-terminus of the respective CaVβ1b isoform and a Flag tag was introduced at the N-terminus of Gem. Cloning was done by using an in-fusion cloning kit (Takara Bio Europe, Gothenburg, Sweden) according to the manufacturer’s manual and verified by sequencing. EGFP was used as reporter gene for CaVβ1b and YFP as reporter for Gem.

Cell culture and transfection

In brief, HEK-293 cells were grown in Petri dishes in Dulbecco’s modified Eagle’s medium (Gibco Thermo Fisher, Waltham, MA, USA) supplemented with 10% FCS (Biochrom GmbH, Berlin Germany). Cells were routinely passaged twice a week and incubated at 37°C under 5% CO2 growth conditions. tsA-201 cells were cultured at 37°C and 5% CO2 in a 60-mm-diameter cell culture dish in 4-ml DMEM GlutaMAX medium (Biochrom GmbH, Berlin Germany) with 10% FCS (Biochrom GmbH, Berlin Germany) and penicillin-streptomycin (Sigma-Aldrich, St. Louis, MO, USA). Cells were routinely passaged twice a week. HEK-293 and tsA-201 cells were transfected using standard calcium phosphate method (Koch et al. 2016). For whole-cell and single-channel recordings, HEK-293 cells were transfected with a 1:0.5:1.5 ratio of CaV1.2 (α1c#77, NM_001129843.2) or CaV2.2 (NM_000718.3), either human WT (NM_000723) or mutant CaVβ-subunit and human CaVα2-δ1-subunit (NM_000722.3). For co-immunoprecipitation experiments, tsA-201 cells were transfected with a 1:2 ratio of either human WT or mutant CaVβ1b_HA-subunit and human Flag-Gem.

Co-immunoprecipitation and Western blot

The Gem and CaVβ co-immunoprecipitation experiments were performed as described previously (Despang et al. 2020). In brief, tsA cells were harvested 48–72 h after transfection in ice cold PBS and lysed in lysis buffer (50-mM Tris; 100-mM NaCl; 10-mM EDTA; 0.4% TritonX-100; 0.4% NP-40; pH: 7.5 with HCl). Protease inhibitor cocktail tablets (Sigma-Aldrich) were added immediately before lysing cells. Cell lysates were incubated for 1h at 4°C with rotation and afterwards for 30 min on ice. Cell lysates were then centrifuged for 15 min to remove cell debris. Lysate of 400μl of was incubated with 50μl (500μg)-Pierce™ Protein A/G Magnetic Beads (Thermo Scientific) and 5μl-anti-FLAG-Antibody M2 (Sigma-Aldrich) or 10-μl anti-HA 3F10 (Roche) at 4°C overnight. Beads were washed two times for 15–30 min in lysis buffer, and then proteins were eluted in 2x Laemmli buffer, incubated at 50°C for 10 min and frozen at −20°C. Elutions were separated using SDS-PAGE, followed by immunoblotting with anti-HA antibody (1:1000, Covance) and anti-FLAG antibody M2 (1:1000, Sigma-Aldrich).

Electrophysiological recordings

Whole-cell recordings were performed as in our previous studies (Breitenkamp et al. 2014; Despang et al. 2020; Graziano et al. 2021). Recordings of EGFP-positive cells were obtained 48–72 h after transfection. Immediately prior to recording, cells kept in 35-mm culture dishes were washed at room temperature (19–23°C) with bath solution. For whole-cell experiments with CaVβ1b and α1C (CaV1.2), the bath solution contained (in mM) 10 BaCl2, 1 MgCl2, 10 HEPES, 65 CsCl, 40 TEA-Cl, 10 Glucose (pH 7.3 with TEA-OH), and the pipette solution (in mM) 140 CsCl, 10 EGTA, 9 HEPES, 1 MgCl2, and 4 MgATP (pH 7.3 with CsOH). For whole-cell experiments with CaVβ1b and α1B (CaV2.2), the bath solution contained (in mM) 20 BaCl2, 1 MgCl2, 10 HEPES, 125 NaCl (pH 7.3 with NaOH), and pipette solution (in mM) 135 CsCl, 10 EGTA, 10 HEPES, and 4 MgATP (pH 7.5 with CsOH). Holding potential was −80 mV. Patch pipettes made from borosilicate glass (1.7-mm diameter and 0.283-mm wall thickness, Hilgenberg GmbH, Malsfeld, Germany) were pulled using a Sutter Instrument P-97 horizontal puller and fire-polished using a Narishige MF-83 microforge (Narishige Scientific Instrument Lab, Tokyo, Japan). Pipette resistance was 2–4 MΩ. Using a holding potential of −80 mV, currents were elicited by applying 500, 1000, or 1500 ms test pulses of −40 mV to +50 mV. Currents were sampled at 10 kHz and filtered at 2 kHz. We used the Clampex software pClamp 10 and an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA, USA).

