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

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

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

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

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

Time-dependent inactivation.

<p>Representative whole-cell Ba²⁺ current traces (a) of Ca_V1.2/α2δ1 co-expressed with β2d_WT -subunit or co-expressed with the ASD mutants β2d_G167S, β2d_S197F or the β2dE7c_WT or its variant β2dE7c_F240L. The currents were elicited from a holding potential of −100 mV by 150 ms (or 500ms) step depolarization of −40 to +50 mV with 20 mM Ba²⁺ (for β2d variants) or 5 mM Ba²⁺ (for β2dE7c variants), respectively. Analysis of the extent of time-dependent inactivation. (<b>b</b>, <b>c</b>) % Inactivation was analyzed as the remaining fraction of whole-cell current that has not inactivated after 150 ms of depolarization. β2d_WT (N = 12), β2d_G167S (N = 8), β2d_S197F (N = 11) (<b>c</b>) Similar analysis for β2dE7c variants β2dE7c_WT (N = 5), β2dE7c_F240L (N = 6). Asterisk (*) marks a statistical significance (P<0.05) compared to the respective WT, (**) marks P<0.01.</p

Current and voltage parameters (mean ± SEM) of the constructs tested.

<p><b>V0.5_act,</b> Voltage of half-maximal activation; <b>V0.5_inact,</b> half-maximal inactivation voltage, dV, slope factor; −<b>β2d,</b> mock transfected cells;</p><p>*indicates P<0.05 versus corresponding WT.</p

Single-channel properties of L-type calcium channels.

<p>(A) Exemplary traces of barium currents show increased single-channel activity in ventricular myocytes of Gα_i3^−/− animals vs. WT mice. (B) The peak ensemble average current is −61±13 fA in Gα_i3^−/− (n = 6) and −44±9 fA in WT mice (n = 7). (C) The open probability within active sweeps is slightly enhanced in Gα_i3^−/− (5.4±1.0% vs. 4.0±0.9% WT) whereas (D) the mean closed time is significantly reduced (Gα_i3^−/− 3.8±0.5 ms vs. 6.6±0.9 ms WT). Unitary amplitude was not different with −0.83±0.02 pA (WT) and −0.79±0.03 pA (Gα_i3^−/−). *p<0.05 vs. WT. Box-and-whisker plots indicate minimum and maximum values as well as 25^th, 50^th and 75^th percentiles.</p

Single-channel gating in WT and Gα_i3^−/− myocytes.

<p>Analysis of single-channel gating parameters in ventricular myocytes from WT (n = 7) and Gα_i3^−/− (n = 6) mice. Recordings with more than one channel were excluded from the analysis. i: unitary current; I_peak: peak ensemble average current; f_active: fraction of traces showing at least one opening; P_open: open probability in active traces; t_open: mean duration of openings; τ_open: dwell time constant of the open state; t_closed: mean duration between two consecutive openings; τ_closed,1: dwell time constant of the fast closed time component; τ_closed,2: dwell time constant of the slow closed time component; proportion: ratio of events conferring to either the fast or the slow closed time component; fl: mean latency until the first opening.</p><p>*: p<0.05 vs. WT.</p

Effects of acute inactivation of G_i/o proteins by PTX incubation of isolated cardiac myocytes.

<p>(A) Effects of PTX on peak L-VDCC current density. PTX treatment by itself did not affect calcium current density. (B) Effects of PTX on steady-state inactivation, as gauged by the midpoint voltage V_0.5 of a Boltzmann function. No change is seen after 3 hours of drug-free incubation compared to 0 hour. PTX leads to a significant leftward shift of V_0.5 in WT (from −19.2±0.7 mV to −21.0±0.7 mV, n = 7–18) and Gα_i3^−/− (from −18.2±0.7 mV to −22.7±1.1 mV, n = 11–13). (C) PTX affects the recovery of the L-VDCC from inactivation. PTX inhibits the channel recovery in Gα_i3^−/− (τ from 189±12 ms to 350±26 ms, n = 5–11). *p<0.05 vs. WT, ^†p<0.05 vs. Gα_i3^−/−, ^¶p<0.05 vs. 3 h without PTX.</p