CaM-dependent modulation of human CaV1.3 whole-cell and single-channel currents by C-terminal CaMKII phosphorylation site S1475.

Phosphorylation enables rapid modulation of voltage-gated calcium channels (VGCC) in physiological and pathophysiological conditions. How phosphorylation modulates human CaV1.3 VGCC, however, is largely unexplored. We characterized modulation of CaV1.3 gating via S1475, the human equivalent of a phosphorylation site identified in the rat. S1475 is highly conserved in CaV1.3 but absent from all other high-voltage activating calcium channel types co-expressed with CaV1.3 in similar tissues. Further, it is located in the C-terminal EF-hand motif, which binds calmodulin (CaM). This is involved in calcium-dependent channel inactivation (CDI). We used amino acid exchanges that mimic either sustained phosphorylation (S1475D) or phosphorylation resistance (S1475A). Whole-cell and single-channel recordings of phosphorylation state imitating CaV1.3 variants in transiently transfected HEK-293 cells revealed functional relevance of S1475 in human CaV1.3. We obtained three main findings: (1) CaV1.3_S1475D, imitating sustained phosphorylation, displayed decreased current density, reduced CDI and (in-) activation kinetics shifted to more depolarized voltages compared with both wildtype CaV1.3 and the phosphorylation-resistant CaV1.3_S1475A variant. Corresponding to the decreased current density, we find a reduced open probability of CaV1.3_S1475D at the single-channel level. (2) Using CaM overexpression or depletion, we find that CaM is necessary for modulating CaV1.3 through S1475. (3) CaMKII activation led to CaV1.3_WT-current properties similar to those of CaV1.3_S1475D, but did not affect CaV1.3_S1475A, confirming that CaMKII modulates human CaV1.3 via S1475. Given the physiological and pathophysiological importance of CaV1.3, our findings on the S1475-mediated interplay of phosphorylation, CaM interaction and CDI provide hints for approaches on specific CaV1.3 modulation under physiological and pathophysiological conditions. KEY POINTS: Phosphorylation modulates activity of voltage-gated L-type calcium channels for specific cellular needs but is largely unexplored for human CaV1.3 channels. Here we report that S1475, a CaMKII phosphorylation site identified in rats, is functionally relevant in human CaV1.3. Imitating phosphorylation states at S1475 alters current density and inactivation in a calmodulin-dependent manner. In wildtype CaV1.3 but not in the phosphorylation-resistant variant S1475A, CaMKII activation elicits effects similar to constitutively mimicking phosphorylation at S1475. Our findings provide novel insights on the interplay of modulatory mechanisms of human CaV1.3 channels, and present a possible target for CaV1.3-specific gating modulation in physiological and pathophysiological conditions.

Inhibitory effects on L- and N-type calcium channels by a novel CaVß1 variant identified in a patient with autism spectrum disorder.

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 ß-subunits, which exist in four isoforms (CaVß1-4). Our previous findings suggested that activation of L-type VGCCs is a common feature of CaVß2 subunit mutations found in ASD patients. In the current study, we functionally characterized a novel CaVß1b variant (p.R296C) identified in an ASD patient. We used whole-cell and single-channel patch clamp to study the effect of CaVß1b_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 CaVß1b-subunits with the RGK-protein Gem. Our data obtained at both, whole-cell and single-channel levels, show that compared to a wild-type CaVß1b, the CaVß1b_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 CaVß1b_R296C variant differing from that attributed to CaVß2 variants found in ASD patients. Further studies have to detail the effects on different VGCC subtypes and on VGCC expression.

Circuits that encode and guide alcohol-associated preference.

A powerful feature of adaptive memory is its inherent flexibility. Alcohol and other addictive substances can remold neural circuits important for memory to reduce this flexibility. However, the mechanism through which pertinent circuits are selected and shaped remains unclear. We show that circuits required for alcohol-associated preference shift from population level dopaminergic activation to select dopamine neurons that predict behavioral choice in Drosophila melanogaster. During memory expression, subsets of dopamine neurons directly and indirectly modulate the activity of interconnected glutamatergic and cholinergic mushroom body output neurons (MBON). Transsynaptic tracing of neurons important for memory expression revealed a convergent center of memory consolidation within the mushroom body (MB) implicated in arousal, and a structure outside the MB implicated in integration of naïve and learned responses. These findings provide a circuit framework through which dopamine neuronal activation shifts from reward delivery to cue onset, and provide insight into the maladaptive nature of memory.