Voltage dependence of Ba2+ whole-cell current inactivation was determined with a two-pulse protocol (a 2500-ms prepulse followed by a 200-ms test pulse at +10mV). For steady-state inactivation, the relative magnitude of inward current elicited during the second pulse was plotted as a function of the voltage of the conditioning first pulse. Data were analyzed using pClamp10 (Molecular Devices, Sunnyvale, CA, USA) and GraphPad 6 Prism. Currents were filtered at 2 kHz. Data were fitted to a Boltzmann function to obtain the half point (V0.5) and slope factor (dV) for the voltage dependence of inactivation. Fits were performed after subtracting the offset from the peak values of the steady-state inactivation data. Offset was defined as a deviation from zero at the end of a fully inactivating current (at the end of the test pulse at +10mV). For voltage dependence of activation, data were fitted by combined Ohm and Boltzmann relation \(\mathrm{I}\left(\mathrm{V}\right)=\left(\mathrm{V}\hbox{--} \mathrm{VR}\right)\ \mathrm{x}\frac{G_{max}}{\left(1+\exp \frac{\left({V}_{0.5}-V\right)}{dV}\right)}\) according to Karmazinova and Lacinova (Karmažínová and Lacinová 2010).

Single-channel recordings were performed at room temperature (19–23°C) using EGFP-positive cells 48–72 h after transfection. The bath solution contained (in mM) 120 K-glutamate, 25 KCl, 2 MgCl2, 10 HEPES, 1 CaCl2, 1 Na-ATP, and 2 EGTA (pH 7.4 with KOH). Patch pipettes were made from borosilicate glass (1.7-mm diameter and 0.283-mm wall thickness, Hilgenberg GmbH, Malsfeld, Germany) using a Narishige PP-83 vertical puller, Sylgard coated and fire-polished using a Narishige MF-83 microforge (Narishige Scientific Instrument Lab, Tokyo, Japan). Pipette resistance was 5–7 MΩ. Pipettes were filled using an internal solution containing (in mM) 110 BaCl2 and 10 HEPES (pH 7.3 with TEA-OH). Single calcium channels were recorded in the cell-attached configuration (depolarizing test pulses of 150 ms at 1.67 Hz from −20mV to +20 mV in 10 mV steps from a holding potential of −100mV). An Axopatch 1D amplifier with pClamp 5.5 software (both from Axon Instruments, Foster City, CA, USA) was used for pulse generation, data acquisition (10 kHz), and filtering (2 kHz, −3 dB, 4-pole Bessel). Experiments were analyzed when channel activity persisted > 180 sweeps using pClamp 10 software (Molecular Devices).

Data analysis

Most experimental data are shown as raw currents, i.e., without capacity or leak subtraction. In a few experiments, for reasons of clarity, the capacitive transients were removed off-line. For calculation of the activation, the leak-current component was obtained from hyperpolarizing voltage steps from Vh and fitted using linear regression. The extrapolated leak current was then subtracted from the current-voltage (I-V) plot before estimation of the reversal potential. For single-channel analysis, leak and capacity currents were digitally subtracted using pClamp 10 software (Molecular Devices). Channel openings and closures were determined using the half-height criterion. The availability (fraction of active sweeps, Factive), open probability (Popen), and mean open time were analyzed for single-channel as well as multi-channel patches. For the latter, these parameters were corrected for the number of channels in the patch (Herzig et al. 2007).

Statistical analysis

Electrophysiological data were obtained in experiments revealed from > 3 individual transfections per plasmid composition and are shown as mean ± SEM. Experiments with WT and mutant were performed on the same day. Data were analyzed using unpaired two-sided Student t tests. Differences were considered statistically significant if p < 0.05.

Ethics approval

The local ethics committee approved the analyses (ref. No. 04-223) as described earlier (Breitenkamp et al. 2014).

