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High voltage‐gated Ca2+ channels (HVCCs) shape the electrical activity and control hormone release in most endocrine cells. HVCCs are multi‐subunit protein complexes formed by the pore‐forming α1 and the auxiliary β, α2δ and γ subunits. Four genes code for the α2δ isoforms. At the mRNA level, mouse chromaffin cells (MCCs) express predominantly the CACNA2D1 gene coding for the α2δ‐1 isoform. Here we show that α2δ‐1 deletion led to ∼60% reduced HVCC Ca2+ influx with slower inactivation kinetics. Pharmacological dissection showed that HVCC composition remained similar in α2δ‐1−/− MCCs compared to wild‐type (WT), demonstrating that α2δ‐1 exerts similar functional effects on all HVCC isoforms. Consistent with reduced HVCC Ca2+ influx, α2δ‐1−/− MCCs showed reduced spontaneous electrical activity with action potentials (APs) having a shorter half‐maximal duration caused by faster rising and decay slopes. However, the induced electrical activity showed opposite effects with α2δ‐1−/− MCCs displaying significantly higher AP frequency in the tonic firing mode as well as an increase in the number of cells firing AP bursts compared to WT. This gain‐of‐function phenotype was caused by reduced functional activation of Ca2+‐dependent K+ currents. Additionally, despite the reduced HVCC Ca2+ influx, the intracellular Ca2+ transients and vesicle exocytosis or endocytosis were unaltered in α2δ‐1−/− MCCs compared to WT during sustained stimulation. In conclusion, our study shows that α2δ‐1 genetic deletion reduces Ca2+ influx in cultured MCCs but leads to a paradoxical increase in catecholamine secretion due to increased excitability. Key points: Deletion of the α2δ‐1 high voltage‐gated Ca2+ channel (HVCC) subunit reduces mouse chromaffin cell (MCC) Ca2+ influx by ∼60% but causes a paradoxical increase in induced excitability.MCC intracellular Ca2+ transients are unaffected by the reduced HVCC Ca2+ influx.Deletion of α2δ‐1 reduces the immediately releasable pool vesicle exocytosis but has no effect on catecholamine (CA) release in response to sustained stimuli.The increased electrical activity and CA release from MCCs might contribute to the previously reported cardiovascular phenotype of patients carrying α2δ‐1 loss‐of‐function mutations. [ABSTRACT FROM AUTHOR]

Key points: Tymothy syndrome (TS) is a multisystem disorder featuring cardiac arrhythmias, autism and adrenal gland dysfunction that originates from a de novo point mutation in the gene encoding the Cav1.2 (CACNA1C) L‐type channel.To study the role of Cav1.2 channel signals in autism, the autistic TS2‐neo mouse has been generated bearing the G406R point‐mutation associated with TS type‐2.Using heterozygous TS2‐neo mice, we report that the G406R mutation reduces the rate of inactivation and shifts leftward the activation and inactivation of L‐type channels, causing marked increase of resting Ca2+ influx ('window' Ca2+ current).The increased 'window current' causes marked reduction of NaV channel density, switches normal tonic firing to abnormal burst firing, reduces mitochondrial metabolism, induces cell swelling and decreases catecholamine release.Overnight incubations with nifedipine rescue NaV channel density, normal firing and the quantity of catecholamine released. We provide evidence that chromaffin cell malfunction derives from altered Cav1.2 channel gating. L‐type voltage‐gated calcium (Cav1) channels have a key role in long‐term synaptic plasticity, sensory transduction, muscle contraction and hormone release. A point mutation in the gene encoding Cav1.2 (CACNA1C) causes Tymothy syndrome (TS), a multisystem disorder featuring cardiac arrhythmias, autism spectrum disorder (ASD) and adrenal gland dysfunction. In the more severe type‐2 form (TS2), the missense mutation G406R is on exon 8 coding for the IS6‐helix of the Cav1.2 channel. The mutation causes reduced inactivation and induces autism. How this occurs and how Cav1.2 gating‐changes alter cell excitability, neuronal firing and hormone release on a molecular basis is still largely unknown. Here, using the TS2‐neo mouse model of TS we show that the G406R mutation altered excitability and reduced secretory activity in adrenal chromaffin cells (CCs). Specifically, the TS2 mutation reduced the rate of voltage‐dependent inactivation and shifted leftward the activation and steady‐state inactivation of L‐type channels. This markedly increased the resting 'window' Ca2+ current that caused an increased percentage of CCs undergoing abnormal action potential (AP) burst firing, cell swelling, reduced mitochondrial metabolism and decreased catecholamine release. The increased 'window' Ca2+ current caused also decreased NaV channel density and increased steady‐state inactivation, which contributed to the increased abnormal burst firing. Overnight incubation with the L‐type channel blocker nifedipine rescued the normal AP firing of CCs, the density of functioning NaV channels and their steady‐state inactivation. We provide evidence that CC malfunction derives from the altered Cav1.2 channel gating and that dihydropyridines are potential therapeutics for ASD. Key points: Tymothy syndrome (TS) is a multisystem disorder featuring cardiac arrhythmias, autism and adrenal gland dysfunction that originates from a de novo point mutation in the gene encoding the Cav1.2 (CACNA1C) L‐type channel.To study the role of Cav1.2 channel signals in autism, the autistic TS2‐neo mouse has been generated bearing the G406R point‐mutation associated with TS type‐2.Using heterozygous TS2‐neo mice, we report that the G406R mutation reduces the rate of inactivation and shifts leftward the activation and inactivation of L‐type channels, causing marked increase of resting Ca2+ influx ('window' Ca2+ current).The increased 'window current' causes marked reduction of NaV channel density, switches normal tonic firing to abnormal burst firing, reduces mitochondrial metabolism, induces cell swelling and decreases catecholamine release.Overnight incubations with nifedipine rescue NaV channel density, normal firing and the quantity of catecholamine released. We provide evidence that chromaffin cell malfunction derives from altered Cav1.2 channel gating. [ABSTRACT FROM AUTHOR]

