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Depolarization drives neuronal plasticity. However, whether depolarization drives sensitization of peripheral nociceptive neurons remains elusive. By high-content screening (HCS) microscopy, we revealed that depolarization of cultured sensory neurons rapidly activates protein kinase A type II (PKA-II) in nociceptors by calcium influx through CaV1.2 channels. This effect was modulated by calpains but insensitive to inhibitors of cAMP formation, including opioids. In turn, PKA-II phosphorylated Ser1928 in the distal C terminus of CaV1.2, thereby increasing channel gating, whereas dephosphorylation of Ser1928 involved the phosphatase calcineurin. Patch-clamp and behavioral experiments confirmed that depolarization leads to calcium- and PKA-dependent sensitization of calcium currents ex vivo and local peripheral hyperalgesia in the skin in vivo. Our data suggest a local activity-driven feed-forward mechanism that selectively translates strong depolarization into further activity and thereby facilitates hypersensitivity of nociceptor terminals by a mechanism inaccessible to opioids.

BackgroundDiagnostic yield in patients with autism spectrum disorder (ASD) has improved over the last years, thanks to the introduction of whole genome arrays and next generation sequencing, but etiology is still unknown for the majority of cases. Among distinct cellular pathways, evidence implicating dysregulation of cellular calcium homeostasis in ASD pathogenesis has been accumulating, and speci c mutations in voltagegated calcium channels found in patients with autism were shown to be functionally relevant. MethodsWhole exome sequencing and Sanger sequencing were performed to identify and con rm variants in a girl with ASD, global developmental delay and precocious puberty, born of rst-degree cousins. Sitedirected mutagenesis was used to generate a human Ca V β 2d calcium channel subunit carrying a CACNB2 mutation. Whole-cell patch-clamp recordings were performed to reveal functional effects of mutant Ca V β 2d on Ba 2+ -currents mediated by L-type (Ca V 1.2) calcium channels in transiently transfected HEK-293 cells. ResultsIn an ASD patient, we identi ed a rare homozygous variant (p.Arg70Cys) in the CACNB2 gene coding for the auxiliary Ca V β 2 subunit of voltage-gated calcium channels. In a recombinant system, the Ca V β 2 variant, which was not previously associated to ASD, was found to alter Ca V 1.2 calcium channel function by signi cantly affecting activation and inactivation of whole-cell Ba 2+ -currents. LimitationsAlthough the evidence of CACNB2 involvement in ASD is slowly accumulating, the number of reported patients is very limited. Deep clinical phenotyping and functional studies in larger sets of subjects will be instrumental to fully understand the penetrance and outcome of CACNB2 variants. ConclusionsThe p.Arg70Cys variant in CACNB2 shows functional consequences similar to other ASD-associated Ca V β 2 mutations. These results support the idea of CACNB2 variations contributing to the development of ASD and hint to a rare form of Mendelian recessive autism with possible speci c comorbidities.

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.