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 CaV1.2 (CACNA1C) and CaVβ2 (CACNB2) were recently identified as risk loci for psychiatric diseases. Here, we present three rare missense mutations of CACNB2 (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 CACNA1C mutation causing Timothy Syndrome, a Mendelian disease presenting with ASD. In conclusion, the results of our first-time biophysical characterization of these three rare CACNB2 missense mutations identified in ASD patients support the hypothesis that calcium channel dysfunction may contribute to autism.

The organic cation transporter 3 (OCT3; synonymous: extraneuronal monoamine transporter, EMT, Slc22a3) encodes an isoform of the organic cation transporters and is expressed widely across the whole brain. OCTs are a family of high-capacity, bidirectional, multispecific transporters of organic cations. These also include serotonin, dopamine and norepinephrine making OCTs attractive candidates for a variety of neuropsychiatric disorders including anxiety disorders. OCT3 has been implicated in termination of monoaminergic signalling in the central nervous system. Interestingly, OCT3 mRNA is however also significantly up-regulated in the hippocampus of serotonin transporter knockout mice where it might serve as an alternative reuptake mechanism for serotonin. The examination of the behavioural phenotype of OCT3 knockout mice thus is paramount to assess the role of OCT3. We have therefore subjected mice lacking the OCT3 gene to a comprehensive behavioural test battery. While cognitive functioning in the Morris water maze test and aggression levels measured with the resident-intruder paradigm were in the same range as the respective control animals, OCT3 knockout animals showed a tendency of increased activity and were significantly less anxious in the elevated plus-maze test and the open field test as compared to their respective wild-type controls arguing for a role of OCT3 in the regulation of fear and anxiety, probably by modulating the serotonergic tone in limbic circuitries.

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.

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Timothy Syndrome (TS) is a multisystem disorder characterized by autism, immune deficiencies, and cardiac arrhythmias. Intriguingly, the underlying defect comes down to a single point mutation (either G402S or G406R) in the IS6 region of Ca V 1.2 channels. These channels are critical conduits of Ca 2þ entry into the heart, smooth muscle and brain. As such, these channels employ two forms of feedback regulationÀvoltage-dependent inactivation (VDI) and Ca 2þ /calmodulin-dependent inactivation (CDI). In TS, these regulatory mechanisms are disrupted, resulting in inappropriate Ca 2þ feedback. Given that the pattern of multisystem pathology differs for the two types of mutant channels, we here undertook in-depth biophysical analysis of the altered inactivation in each of these constructs. As reported, both mutants exhibited strongly attenuated VDI. Rather surprisingly, however, both constructs also demonstrated a clear reduction of CDI, in contrast to a previous study reporting selective weakening of VDI (PNAS105:11987). Further analysis revealed that the CDI deficits in the two mutants may arise from very different mechanisms. For G406R, voltagedependent activation is strongly shifted to more negative potentials, while estimated maximal open probability (P O/max ) at saturating depolarization was only slightly altered. According to an allosteric mechanism of CDI (Biophys J 96:222a), this favoring of channel activation would reduce CDI, because opening would be enhanced even within inactivated channels (i.e., the current decrease seen upon channel inactivation would be lessened). By contrast, the G402S mutation caused a marked depolarizing shift in voltage-dependent activation, with largely unchanged P O/max . This outcome would sharply diminish channel opening at physiological voltages, yielding attenuated CDI via decreased entry into inactivated states. Recognizing these divergent mechanisms of CDI disruption may shed light on the differing disease phenotypes elaborated by the two mutations, and ultimately prove beneficial in tailoring treatments for each TS population.

Voltage-gated calcium channel (VGCC) subunits have been genetically associated with autism spectrum disorders (ASD). The properties of the pore-forming VGCC subunit are modulated by auxiliary β-subunits, which exist in four isoforms (CaVβ1-4). Our previous findings suggested that activation of L-type VGCCs is a common feature of CaVβ2 subunit mutations found in ASD patients. In the current study, we functionally characterized a novel CaVβ1b variant (p.R296C) identified in an ASD patient. We used whole-cell and single-channel patch clamp to study the effect of CaVβ1b_R296C on the function of L- and N-type VGCCs. Furthermore, we used co-immunoprecipitation followed by Western blot to evaluate the interaction of the CaVβ1b-subunits with the RGK-protein Gem. Our data obtained at both, whole-cell and single-channel levels, show that compared to a wild-type CaVβ1b, the CaVβ1b_R296C variant inhibits L- and N-type VGCCs. Interaction with and modulation by the RGK-protein Gem seems to be intact. Our findings indicate functional effects of the CaVβ1b_R296C variant differing from that attributed to CaVβ2 variants found in ASD patients. Further studies have to detail the effects on different VGCC subtypes and on VGCC expression.

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change in the pore size due to the intrinsic heterogeneity in the fluorescently labeled MscL pentamers where various stoichiometry of the donors and acceptors are present. Furthermore, we have found that sticking the channel to a surface alters the function of them. To get around these two problems, we performed single-molecule FRET experiments using an Alternating Laser EXcitation (ALEX) apparatus. By using alternating lasers of wavelengths 488 nm and 561 nm to excite directly the donors (Alexa488) and acceptors (Alexa568) present in single diffusing MscL molecules which are incorporated in 50 nm liposomes, both the distance-based FRET efficiency E and stoichiometrybased ratio S could be measured. Our single-molecule experiments show that the addition of an asymmetric lipid (LPC) to the liposomes opens the channels, consistent with the results from ensemble measurements.