Results

Description and genetic assessment of the CaVβ1b mutation

We refer to the same probes analyzed previously (Breitenkamp et al. 2014). In a mutation screening involving 155 ASD patients, we found a rare missense mutation in the candidate gene CACNB1 leading to an arginine-to-cysteine exchange (p.R296C) in the highly conserved GK-domain of the CaVβ1 subunit (NP_000714.3). The variant was found in a male patient (AGRE ID: AU09935301), but in none of 259 matching controls. The mutation was present in all splice variants of the patient’s CACNB1 gene. No other pathogenic or suspicious mutations known to contribute to neurodevelopmental disorders were found. No data on the presence of the CACNB1 mutation in his parents are available, but they were not diagnosed with ASD.

The variant (chr17:37340296G>A; GRCh37) is listed in dbSNP (rs746198242) and in the gnomAD and ExAC databases with an allele frequency of 1.6 x 10-5 and 1.7 x 10-5, respectively. The here presented CACNB1 variant p.R296C is not listed in ClinVar (https://www.ncbi.nlm.nih.gov/clinvar) and has not been linked to ASD so far. According to the web-based program “PolyPhen2” (Adzhubei et al. 2010), the p.R296C missense variant is classified as damaging with 100% probability. According to standards and guidelines for the international interpretation of sequence variants (Richards et al. 2015), the rare frequency, the location of the variant in an essential and highly conserved protein domain, and the predictions render this variant a “variant of uncertain significance” (class 3, due to AGMC criteria PM2, PM1 and PP3). In the following, we tested for putative functional effects in electrophysiological patch-clamp experiments. Unlike CaVβ1a and CaVβ1c, the splice variant CaVβ1b is expressed in the brain and is upregulated during ontogenesis (Buraei and Yang 2010). Therefore, we subcloned the mutation into the splice form CaVβ1b (NM_000723).

Functional characterization of the CaVβ1b variant

Whole-cell recordings

When co-expressing CaV1.2 with the CaVβ1b_R296C mutant, whole-cell current density was decreased compared to the wild-type CaVβ1b (e.g., 21.0 ± 2.5 vs. 11.7 ± 1.6 pA/pF at 0 mV, p=0.003; Figs. 1a and b) along with a shift in the half-maximal activation potential V0.5act to more positive values (−13.3 ± 1.9 vs. −8.0 ± 1.5 mV, p=0.038; Fig. 1c). Furthermore, time-dependent inactivation was increased at 150 ms (e.g., 25 ± 4 vs. 39 ± 4 % at +10 mV, p=0.015) and 300 ms (e.g. 52 ± 4 vs. 65 ± 4 % at +10 mV, p=0.03) (Figs. 2a and b). No statistically significant differences were found at 500 ms, 1000 ms, and 1500 ms (not shown). Analysis of steady-state inactivation revealed a non-significant (p=0.071) shift of CaVβ1b_R296C to more positive potentials compared to CaVβ1b_WT (Fig. 2c). Similar to CaV1.2, the current density was decreased when CaV2.2 was co-expressed with CaVβ1b_R296C compared to CaVβ1b_WT, although the difference was only statistically significant at 0 mV (p=0.045). V0.5act was shifted to more positive values by CaVβ1b_R296C (−1.1 ± 2.4 vs. 6.2 ± 1.6 mV, p=0.017; Fig. 3c). No difference in time-dependent inactivation or steady-state inactivation was found between mutant and wild-type CaVβ1b subunits (not shown).

Fig. 1
figure 1

Whole-cell currents mediated by CaV1.2 co-expressed with CaVα2-δ1 and either the wild-type (CaVβ1b_WT) or mutant (CaVβ1b_R296C) form of CaVβ1b. (a) Exemplary traces of whole-cell recordings with either CaVβ1b_WT (left) or CaVβ1b_R296C (right). (b) IV-relationships of CaVβ1b_WT (n=18) and CaVβ1b_R296C (n=18). Starting from a holding potential of −80 mV, currents were elicited at test potentials from −40 to +50 mV in 10 mV increments using 10 mM Ba2+ as charge carrier. (c) Test potential of the half-maximal activation (V0.5act). Data were obtained using patch-clamp recordings in the whole-cell configuration. *,**: p<0.05 and p<0.01 in Student’s t tests

