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α 2 δ proteins serve as auxiliary subunits of voltage-gated calcium channels and regulate channel membrane expression and current properties. Besides their channel function, α 2 δ proteins regulate synapse formation, differentiation, and synaptic wiring. Considering these important functions, it is not surprising that CACNA2D1-4, the genes encoding for α 2 δ-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 α 2 δ-2, a homozygous mutation previously identified in two siblings with DEE. Importantly, we analyzed both calcium channel-dependent as well as synaptic functions of α 2 δ-2. Our data show that the corresponding p.R596P mutation in mouse α 2 δ-2 drastically decreases membrane expression and synaptic targeting of α 2 δ-2. This defect correlates with altered biophysical properties of postsynaptic Ca V 1.3 channel but has no effect on presynaptic Ca V 2.1 channels upon heterologous expression in tsA201 cells. However, homologous expression of α 2 δ-2_R596P in primary cultures of hippocampal neurons affects the ability of α 2 δ-2 to induce a statistically significant increase in the presynaptic abundance of endogenous Ca V 2.1 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 GABA A receptors and presynaptic synapsin clustering in glutamatergic synapses. Lastly, the α 2 δ-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 α 2 δ mutations not only in the context of channelopathies but also synaptopathies., (© 2024 The Author(s). Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

Synapses provide the main route of signal transduction within neuronal networks. Many factors regulate critical synaptic functions. These include presynaptic calcium channels, triggering neurotransmitter release, and postsynaptic ionotropic receptors, mediating excitatory and inhibitory postsynaptic potentials. The key features of synaptic transmission and plasticity can be studied in primary cultured hippocampal neurons. Here, we describe a protocol for the preparation and electrophysiological analysis of paired hippocampal neurons. This model system allows the selective genetic manipulation of one neuron in a simple neuronal network formed by only two hippocampal neurons. Bi-directionally analyzing synaptic transmission and short-term synaptic plasticity allows the analysis of both pre- and postsynaptic effects on synaptic transmission. For example, with one single paired network synaptic responses induced by both, a wild-type neuron and a genetically modified neuron can be directly compared. Ultimately, this protocol allows experimental modulation and hence investigation of synaptic mechanisms and thereby improves previously developed methods of studying synaptic transmission and plasticity in ex vivo cultured neurons. Key features Preparation of ex vivo paired cultured hippocampal neurons. Bi-directional electrophysiological recordings of synaptic transmission and plasticity. Genetic modulation of synaptic network formation (demonstrated by presynaptic viral overexpression of the auxiliary calcium channel α 2 δ-2 subunit). Graphical overview., Competing Interests: Competing interestsWe declare neither competing interests nor conflicts of interest., (©Copyright : © 2023 The Authors; This is an open access article under the CC BY license.)

Presynaptic α 2 δ subunits of voltage-gated calcium channels regulate channel abundance and are involved in glutamatergic synapse formation. However, little is known about the specific functions of the individual α 2 δ isoforms and their role in GABAergic synapses. Using primary neuronal cultures of embryonic mice of both sexes, we here report that presynaptic overexpression of α 2 δ-2 in GABAergic synapses strongly increases clustering of postsynaptic GABA A Rs. Strikingly, presynaptic α 2 δ-2 exerts the same effect in glutamatergic synapses, leading to a mismatched localization of GABA A Rs. This mismatching is caused by an aberrant wiring of glutamatergic presynaptic boutons with GABAergic postsynaptic positions. The trans-synaptic effect of α 2 δ-2 is independent of the prototypical cell-adhesion molecules α-neurexins (α-Nrxns); however, α-Nrxns together with α 2 δ-2 can modulate postsynaptic GABA A R abundance. Finally, exclusion of the alternatively spliced exon 23 of α 2 δ-2 is essential for the trans-synaptic mechanism. The novel function of α 2 δ-2 identified here may explain how abnormal α 2 δ subunit expression can cause excitatory-inhibitory imbalance often associated with neuropsychiatric disorders. SIGNIFICANCE STATEMENT Voltage-gated calcium channels regulate important neuronal functions such as synaptic transmission. α 2 δ subunits modulate calcium channels and are emerging as regulators of brain connectivity. However, little is known about how individual α 2 δ subunits contribute to synapse specificity. Here, we show that presynaptic expression of a single α 2 δ variant can modulate synaptic connectivity and the localization of inhibitory postsynaptic receptors. Our findings provide basic insights into the development of specific synaptic connections between nerve cells and contribute to our understanding of normal nerve cell functions. Furthermore, the identified mechanism may explain how an altered expression of calcium channel subunits can result in aberrant neuronal wiring often associated with neuropsychiatric disorders such as autism or schizophrenia., (Copyright © 2019 Geisler et al.)

