Autism associated mutations in β₂ subunit of voltage-gated calcium channels constitutively activate gene expression

Highlights

• Cavβ_2a^G113S, and Cavβ_2a^S143F corresponding to human autistic Cavβ_2d mutants, mediate ET coupling via the Ras/ERK/CREB pathway similar to wtCavβ_2a.

• The Cavβ_2a^G113S, and Cavβ_2a^S143F mutants, as opposed to wtCavβ_2a, elicit spontaneous activation of ERK1/2 and CREB.

• Enhanced spontaneous channel activity appears a common molecular mechanism of Ca²⁺-channel-mediated neurodevelopmental disorders.

• wt Cavβ_2a, Cavβ_2a^G113S, Cavβ_2a^S1143F and Cavβ_2a ^DDD/AAA selectively interact with RasGRF2 and RhoGEF

• The triple-D -mutant within the guanylate kinase domain inhibits ET coupling.

Abstract

Membrane depolarization triggers gene expression through voltage-gated calcium channels (VGCC) in a process called Excitation-transcription (ET) coupling. Mutations in the channel subunits α₁1.2, or β_2d, are associated with neurodevelopmental disorders such as ASD. Here, we found that two mutations S143F and G113S within the rat Cavβ_2a corresponding to autistic related mutations Cavβ_2d^S197F and Cavβ_2d^G167S in the human Cavβ_2d, activate ET-coupling via the RAS/ERK/CREB pathway. Membrane depolarization of HEK293 cells co-expressing α₁1.2 and α_2δ with Cavβ_2a^S143F or Cavβ_2a^G113S triggers constitutive transcriptional activation, which is correlated with facilitated channel activity. Similar to the Timothy-associated autistic mutation α₁1.2^G406R, constitutive gene activation is attributed to a hyperpolarizing shift in the activation kinetics of Cav1.2. Pulldown of RasGRF2 and RhoGEF by wt and the Cavβ_2a autistic mutants is consistent with Cavβ₂/Ras activation in ET coupling and implicates Rho signaling as yet another molecular pathway activated by Cavα₁1.2/Cavβ2 . Facilitated spontaneous channel activity preceding enhanced gene activation via the Ras/ERK/CREB pathway, appears a general molecular mechanism for Ca²⁺ channel mediated ASD and other neurodevelopmental disorders.

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 α₁1.2^G406R 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 Ca_Vβ_2d 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 Ca_Vβ_2d [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 Ca_Vβ_1b, which inhibits both the L-and N-type VGCCs [40].

In the present report we studied the Cavβ_2a autistic mutations, which correspond to G167S and S197F in Ca_Vβ_2d, 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.