Abstract Background and Purpose Pathogenic gain‐of‐function mutations in Ca v 1.3 L‐type voltage‐gated Ca 2+ ‐channels ( CACNA1D ) cause neurodevelopmental disorders with or without endocrine symptoms. We aimed to confirm a pathogenic gain‐of function phenotype of CACNA1D de novo missense mutations A749T and L271H, and investigated the molecular mechanism causing their enhanced sensitivity for the Ca 2+ ‐channel blocker isradipine, a potential therapeutic for affected patients. Experimental Approach Wildtype and mutant channels were expressed in tsA‐201 cells and their gating analysed using whole‐cell and single‐channel patch‐clamp recordings. The voltage‐dependence of isradipine action was quantified using protocols inducing variable fractions of inactivated channels. The molecular basis for altered channel gating in the mutants was investigated using in silico modelling and molecular dynamics simulations. Key Results Both mutations were confirmed pathogenic due to characteristic shifts of voltage‐dependent activation and inactivation towards negative potentials (~20 mV). At negative holding potentials both mutations showed significantly higher isradipine sensitivity compared to wildtype. The affinity for wildtype and mutant channels increased with channel inactivation as predicted by the modulated receptor hypothesis (30‐ to 40‐fold). The IC 50 was indistinguishable for wildtype and mutants when >50% of channels were inactivated. Conclusions and Implications Mutations A749T and L271H induce pathogenic gating changes. Like wildtype, isradipine inhibition is strongly voltage‐dependent. Our data explains their apparent higher drug sensitivity at a given negative voltage by the availability of more inactivated channels due to their more negative inactivation voltage range. Low nanomolar isradipine concentrations will only inhibit Ca v 1.3 channels in neurons during prolonged depolarized states without selectivity for mutant channels.
Abstract Substance use disorders are chronic relapsing disorders often impelled by enduring memories and persistent cravings. Alcohol, as well as other addictive substances, remolds neural circuits important for memory to establish obstinate preference despite aversive consequences. How pertinent circuits are selected and shaped to result in these unchanging, inflexible memories is unclear. Using neurogenetic tools available in Drosophila melanogaster we define how circuits required for alcohol associated preference shift from population level dopaminergic activation to select dopamine neurons that predict behavioral choice. During memory expression, these dopamine neurons directly, and indirectly via the mushroom body (MB), modulate the activity of interconnected glutamatergic and cholinergic output neurons. Transsynaptic tracing of these output neurons revealed at least two regions of convergence: 1) a center of memory consolidation within the MB implicated in arousal, and 2) a structure outside the MB implicated in integration of naïve and learned responses. These findings provide a circuit framework through which dopamine neuron activation shifts from reward delivery to cue onset, and provides insight into the inflexible, maladaptive nature of alcohol associated memories.
Abstract Phosphorylation enables rapid modulation of voltage‐gated calcium channels (VGCC) in physiological and pathophysiological conditions. How phosphorylation modulates human Ca V 1.3 VGCC, however, is largely unexplored. We characterized modulation of Ca V 1.3 gating via S1475, the human equivalent of a phosphorylation site identified in the rat. S1475 is highly conserved in Ca V 1.3 but absent from all other high‐voltage activating calcium channel types co‐expressed with Ca V 1.3 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 Ca V 1.3 variants in transiently transfected HEK‐293 cells revealed functional relevance of S1475 in human Ca V 1.3. We obtained three main findings: (1) Ca V 1.3_S1475D, imitating sustained phosphorylation, displayed decreased current density, reduced CDI and (in‐) activation kinetics shifted to more depolarized voltages compared with both wildtype Ca V 1.3 and the phosphorylation‐resistant Ca V 1.3_S1475A variant. Corresponding to the decreased current density, we find a reduced open probability of Ca V 1.3_S1475D at the single‐channel level. (2) Using CaM overexpression or depletion, we find that CaM is necessary for modulating Ca V 1.3 through S1475. (3) CaMKII activation led to Ca V 1.3_WT‐current properties similar to those of Ca V 1.3_S1475D, but did not affect Ca V 1.3_S1475A, confirming that CaMKII modulates human Ca V 1.3 via S1475. Given the physiological and pathophysiological importance of Ca V 1.3, our findings on the S1475‐mediated interplay of phosphorylation, CaM interaction and CDI provide hints for approaches on specific Ca V 1.3 modulation under physiological and pathophysiological conditions. image Key points Phosphorylation modulates activity of voltage‐gated L‐type calcium channels for specific cellular needs but is largely unexplored for human Ca V 1.3 channels. Here we report that S1475, a CaMKII phosphorylation site identified in rats, is functionally relevant in human Ca V 1.3. Imitating phosphorylation states at S1475 alters current density and inactivation in a calmodulin‐dependent manner. In wildtype Ca V 1.3 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 Ca V 1.3 channels, and present a possible target for Ca V 1.3‐specific gating modulation in physiological and pathophysiological conditions.
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.
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 CaVβ2 subunits of VGCCs. From this, we derived the hypothesis that several CaVβ2-mutations associated with ASD show common features sensitizing LTCCs and/or enhancing currents. Using a CaVβ2d 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 CaVβ2 mutations (G167S, S197F) as well as a recently identified point mutation (V2D). Furthermore, the interaction of the mutated CaVβ2d 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 CaVβ2 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: CaVβ2d_G167S > CaVβ2d_V2D > CaVβ2d_S197F). In conclusion, patch-clamp recordings of ASD-associated CaVβ2d 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 CaVβ2d mutations analyzed might contribute to the complex pathophysiology of ASD and by this indicate a possible underlying molecular mechanism.
Abstract 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 (Ca V β 1-4 ). Our previous findings suggested that activation of L-type VGCCs is a common feature of Ca V β 2 subunit mutations found in ASD patients. In the current study, we functionally characterized a novel Ca V β 1b variant (p.R296C) identified in an ASD patient. We used whole-cell and single-channel patch clamp to study the effect of Ca V β 1b_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 Ca V β 1b -subunits with the RGK-protein Gem. Our data obtained at both, whole-cell and single-channel levels, show that compared to a wild-type Ca V β 1b , the Ca V β 1b_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 Ca V β 1b_R296C variant differing from that attributed to Ca V β 2 variants found in ASD patients. Further studies have to detail the effects on different VGCC subtypes and on VGCC expression.
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