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Fig. 2
figure 2

Time-dependent inactivation behavior of CaV1.2 co-expressed with CaVα2-δ1 and either the wild-type (CaVβ1b_WT; n=18) or mutant (CaVβ1b_R296C; n=18) form of CaVβ1b, analyzed as the fraction of peak current inactivated after 150 ms (a) or 300 ms (b) of depolarization. (c) Voltage-dependent steady-state inactivation behavior of CaVβ1b_WT (n=14) compared to CaVβ1b_R296C (n=13). Data were obtained using patch-clamp recordings in the whole-cell configuration. *: p<0.05 in Student’s t tests

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Fig. 3
figure 3

Whole-cell currents mediated by CaV2.2 co-expressed with CaVα2-δ1 and either the wildtype (CaVβ1b_WT) or mutant (CaVβ1b_R296C) form of CaVβ1b. (a) Exemplary traces of whole-cell recordings with either CaVβ1b_WT (left) or CaVβ1b_R296C (right). (b) IV-relationships of CaVβ1b_WT (n=19) and CaVβ1b_R296C (n=21). Starting from a holding potential of -80 mV, currents were elicited at test potentials from -40 to +50 mV in 10 mV increments using 20 mM Ba2+ as charge carrier. (c) Test potential of the half-maximal activation (V0.5act). Data were obtained using patch-clamp recordings in the whole-cell configuration. *: p<0.05 in Student’s t tests

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Single-channel recordings

At the single-channel level, CaVβ1b_R296C showed statistically significant inhibitory effects on both CaV1.2 and CaV2.2. Fraction of active sweeps (Factive) and open probability (Popen) of CaV1.2 were significantly reduced when co-expressing CaVβ1b_R296C instead of the CaVβ1b_WT (Figs. 4 ba and bb). The latency until a first opening and the extent of inactivation were increased (Figs. 4 bc and bd). Similar effects of CaVβ1b_R296C were seen on the activity of single CaV2.2 reaching statistical significance for fraction of active sweeps and first latency (Figs. 4 ca and cc).

Fig. 4
figure 4

Gating of single L-type (CaV1.2, left) and N-type (CaV2.2, right) channels in the presence of a wildtype (CaVβ1b_WT) or the mutant (CaVβ1b_R296C) form of CaVβ1b. (a) Exemplary traces. Top: pulse protocol (150 ms duration, holding potential of -100 mV, test pulse to +10 mV). Middle: ten consecutive traces for each channel complex. Bottom: ensemble average currents of the respective experiments. (b, c) Single-channel gating parameters of human CaV1.2 (ba-bd; n=9–10) or CaV2.2 (ca-cd; n=5–6) transiently expressed in HEK-293 cells and co-transfected with CaVβ1b_WT or CaVβ1b_R296C and a human CaVα2-δ1. Statistical analyses revealed a significant decrease in the fraction of active sweeps and an increase in first latency in the presence of CaVβ1b_R296C (ba, bc, ca, cc). The reduction of the open probability was statistically significant with CaV1.2 (bb) but slightly missed statistical significance with CaV2.2 (cb; p=0.07). Inactivation was significantly increased when co-expressing CaVβ1b_R296C together with CaV1.2 (bd), but unaffected with CaV2.2 (cd). Data were obtained using patch-clamp recordings in the cell-attached configuration using 110mM Ba2+ as charge carrier. *: p<0.05 in Student’s t tests

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Effects of Gem co-expression on whole-cell and single-channel activity

Gem is an RGK-protein that interacts with auxiliary CaVβ subunits and inhibits VGCCs (Yang and Colecraft 2013). As in our previous study (Despang et al. 2020), we analyzed the interaction of Gem with the mutated CaVβ1b and the effects on CaV1.2-mediated Ba2+ currents. Similar to our previous findings with CaVβ2d, co-immunoprecipitation experiments showed an interaction of Gem with both mutated and wild-type CaVβ1b subunits (Fig. 5a). In whole-cell experiments, we saw an almost complete inhibition of Ba2+ currents compared to experiments without Gem transfection (Figs. 5b and c). Gem reduced whole-cell currents similarly in the presence of CaVβ1b_WT or CaVβ1b_R296C (Fig. 5c). Corresponding to our findings at the whole-cell level, we found Gem to reduce single-channel activity, e.g., by lowering the fraction of active sweeps or open probability (Fig. 6).