Sprouty proteins act as negative feedback inhibitors of fibroblast growth factor (FGF) signaling. FGFs belong to the neurotrophic factors and are involved in axonal growth during development and repair. We investigated the expression of Sprouty isoforms in hippocampal neurons as well as the regulation of Sprouty2 and -4 during development and their role in axon growth. Sprouty2 and -4 were located in the nucleus, the cytoplasm, in dendrites, and axons of hippocampal neurons concentrated in growth cones. During development in vivo and differentiation in vitro, expression of Sprouty2 and -4 was gradually downregulated in hippocampal neurons. Between 5 and 24 days in culture expression of both Sprouty isoforms was reduced by 70%. In vivo expression of Sprouty2 was reduced by 79% and of Sprouty4 by 93% on postnatal day 14 compared to embryonic day 16.5. Downregulation of Sprouty2 and -4 by shRNAs strongly promoted elongative axon growth by cultured hippocampal neurons, which was further increased by FGF-2 treatment. In addition, FGF-2 reduced expression of Sprouty2 by 33% and of Sprouty4 by 44%. Together, our results imply that Sprouty2 and -4 are downregulated in the hippocampus during postnatal brain development and that they can act as regulators of developmental axon growth. © 2011 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Sequences in the cytoplasmic II–III loop of CaV2 voltage-gated calcium channels, termed the synaptic protein interaction (synprint) site, are considered important for the functional incorporation of presynaptic calcium channels into the synaptic vesicle fusion apparatus. Two novel CaV2.2 splice variants lack large parts of the cytoplasmic II-III loop (Δ1 R756-L1139, Δ2 K737-A1001) including the synprint protein–protein interaction domain. Here we expressed green fluorescent protein (GFP)-α1B subunit fusion constructs of CaV2.2 splice variants in mouse hippocampal neurons to study their distribution in distinct neuronal compartments and to address the question of whether and how the synprint site functions in the presynaptic targeting of N-type calcium channels. Similar to full-length GFP-α1B but divergent from the somatodendritic α1C-HA (CaV1.2) channel type, the splice variants GFP-α1B-Δ1 and GFP-α1B-Δ2 were targeted into the axons. Nevertheless, their ability to form bona fide presynaptic clusters was almost abolished for GFP-α1B-Δ1 and significantly reduced for GFP-α1B-Δ2. Thus, the synprint site is important for normal synaptic targeting of CaV2.2 but not essential. Conversely, insertion of the synprint site into the II–III loop of α1C-HA did not restore axonal targeting or synaptic clustering. Together these results indicate that protein–protein interactions with the synprint site must cooperate with other targeting mechanisms in the incorporation of CaV2.2 into presynaptic specializations of hippocampal neurons but are neither necessary nor sufficient for axonal targeting. The unique targeting properties of the splice variants lacking the synprint site are suggestive of specific functions of these calcium channels apart from activating fast synaptic transmission. [ABSTRACT FROM AUTHOR]

