Autism associated mutations in β2 subunit of voltage-gated calcium channels constitutively activate gene expression
Graphical abstract
Introduction
Neuronal activity regulates gene transcription and affects long-term plasticity associated with learning and memory. A dysregulation of nuclear signaling pathways in neurons is manifested in neurodevelopmental disorders as autism spectrum disorders (ASD).
Major neuronal functions in the brain such as synaptogenesis, neuronal differentiation, and cell migration are regulated by the voltage-gated calcium channels (VGCCs) by means of different signaling pathways [1,2].
The VGCC is composed of four subunits, the transmembrane α11 subunit (Cavα), the extracellular α2δ (Cavα2δ), the intracellular β (Cavβ), and a transmembrane γ (Cavγ) subunit. The VGCC activates genes during membrane depolarization in a process called Excitation transcription (ET) coupling. ET coupling via the Ras/ERK/CREB pathway has been shown to be mediated non-ionotropically (independent of Ca2+ influx) during Ca2+ binding to Cav1.2 [3], [4], [5], [6], [7]. Accordingly, a signal initiated during membrane depolarization is propagated from the α11.2 to the Cavβ2 subunit. Cavβ2 then activates H-Ras consistent with the importance of Ras in gene regulation.
The majority of VGCC mutations that are tightly associated with long-term activity-dependent gene expression occur within pathogenic variants in genes encoding the pore-forming α1-subunits of Cav1.2 (cardiac arrhythmias and psychiatric diseases, autism), Cav1.3 (autism spectrum disorder epilepsy, primary aldosteronism) Cav1.4 (X-linked retinal disorders), Cav2.1 (familial hemiplegic migraine, epilepsy), Cav3.1 (ataxia) and Cav3.2 (epilepsy) [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21].
Among the most studied mutations is the rare autosomal mutation in the pore-forming subunit α11.2 of the gene CACNA1C in patients with Timothy Syndrome (TS). This single G406R mutation is involved in neurological and cognitive disorders including ASD and has been tightly associated with long-term activity-dependent gene expression [22,23].
The α11.2G406R mutation causes specific gain-of-function changes in Cav1.2 channel gating, a hyperpolarizing shift in voltage activation, a slowed voltage-dependent inactivation, and a depolarizing shift in the steady-state inactivation [5,[22], [23], [24], [25], [26], [27], [28]], (see reviews [29,30]).
The robust hyperpolarizing shift in the voltage dependence of activation, which was exhibited by the pathogenic de novo missense Timothy-channel autism mutation G406R, was correlated with Ca2+-influx independent-enhanced gene activation at rest. The enhanced spontaneous transcription by facilitation of channel activity was hypothesized to be responsible for the neurodevelopmental disorders associated with this mutant [5].
The Cavβ subunit encoded by CACNB2 interacts with the α1 1.2 subunits of CACNA1C (Cav1.2), CACNA1D (Cav1.3) and CACNA1S (Cav1.1). It regulates the trafficking of the pore forming α1 subunit, and the activation and inactivation kinetics of the channel [31], [32], [33], [34].
Similar to CACNA1C, mutations in CACNB2 have been shown to be frequent in the Genome-wide association studies (GWAS) of bipolar disorder in a Han Chinese population [35]. Also, specific single-nucleotide polymorphism (SNPs) within the intracellular CACNB2, have been shown to be associated with a range of both childhood onset and adult onset of psychiatric disorders [36,37].
Rare missense mutations, G167S, S197F and F240L within the SH3 and HOOK-domains of CaVβ2d have also been found to be associated with ASD patients [10]. Whole-cell recordings showed that the two mutations G167S, S197F were associated with a decrease in the rate of channel inactivation, while the F240L mutation, showed an increase in the rate of inactivation. Both G167S, and S197F mutations exhibit a higher single Cav1.2 channel activity compared to wt CaVβ2d [38].
Two other autistic related mutations V2D [38] and R70C [39], showed a reduction in whole-cell currents inactivation of the L-type calcium channel. A more recent report revealed yet another mutation in an ASD individual, the R286C variant of CaVβ1b, which inhibits both the L-and N-type VGCCs [40].
In the present report we studied the Cavβ2a autistic mutations, which correspond to G167S and S197F in CaVβ2d, and to a triple D344A/D320A/D322A mutant. Their effects on Cav1.2 mediated gene activation were examined by monitoring depolarization-triggered activation of the Ras/ERK/CREB pathway in HEK293 cells co-expressing α11.2 and α2δ. Here we found that the asutistic mutants exhibited an increase in spontaneous gene activation consistent with the observed enhanced spontaneous basal transcription activity elicited by the autistic Timothy channel [5]. These results reveals a common general molecular mechanism by which CACNA1C and CACNB2 missense autistic mutations could contribute to autism associated neurodevelopmental disorders.
Section snippets
Materials
Antibodies/reagents Company Catalog number Anti-RASGRF2 antibody – C-terminal Abcam
Cambridge, UKCat# ab226973 Phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) Cell Signaling,
Danvers, MA USACat# 9101 Mouse mAb; ERK2 (Santa Cruz, USA) Cat #PA5-29636 Purified mouse anti-β-Catenin mAB BD biosciences Franklin Lakes, New Jersey, USA Cat# 610154 PageRuler™ Unstained Protein Ladder Thermo Scientific™
Waltham, Massachusetts, U.S.ACat# 26614 Xpert 2 Prestained Protein Marker Gendepot
Texas, U.S.ACat# P8503 Anti-mouse
Expression of Cavβ2 mutants in HEK293 cells
The intracellular Cavβ subunits encoded by four different genes (Cavβ1–4) have a conserved core region formed by Src homology 3 (SH3) and guanylate kinase (GK) domains. These domains are connected by a HOOK region, which is flanked by non-conserved amino acid carboxy-termini (Fig. 1, upper ).The cDNAs of wt α11.2, and α2δ subunits were co-expressed in HEK293 cells with the cDNA of GFP-tagged wtCavβ2a, Cavβ2aG113S, GFP-Cavβ2aS143F, or GFP-Cavβ2aDDD/AAA and expression was monitored by
Exploring the molecular mechanism of the Cavβ autistic mutants
One mechanism of regulating gene expression programs is by means of the Cav1.2, which couples electrical signal to activation of intracellular signaling pathways [48,49].
Although most mutations associated with neurodevelopmental disorders are found in the α1 pore forming subunit of the VGCC, recent studies have shown neurodegenerative disorders to be associated with mutations in the intracellular Cavβ subunit of the channel, [10,[36], [37], [38],40].
In previous studies we have shown that Cavβ2a
Data availability statement
This study did not generate any unique datasets or code. Data reported in this paper is available from the lead contact.
CRediT authorship contribution statement
M.T, D.A, and E.S. designed the experiments. MT and D.A wrote the manuscript. M.S, E.S. T.T.C. performed the experiments and analyzed the data. All authors edited the manuscript.
Conceptualization, D.A, M.T., methodology, M.T, E. S, T.T; analysis, M.T., E.S, T.T; D.A writing original draft; writing review & editing, DA., MT., E.S., T.T; supervision, D.A.
Declaration of Competing Interest
The authors declare no competing interests.
Acknowledgments
The authors acknowledge Dr. A Citri for providing us with mice brain, and to Y. Ariav for initial experiments
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