Autism-associated mutations in the CaVß2 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 CaVß2 subunits of VGCCs. From this, we derived the hypothesis that several CaVß2-mutations associated with ASD show common features sensitizing LTCCs and/or enhancing currents. Using a CaVß2d backbone, we performed extensive whole-cell and single-channel patch-clamp analyses of Ba2+ currents carried by Cav1.2 pore subunits co-transfected with the previously described CaVß2 mutations (G167S, S197F) as well as a recently identified point mutation (V2D). Furthermore, the interaction of the mutated CaVß2d subunits with the RGK protein Gem was analyzed by co-immunoprecipitation 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 CaVß2 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: CaVß2d_G167S > CaVß2d_V2D > CaVß2d_S197F). In conclusion, patch-clamp recordings of ASD-associated CaVß2d mutations revealed differential modulation of Ba2+ currents carried by CaV1.2 suggesting kind of an "electrophysiological fingerprint" each. The increase in current finally observed with all CaVß2d mutations analyzed might contribute to the complex pathophysiology of ASD and by this indicate a possible underlying molecular mechanism.

Test-retest reliability of the diagnosis of schizoaffective disorder in childhood and adolescence - A systematic review and meta-analysis.

OBJECTIVES: Reliability of schizoaffective disorder (SAD) diagnoses is low in adults but unclear in children and adolescents (CAD). We estimate the test-retest reliability of SAD and its key differential diagnoses (schizophrenia, bipolar disorder, and unipolar depression). METHODS: Systematic literature search of Medline, Embase, and PsycInfo for studies on test-retest reliability of SAD, in CAD. Cohen's kappa was extracted from studies. We performed meta-analysis for kappa, including subgroup and sensitivity analysis (PROSPERO protocol: CRD42013006713). RESULTS: Out of > 4000 records screened, seven studies were included. We estimated kappa values of 0.27 [95%-CI: 0.07 0.47] for SAD, 0.56 [0.29; 0.83] for schizophrenia, 0.64 [0.55; 0.74] for bipolar disorder, and 0.66 [0.52; 0.81] for unipolar depression. In 5/7 studies kappa of SAD was lower than that of schizophrenia; similar trends emerged for bipolar disorder (4/5) and unipolar depression (2/3). Estimates of positive agreement of SAD diagnoses supported these results. LIMITATIONS: The number of studies and patients included is low. CONCLUSIONS: The point-estimate of the test-retest reliability of schizoaffective disorder is only fair, and lower than that of its main differential diagnoses. All kappa values under study were lower in children and adolescents samples than those reported for adults. Clinically, schizoaffective disorder should be diagnosed in strict adherence to the operationalized criteria and ought to be re-evaluated regularly. Should larger studies confirm the insufficient reliability of schizoaffective disorder in children and adolescents, the clinical value of the diagnosis is highly doubtful.

CDK5RAP2 interaction with components of the Hippo signaling pathway may play a role in primary microcephaly.

Autosomal recessive primary microcephaly (MCPH) is characterized by a substantial reduction in brain size but with normal architecture. It is often linked to mutations in genes coding for centrosomal proteins; however, their role in brain size regulation is not completely understood. By combining homozygosity mapping and whole-exome sequencing in an MCPH family from Pakistan, we identified a novel mutation (XM_011518861.1; c.4114C > T) in CDK5RAP2, the gene associated with primary microcephaly-3 (MCPH3), leading to a premature stop codon (p.Arg1372*). CDK5RAP2 is a component of the pericentriolar material important for the microtubule-organizing function of the centrosome. Patient-derived primary fibroblasts had strongly decreased CDK5RAP2 amounts, showed centrosomal and nuclear abnormalities and exhibited changes in cell size and migration. We further identified an interaction of CDK5RAP2 with the Hippo pathway components MST1 kinase and the transcriptional regulator TAZ. This finding potentially provides a mechanism through which the Hippo pathway with its roles in the regulation of centrosome number is linked to the centrosome. In the patient fibroblasts, we observed higher levels of TAZ and YAP. However, common target genes of the Hippo pathway were downregulated as compared to the control with the exception of BIRC5 (Survivin), which was significantly upregulated. We propose that the centrosomal deficiencies and the altered cellular properties in the patient fibroblasts can also result from the observed changes in the Hippo pathway components which could thus be relevant for MCPH and play a role in brain size regulation and development.