In central nervous system neurons L-type Ca2+ channels are involved in developmental processes, the integration and conduction of postsynaptic electric activity and synaptic plasticity. However, little is known about the channel isoforms underlying each of these functions or about the exact localization and targeting properties of the major L-type channel isoform α1C (Cav1.2) in neurons. We addressed these questions using high-resolution immunofluorescence analysis of the endogenous α1C and epitope-tagged recombinant channel isoforms expressed in mouse hippocampal neurons. Endogenous α1C and surface-expressed hemagglutinin (HA)tagged α1C-HA were localized in small clusters distributed between the axon initial segment and the apical branches of the dendritic tree. The average cluster size was estimated to be eight channels per α1C-HA cluster. Analysis of the subcellular localization of α1C-HA clusters relative to known synaptic markers suggested the existence of two distinct populations of α1C clusters, extrasynaptic and synaptic, the latter associated with glutamatergic synapses in dendritic spines. Both glutamatergic and GABAergic neurons expressed α1C in the soma and dendrites. In contrast to the N-type channel GFP-α1B, GFP-α1C was excluded from distal axons and nerve terminals of mature neurons. In developing neurons, however, α1C and α1C-HA were robustly expressed in the growth cone, indicating that specific targeting properties of neuronal compartments change during differentiation. Synaptic and extrasynaptic localizations of α1C correspond to putative roles of L-type Ca2+ currents in synaptic modulation and in the propagation of dendritic Ca2+ spikes, respectively. The transiently expressed α1C in the growth cone may be involved in neurite extension and axonal pathfinding. [ABSTRACT FROM AUTHOR]

α2δ proteins serve as auxiliary subunits of voltage‐gated calcium channels and regulate channel membrane expression and current properties. Besides their channel function, α2δ proteins regulate synapse formation, differentiation, and synaptic wiring. Considering these important functions, it is not surprising that CACNA2D1‐4, the genes encoding for α2δ‐1 to ‐4 isoforms, have been implicated in neurological, neurodevelopmental, and neuropsychiatric disorders. Mutations in CACNA2D2 have been associated with developmental and epileptic encephalopathy (DEE) and cerebellar atrophy. In our present study, we performed a detailed functional characterization of the p.R593P mutation in α2δ‐2, a homozygous mutation previously identified in two siblings with DEE. Importantly, we analyzed both calcium channel‐dependent as well as synaptic functions of α2δ‐2. Our data show that the corresponding p.R596P mutation in mouse α2δ‐2 drastically decreases membrane expression and synaptic targeting of α2δ‐2. This defect correlates with altered biophysical properties of postsynaptic CaV1.3 channel but has no effect on presynaptic CaV2.1 channels upon heterologous expression in tsA201 cells. However, homologous expression of α2δ‐2_R596P in primary cultures of hippocampal neurons affects the ability of α2δ‐2 to induce a statistically significant increase in the presynaptic abundance of endogenous CaV2.1 channels and presynaptic calcium transients. Moreover, our data demonstrate that in addition to lowering membrane expression, the p.R596P mutation reduces the trans‐synaptic recruitment of GABAA receptors and presynaptic synapsin clustering in glutamatergic synapses. Lastly, the α2δ‐2_R596P reduces the amplitudes of glutamatergic miniature postsynaptic currents in transduced hippocampal neurons. Taken together, our data strongly link the human biallelic p.R593P mutation to the underlying severe neurodevelopmental disorder and highlight the importance of studying α2δ mutations not only in the context of channelopathies but also synaptopathies. [ABSTRACT FROM AUTHOR]

Abstract In the central nervous system small conductance Ca2+ -activated K+ (SK) channels are important for generating the medium/slow afterhyperpolarization seen after single or trains of action potentials. Three SK channel isoforms (SK1,-2,-3) are differentially distributed throughout the brain, but little is known about their specific expression in particular neuronal compartments. In the hippocampus SK3 was found in the neuropil, predominantly in the terminal field of the mossy fibres and in fine varicose fibres, but excluded from the pyramidal and granule cell layers. Because this expression pattern suggested a presynaptic localization, we examined the subcellular distribution of SK3 in cultured hippocampal neurons using high-resolution immunofluorescence analysis. SK3 was localized in a punctate, synaptic pattern. The SK3 clusters were precisely colocalized with the presynaptic marker synapsin and at close range (0.4–0.5 µm) from NMDA-receptors and PSD-95. This arrangement is consistent with a localization of SK3 in the presynaptic nerve terminal, but not restricted to the synaptic membrane proper. In agreement with the increasing expression of SK3 during early postnatal development in vivo , the fraction of synapses containing SK3 increased from 14% to 57% over a six-week culture period. SK3-containing synapses were equally observed on spiny, glutamatergic and smooth GABAergic neurons. In contrast to its close association with NMDA-receptors and PSD-95, SK3 was rarely associated with GABAA -receptor clusters. Thus, SK3 is a presynaptic channel in excitatory hippocampal synapses, with no preference for glutamatergic or GABAergic postsynaptic neurons, and is probably involved in regulating neurotransmitter release. [ABSTRACT FROM AUTHOR]