Fig. 5
figure 5

CaVβ-Gem interaction and Gem effects on CaV1.2-mediated whole-cell currents in the presence of either the wild-type (CaVβ1b_WT) or mutant (CaVβ1b_R296C) form of CaVβ1b. (a) Gem binds to both, CaVβ1b_WT and CaVβ1b_R296C. For co-immunoprecipitation experiments, tsA-201 cells were co-transfected with cDNA of Flag-tagged Gem and HA-tagged CaVβ1b subunits as indicated. Flag-tagged Gem proteins were immunoprecipitated (IP) and bound CaVβ1b protein was detected by Western-blot. Interaction was confirmed by reciprocal immunoprecipitation. Cell lysates were blotted with a HA- or Flag-antibody to verify CaVβ1b and Gem expression, respectively. All experiments were repeated at least three times. (b) Exemplary traces of whole-cell recordings in HEK293 cells with expression of CaVβ1b_WT at a test potential of 0 mV. Exemplary traces are shown as overlay to better visualize the reduction of currents in the presence of Gem. (c) IV-relationships in Gem-transfected HEK293 cells with expression of either CaVβ1b_WT or CaVβ1b_R296C (solid curves). For comparison, IV-relationships without co-transfection of Gem is shown as dashed curves (taken from Fig. 1). Starting from a holding potential of −80 mV, currents were elicited at test potentials from −40 to +50 mV in 10 mV increments using 10 mM Ba2+ as charge carrier. Data were obtained using patch-clamp recordings in the whole-cell configuration

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Fig. 6
figure 6

Effects of Gem on single-channel behavior of L-type (CaV1.2) calcium channels in the presence of either the wild-type (CaVβ1b_WT) or the mutant (CaVβ1b_R296C) form of CaVβ1b. (a) Ten representative consecutive traces obtained at a test potential of +10 mV in Gem-transfected HEK293 cells expressing either CaVβ1b_WT (left) or CaVβ1b_R296C (right). Fraction of active sweeps (b) and open probability (c) indicate similarly reduced activity of single CaV1.2 when Gem was co-expressed together with either CaVβ1b_WT or CaVβ1b_R296C. Number of experiments are indicated in the figure. Asterisks indicate p<0.05 in Student’s t tests. Data were obtained using patch-clamp recordings in the cell-attached configuration (charge carrier: 110 mM Ba2+)

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Discussion

In the last years, the evidence for the involvement of VGCCs in ASD has been growing and actually for most VGCC genes evidence for an association with ASD was found (Breitenkamp et al. 2015). Our group focuses on auxiliary CaVβ subunits of VGCCs and their putative contribution to this complex-genetic disease. To our knowledge, this is the first report of a mutation in CACNB1 identified in an ASD patient. We previously described different mutations of the CaVβ2 isoform which led to an increase of CaV1.2 activity both at the single-channel and the whole-cell level (Breitenkamp et al. 2014; Despang et al. 2020). In a more recent study, we could show that another mutation in CACNB2 led to a reduced inactivation of whole-cell currents, which also indicates increased CaV1.2 activity (Graziano et al. 2021). Taken together, the increase in CaV1.2 activity appears to be a common feature among CaVβ2 variants found in ASD patients. In contrast, the CACNB1 mutation described here results in a CaVβ1 variant that, compared to a wild-type CaVβ1, decreases current density and shifts half-maximal activation to more positive potentials when co-expressed with CaV1.2 or CaV2.2 and it increases inactivation of CaV1.2. These findings are consistent with the reduced single-channel activity of both, L-type (CaV1.2) and N-type (CaV2.2) VGCCs. CaVβ1b_R296C thus can be considered a loss-of-function mutation.

To our knowledge, reduced single-channel activity has not been described in the context of ASD-associated VGCC variants so far. Even more, the only (electrophysiological) loss-of-function variant of CaVβ1 we are aware of was described in the context of Brugada syndrome, a cardiac disease associated with arrhythmia (Antzelevitch et al. 2007). Interestingly, there are CaVβ2 variants associated with Brugada syndrome that lead to decreased whole-cell currents (CaVβ2b_S481L) or accelerated inactivation of CaV1.2 (CaVβ2b_T11I) (Antzelevitch et al. 2007; Cordeiro et al. 2009). The affected CaVβ2b subunit is the most abundant CaVβ subunit in the heart but is also expressed in the brain (Hullin et al. 2007; Buraei and Yang 2010). Nevertheless, the patients described in the context of Brugada syndrome did not show a neurodevelopmental phenotype. Like CaVβ2b, CaVβ1b is one of several CaVβ subunits expressed in the brain (in addition to, e.g., CaVβ2a,e, CaVβ3, and CaVβ4) (Buraei and Yang 2010). Similar to CaVβ2 variants found in ASD patients (Despang et al. 2020), the CaVβ1b variant described here shows a heterozygous expression profile. It has been shown that different CaVβ isoforms can compete for the interaction with the CaV1.2 subunit by this modulating VGCC activity dynamically on a minute timescale (Jangsangthong et al. 2011). In another study, co-expression of CaVβ2a and CaVβ3 resulted in two biophysical distinct channel populations (Jones et al. 1998). Thus, we cannot exclude that the inhibitory effects of the CaVβ1b_R296C variant observed in the current study might be (partially) compensated by other CaVβ isoforms and/or splice variants in a more physiological expression system.