L-type voltage-gated Ca V 1.2 calcium channels (Ca V 1.2) are key regulators of neuronal excitability, synaptic plasticity, and excitation-transcription coupling. Surface-exposed Ca V 1.2 distributes in clusters along the dendrites of hippocampal neurons. A permanent exchange between stably clustered and laterally diffusive extra-clustered channels maintains steady-state levels of Ca V 1.2 at dendritic signaling domains. A dynamic equilibrium between anchored and diffusive receptors is a common feature among ion channels and is crucial to modulate signaling transduction. Despite the importance of this fine regulatory system, the molecular mechanisms underlying the surface dynamics of Ca V 1.2 are completely unexplored. Here, we examined the dynamic states of Ca V 1.2 depending on phosphorylation on Ser-1700 and Ser-1928 at the channel C terminus. Phosphorylation at these sites is strongly involved in Ca V 1.2-mediated nuclear factor of activated T cells (NFAT) signaling, long-term potentiation, and responsiveness to adrenergic stimulation. We engineered Ca V 1.2 constructs mimicking phosphorylation at Ser-1700 and Ser-1928 and analyzed their behavior at the membrane by immunolabeling protocols, fluorescence recovery after photobleaching, and single particle tracking. We found that the phosphomimetic S1928E variant increases the mobility of Ca V 1.2 without altering the steady-state maintenance of cluster in young neurons and favors channel stabilization later in differentiation. Instead, mimicking phosphorylation at Ser-1700 promoted the diffusive state of Ca V 1.2 irrespective of the differentiation stage. Together, these results reveal that phosphorylation could contribute to the establishment of channel anchoring mechanisms depending on the neuronal differentiation state. Finally, our findings suggest a novel mechanism by which phosphorylation at the C terminus regulates calcium signaling by tuning the content of Ca V 1.2 at signaling complexes., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Spinocerebellar ataxia type 6 (SCA6) is an autosomal-dominant neurodegenerative disorder that is caused by a CAG trinucleotide repeat expansion in the CACNA1A gene. As one of the few bicistronic genes discovered in the human genome, CACNA1A encodes not only the α1A subunit of the P/Q type voltage-gated Ca 2+ channel Ca V 2.1 but also the α1ACT protein, a 75 kDa transcription factor sharing the sequence of the cytoplasmic C-terminal tail of the α1A subunit. Isoforms of both proteins contain the polyglutamine (polyQ) domain that is expanded in SCA6 patients. Although certain SCA6 phenotypes appear to be specific for Purkinje neurons, other pathogenic effects of the SCA6 polyQ mutation can affect a broad spectrum of central nervous system (CNS) neuronal subtypes. We investigated the expression and function of CACNA1A gene products in human neurons derived from induced pluripotent stem cells from two SCA6 patients. Expression levels of CACNA1A encoding α1A subunit were similar between SCA6 and control neurons, and no differences were found in the subcellular distribution of Ca V 2.1 channel protein. The α1ACT immunoreactivity was detected in the majority of cell nuclei of SCA6 and control neurons. Although no SCA6 genotype-dependent differences in Ca V 2.1 channel function were observed, they were found in the expression levels of the α1ACT target gene Granulin (GRN) and in glutamate-induced cell vulnerability.

