Voltage-gated Calcium Channels and Autism Spectrum Disorders

Autism spectrum disorder is a complex-genetic disease and its etiology is unknown for the majority of cases. So far, more than one hundred different susceptibility genes were detected. Voltage-gated calcium channels are among the candidates linked to autism spectrum disorder by results of genetic studies. Mutations of nearly all pore-forming and some auxiliary subunits of voltage gated calcium channels have been revealed from investigations of autism spectrum disorder patients and populations. Though there are only few electrophysiological characterizations of voltage-gated calcium channel mutations found in autistic patients these studies suggest their functional relevance. In summary, both genetic and functional data suggest a potential role of voltage-gated calcium channels in autism spectrum disorder. Future studies require refinement of the clinical and systems biological concepts of autism spectrum disorder and an appropriate holistic approach at the molecular level, e.g. regarding all facets of calcium channel functions

Autism-associated mutations in the Ca-v beta(2) calcium-channel subunit increase Ba2+-currents and lead to differential modulation by the RGK-protein Gem

Voltage-gated calcium-channels (VGCCs) are heteromers consisting of several subunits. Mutations in the genes coding for VGCC subunits have been reported to be associated with autism spectrum disorder (ASD). In a previous study, we identified electrophysiologically relevant missense mutations of Ca-v beta(2) subunits of VGCCs. From this, we derived the hypothesis that several Ca-v beta(2)-mutations associated with ASD show common features sensitizing LTCCs and/or enhancing currents. Using a Ca-v beta(2d) backbone, we performed extensive whole-cell and single-channel patch-clamp analyses of Ba2+ currents carried by Ca(v)1.2 pore subunits co-transfected with the previously described Ca-v beta(2) mutations (G167S, S197F) as well as a recently identified point mutation (V2D). Furthermore, the interaction of the mutated Ca-v beta(2) subunits with the RGK protein Gem was analyzed by coimmunoprecipitation assays and electrophysiological studies. Patch-clamp analyses revealed that all mutations increase Ba2+ currents, e.g. by decreasing inactivation or increasing fraction of active sweeps. All Ca-v beta(2) mutations interact with Gem, but differ in the extent and characteristics of modulation by this RGK protein (e.g. decrease of fraction of active sweeps: Ca-v beta(2d_G167S) > Ca-v beta(2d_v2D) > Ca-v beta(2d_S197F). In conclusion, patch-clamp recordings of ASD-associated Ca-v beta(2d) mutations revealed differential modulation of Ba2+ currents carried by Ca(v)1.2 suggesting kind of an electrophysiological fingerprint each. The increase in current finally observed with all Ca-v beta(2d) mutations analyzed might contribute to the complex pathophysiology of ASD and by this indicate a possible underlying molecular mechanism

Rare Mutations of <i>CACNB2</i> Found in Autism Spectrum Disease-Affected Families Alter Calcium Channel Function

<div><p>Autism Spectrum Disorders (ASD) are complex neurodevelopmental diseases clinically defined by dysfunction of social interaction. Dysregulation of cellular calcium homeostasis might be involved in ASD pathogenesis, and genes coding for the L-type calcium channel subunits Ca_V1.2 (<i>CACNA1C)</i> and Ca_Vβ2 (<i>CACNB2</i>) were recently identified as risk loci for psychiatric diseases. Here, we present three rare missense mutations of <i>CACNB2</i> (G167S, S197F, and F240L) found in ASD-affected families, two of them described here for the first time (G167S and F240L). All these mutations affect highly conserved regions while being absent in a sample of ethnically matched controls. We suggest the mutations to be of physiological relevance since they modulate whole-cell Ba²⁺ currents through calcium channels when expressed in a recombinant system (HEK-293 cells). Two mutations displayed significantly decelerated time-dependent inactivation as well as increased sensitivity of voltage-dependent inactivation. In contrast, the third mutation (F240L) showed significantly accelerated time-dependent inactivation. By altering the kinetic parameters, the mutations are reminiscent of the <i>CACNA1C</i> mutation causing Timothy Syndrome, a Mendelian disease presenting with ASD. In conclusion, the results of our first-time biophysical characterization of these three rare <i>CACNB2</i> missense mutations identified in ASD patients support the hypothesis that calcium channel dysfunction may contribute to autism.</p></div

Splice scheme of human <i>CACNB2</i> resulting in 9 splice variants of the Ca_Vβ2-subunit with the localization of the three mutations.

<p>Spliced exons are shown in light grey and conserved exons in dark grey. All nine splice variants express the mutation-carrying exon 5, while three of variants contain the localization of the third mutation in exon 7c <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095579#pone.0095579-Buraei1" target="_blank">[10]</a>.</p

Voltage-dependent steady-state inactivation (a, b) of Ba²⁺ currents through L-type calcium channels.

<p>The ASD mutants p.G167S (N = 4) and p.S197F (N = 6) showed a significantly flattened slope of voltage-dependent inactivation compared to β2d_WT (N = 7). The third mutation β2dE7c_F240L. (N = 5) did not obviously differ from its corresponding β2dE7c_WT (N = 2). Half-inactivation potentials (V0.5_inact) (<b>c</b>) and the slope factors dV (<b>d</b>) were obtained from the fits of individual experiments using the Boltzmann equation and averaging the results. Asterisk (*) marks a statistical significance (P<0.05) compared to the respective WT.</p

I–V relationships.

<p>Currents were elicited from −40 to +60 mV in 10 mV increments with 5 mM or 20 mM Ba²⁺, respectively. The holding potential was −100 mV. Current density of the variants p.G167S (N = 11) and p.S197F (N = 8) in (<b>a</b>) and p.F240L (N = 5) in (<b>b</b>) were compared with their respective WTs (β2d_WT: N = 9 and β2dE7c_WT: N = 5).</p

Clinical Features of ASD patients with rare mutations in <i>CACNB2</i>.

<p><b>ADI-R</b>, Autism Diagnostic Interview-Revised; <b>ADOS</b>, Diagnostic Observation Schedule-Generic; <b>MRI,</b> magnetic resonance imaging.</p