The mutation described in this study lies within the GK-domain of CaVβ1, a region well established as a protein-protein interaction site, especially for the intramolecular interactions with the SH3-domain critically involved in the function of CaVβ subunits (Buraei and Yang 2010). Alterations affecting these domains have been shown to change the effects of CaVβ subunits on VGCC gating (Chen et al. 2009). Furthermore, GK-GK domain or SH3-SH3 domain interactions are involved in CaVβ dimerization (Lao et al. 2010; Miranda-Laferte et al. 2011). Of note, the formation of SH3-SH3 dimers is probably mediated by a cysteine residue (Miranda-Laferte et al. 2011). Whether the introduction of a cysteine in the GK-domain of our CaVβ1b_R296C variant has a similar effect should be investigated in future studies. According to the web-based prediction program DiANNA 1.1 (Ferrè and Clote 2005), the exchange from arginine to cysteine results in an additional disulfide bond and by this a potentially misfolded tertiary structure of the mutant compared to the wild-type CaVβ1b.

It has been shown that the CaVβ1b subunit prevents CaV1.2 from degradation and facilitates its export from the endoplasmic reticulum (Altier et al. 2011). Similarly, it prevented CaV2.2 from proteasomal degradation (Page et al. 2016). In this context, a recent study could show that the CaVβ subunit is imperative for the anterograde transport of CaV1.2 channels to the plasma membrane, while the retrograde transport seems to be CaVβ independent (Conrad et al. 2021). Thus, we cannot exclude that beneath single-channel activity, VGCC expression might be affected by the examined CaVβ variant. RGK-proteins in general are known VGCC inhibitors by directly inhibiting the activity of VGCCs or altering number of channels at the membrane (Buraei et al. 2015). While some of the RKG-proteins can regulate VGCC activity by directly interacting with the pore-forming subunit of VGCC complexes, Gem utilizes a solely CaVβ-dependent mode of action (Puckerin et al. 2016, 2018). In the present study, co-expression of the RGK-protein Gem similarly reduced whole-cell currents mediated by VGCCs co-expressed with either CaVβ1b_R296C or CaVβ1b_WT. In our previous study, CaVβ2 variants found in ASD patients showed a differential modulation of CaV1.2 compared to each other and to the respective wild-type CaVβ2 (Despang et al. 2020). Of note, this was also seen in differences between Gem-mediated inhibitory effects. RGK-proteins have been proposed to have potentials as therapeutic tools for a differential and specific modulation of VGCCs (Colecraft 2020). It is tempting to speculate that RGK proteins might be a tool to differentially compensate for changes of VGCC-mediated currents that are due to mutated CaVβ subunits.

The alterations caused by co-expressing the CaVβ1b_R296C variant appear to be less pronounced on CaV2.2 compared to CaV1.2, both in respect to whole-cell currents and single-channel gating. Differences between modulation of CaV1 and CaV2 VGCCs by CaVβ subunits have already been described (Buraei and Yang 2010). Future studies should address this in more detail.

Conclusion

While evidence for CACNB2 involvement in ASD is growing, this is the first report of a CACNB1 variant found in an ASD patient that shows functional consequences, at least at the molecular level. Interestingly, the effects of the CaVβ1b_R296C described here are opposite to those described for CaVβ2 variants previously, i.e. an inhibitory instead of a stimulatory effect compared to the respective WT CaVβ isoform. This might indicate differential roles of different CaVβ isoforms in the context of ASD. However, the electrophysiological effects we have described at the molecular and cellular level need further investigation in models of more pathophysiological relevance.