Sequences in the cytoplasmic II-III loop of CaV2 voltage-gated calcium channels, termed the synaptic protein interaction (synprint) site, are considered important for the functional incorporation of presynaptic calcium channels into the synaptic vesicle fusion apparatus. Two novel CaV2.2 splice variants lack large parts of the cytoplasmic II-III loop (Delta1 R756-L1139, Delta2 K737-A1001) including the synprint protein-protein interaction domain. Here we expressed green fluorescent protein (GFP)-alpha1B subunit fusion constructs of CaV2.2 splice variants in mouse hippocampal neurons to study their distribution in distinct neuronal compartments and to address the question of whether and how the synprint site functions in the presynaptic targeting of N-type calcium channels. Similar to full-length GFP-alpha1B but divergent from the somatodendritic alpha1C-HA (CaV1.2) channel type, the splice variants GFP-alpha1B-Delta1 and GFP-alpha1B-Delta2 were targeted into the axons. Nevertheless, their ability to form bona fide presynaptic clusters was almost abolished for GFP-alpha1B-Delta1 and significantly reduced for GFP-alpha1B-Delta2. Thus, the synprint site is important for normal synaptic targeting of CaV2.2 but not essential. Conversely, insertion of the synprint site into the II-III loop of alpha1C-HA did not restore axonal targeting or synaptic clustering. Together these results indicate that protein-protein interactions with the synprint site must cooperate with other targeting mechanisms in the incorporation of CaV2.2 into presynaptic specializations of hippocampal neurons but are neither necessary nor sufficient for axonal targeting. The unique targeting properties of the splice variants lacking the synprint site are suggestive of specific functions of these calcium channels apart from activating fast synaptic transmission.

In central nervous system neurons L-type Ca2+ channels are involved in developmental processes, the integration and conduction of postsynaptic electric activity and synaptic plasticity. However, little is known about the channel isoforms underlying each of these functions or about the exact localization and targeting properties of the major L-type channel isoform alpha1C (CaV1.2) in neurons. We addressed these questions using high-resolution immunofluorescence analysis of the endogenous alpha1C and epitope-tagged recombinant channel isoforms expressed in mouse hippocampal neurons. Endogenous alpha1C and surface-expressed hemagglutinin (HA)-tagged alpha1C-HA were localized in small clusters distributed between the axon initial segment and the apical branches of the dendritic tree. The average cluster size was estimated to be eight channels per alpha1C-HA cluster. Analysis of the subcellular localization of alpha1C-HA clusters relative to known synaptic markers suggested the existence of two distinct populations of alpha1C clusters, extrasynaptic and synaptic, the latter associated with glutamatergic synapses in dendritic spines. Both glutamatergic and GABAergic neurons expressed alpha1C in the soma and dendrites. In contrast to the N-type channel GFP-alpha1B, GFP-alpha1C was excluded from distal axons and nerve terminals of mature neurons. In developing neurons, however, alpha1C and alpha1C-HA were robustly expressed in the growth cone, indicating that specific targeting properties of neuronal compartments change during differentiation. Synaptic and extrasynaptic localizations of alpha1C correspond to putative roles of L-type Ca2+ currents in synaptic modulation and in the propagation of dendritic Ca2+ spikes, respectively. The transiently expressed alpha1C in the growth cone may be involved in neurite extension and axonal pathfinding.

In the central nervous system small conductance Ca2+-activated K+ (SK) channels are important for generating the medium/slow afterhyperpolarization seen after single or trains of action potentials. Three SK channel isoforms (SK1,-2,-3) are differentially distributed throughout the brain, but little is known about their specific expression in particular neuronal compartments. In the hippocampus SK3 was found in the neuropil, predominantly in the terminal field of the mossy fibres and in fine varicose fibres, but excluded from the pyramidal and granule cell layers. Because this expression pattern suggested a presynaptic localization, we examined the subcellular distribution of SK3 in cultured hippocampal neurons using high-resolution immunofluorescence analysis. SK3 was localized in a punctate, synaptic pattern. The SK3 clusters were precisely colocalized with the presynaptic marker synapsin and at close range (0.4-0.5 microm) from NMDA-receptors and PSD-95. This arrangement is consistent with a localization of SK3 in the presynaptic nerve terminal, but not restricted to the synaptic membrane proper. In agreement with the increasing expression of SK3 during early postnatal development in vivo, the fraction of synapses containing SK3 increased from 14% to 57% over a six-week culture period. SK3-containing synapses were equally observed on spiny, glutamatergic and smooth GABAergic neurons. In contrast to its close association with NMDA-receptors and PSD-95, SK3 was rarely associated with GABAA-receptor clusters. Thus, SK3 is a presynaptic channel in excitatory hippocampal synapses, with no preference for glutamatergic or GABAergic postsynaptic neurons, and is probably involved in regulating neurotransmitter release.