Dendritic spines are the postsynaptic compartments of glutamatergic synapses in the brain. Their number and shape are subject to change in synaptic plasticity and neurological disorders including autism spectrum disorders and Parkinson's disease. The L-type calcium channel Ca V 1.3 constitutes an important calcium entry pathway implicated in the regulation of spine morphology. Here we investigated the importance of full-length Ca V 1.3 L and two C-terminally truncated splice variants (Ca V 1.3 42A and Ca V 1.3 43S ) and their modulation by densin-180 and shank1b for the morphology of dendritic spines of cultured hippocampal neurons. Live-cell immunofluorescence and super-resolution microscopy of epitope-tagged Ca V 1.3 L revealed its localization at the base-, neck-, and head-region of dendritic spines. Expression of the short splice variants or deletion of the C-terminal PDZ-binding motif in Ca V 1.3 L induced aberrant dendritic spine elongation. Similar morphological alterations were induced by co-expression of densin-180 or shank1b with Ca V 1.3 L and correlated with increased Ca V 1.3 currents and dendritic calcium signals in transfected neurons. Together, our findings suggest a key role of Ca V 1.3 in regulating dendritic spine structure. Under physiological conditions it may contribute to the structural plasticity of glutamatergic synapses. Conversely, altered regulation of Ca V 1.3 channels may provide an important mechanism in the development of postsynaptic aberrations associated with neurodegenerative disorders.

The β subunits of voltage-gated calcium channels regulate surface expression and gating of CaV1 and CaV2 α1 subunits and thus contribute to neuronal excitability, neurotransmitter release, and calcium-induced gene regulation. In addition, certain β subunits are targeted into the nucleus, where they interact directly with the epigenetic machinery. Whereas their involvement in this multitude of functions is reflected by a great molecular heterogeneity of β isoforms derived from four genes and abundant alternative splicing, little is known about the roles of individual β variants in specific neuronal functions. In the present study, an alternatively spliced β4 subunit lacking the variable N terminus (β4e) is identified. It is highly expressed in mouse cerebellum and cultured cerebellar granule cells (CGCs) and modulates P/Q-type calcium currents in tsA201 cells and CaV2.1 surface expression in neurons. Compared with the other two known full-length β4 variants (β4a and β4b), β4e is most abundantly expressed in the distal axon, but lacks nuclear-targeting properties. To determine the importance of nuclear targeting of β4 subunits for transcriptional regulation, we performed whole-genome expression profiling of CGCs from lethargic (β4-null) mice individually reconstituted with β4a, β4b, and β4e. Notably, the number of genes regulated by each β4 splice variant correlated with the rank order of their nuclear-targeting properties (β4b > β4a > β4e). Together, these findings support isoform-specific functions of β4 splice variants in neurons, with β4b playing a dual role in channel modulation and gene regulation, whereas the newly detected β4e variant serves exclusively in calcium-channel-dependent functions.

Elevation of cytosolic level of Ca(2+) was measured by spatial screening of freshly isolated dorsal root ganglion neurons loaded with Fura-2AM after subjecting them to a moderate hypoxic solution (pO(2)=10-40 mmHg). Short exposure of neurons to hypoxia resulted in a reversible elevation of intracellular Ca(2+) to about 120% in the cell center and to 80% in the cell periphery. Such elevation could be almost completely eliminated by removal of Ca(2+) or Na(+) from external medium or application of nifedipine, an L-type calcium channel blocker. Remarkable antihypoxic efficiency (58%) was achieved by preapplication of mitochondrial protonophore CCCP. A conclusion is made that in sensory neurons the hypoxia-induced elevation of cytosolic Ca(2+) is induced by combined changes of function in three cell substructures: voltage-operated L-type Ca(2+) and Na(+) channels and Ca(2+) accumulation by mitochondria. Mitochondria are important for spatial difference in the hypoxia-induced Ca(2+) elevation due to their specific location in these neurons.

Hypoxia is the main reason leading to neuronal death during different forms of brain diseases. The main phenomenon observed at hypoxia is excessive growth of intraneuronal Ca2+ concentration leading to irreversible cell damage. Despite extensive studies of this process, the intracellular mechanisms responsible for disturbance in Ca2+ are still unclear. The aim of present investigations was to explore these mechanisms. Ca2+ was measured by spatial screening of isolated dorsal root ganglion (sensory) neurons loaded with fluorescent dye Fura-2AM after exposing them hypoxic solution. Hypoxia resulted in a reversible elevation of Ca2+, which could be partly prevented by several pharmacological agents. We concluded that in sensory neurons hypoxia-induced elevation of cytosolic Ca2+ is induced by primary changes in ionic channels and secondary in function of mitochondria.