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VGCCs are multisubunit complexes that play a crucial role in neuronal signaling. Auxiliary α2δ subunits of VGCCs modulate trafficking and biophysical properties of the pore-forming α1 subunit and trigger excitatory synaptogenesis. Alterations in the expression level of α2δ subunits were implicated in several syndromes and diseases, including chronic neuropathic pain, autism, and epilepsy. However, the contribution of distinct α2δ subunits to excitatory/inhibitory imbalance and aberrant network connectivity characteristic for these pathologic conditions remains unclear. Here, we show that α2δ1 overexpression enhances spontaneous neuronal network activity in developing and mature cultures of hippocampal neurons. In contrast, overexpression, but not downregulation, of α2δ3 enhances neuronal firing in immature cultures, whereas later in development it suppresses neuronal activity. We found that α2δ1 overexpression increases excitatory synaptic density and selectively enhances presynaptic glutamate release, which is impaired on α2δ1 knockdown. Overexpression of α2δ3 increases the excitatory synaptic density as well but also facilitates spontaneous GABA release and triggers an increase in the density of inhibitory synapses, which is accompanied by enhanced axonaloutgrowth in immature interneurons. Together, our findings demonstrate that α2δ1 and α2δ3 subunits play distinct but complementary roles in driving formation of structural and functional network connectivity during early development. An alteration in α2δ surface expression during critical developmental windows can therefore play a causal role and have a profound impact on the excitatory-to-inhibitory balance and network connectivity. SIGNIFICANCE STATEMENT The computational capacity of neuronal networks is determined by their connectivity. Chemical synapses are the main interface for transfer of information between individual neurons. The initial formation of network connectivity requires spontaneous electrical activity and the calcium channel-mediated signaling. We found that, in early development, auxiliary α2δ3 subunits of calcium channels foster presynaptic release of GABA, trigger formation of inhibitory synapses, and promote axonal outgrowth in inhibitory interneurons. In contrast, later in development, α2δ1 subunits promote the glutamatergic neurotransmission and synaptogenesis, as well as strongly enhance neuronal network activity. We propose that formation of connectivity in neuronal networks is associated with a concerted interplay of α2δ1 and α2δ3 subunits of calcium channels., (Copyright © 2020 Bikbaev, Ciuraszkiewicz-Wojciech et al.)

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.)

Voltage-gated Ca v 1.2 and Ca v 1.3 (L-type) Ca 2+ channels regulate neuronal excitability, synaptic plasticity, and learning and memory. Densin-180 (densin) is an excitatory synaptic protein that promotes Ca 2+ -dependent facilitation of voltage-gated Ca v 1.3 Ca 2+ channels in transfected cells. Mice lacking densin (densin KO) exhibit defects in synaptic plasticity, spatial memory, and increased anxiety-related behaviors-phenotypes that more closely match those in mice lacking Ca v 1.2 than Ca v 1.3. Therefore, we investigated the functional impact of densin on Ca v 1.2. We report that densin is an essential regulator of Ca v 1.2 in neurons, but has distinct modulatory effects compared with its regulation of Ca v 1.3. Densin binds to the N-terminal domain of Ca v 1.2, but not that of Ca v 1.3, and increases Ca v 1.2 currents in transfected cells and in neurons. In transfected cells, densin accelerates the forward trafficking of Ca v 1.2 channels without affecting their endocytosis. Consistent with a role for densin in increasing the number of postsynaptic Ca v 1.2 channels, overexpression of densin increases the clustering of Ca v 1.2 in dendrites of hippocampal neurons in culture. Compared with wild-type mice, the cell surface levels of Ca v 1.2 in the brain, as well as Ca v 1.2 current density and signaling to the nucleus, are reduced in neurons from densin KO mice. We conclude that densin is an essential regulator of neuronal Ca v 1 channels and ensures efficient Ca v 1.2 Ca 2+ signaling at excitatory synapses. SIGNIFICANCE STATEMENT The number and localization of voltage-gated Ca v Ca 2+ channels are crucial determinants of neuronal excitability and synaptic transmission. We report that the protein densin-180 is highly enriched at excitatory synapses in the brain and enhances the cell surface trafficking and postsynaptic localization of Ca v 1.2 L-type Ca 2+ channels in neurons. This interaction promotes coupling of Ca v 1.2 channels to activity-dependent gene transcription. Our results reveal a mechanism that may contribute to the roles of Ca v 1.2 in regulating cognition and mood., (Copyright © 2017 the authors 0270-6474/17/374679-13$15.00/0.)

The auxiliary subunit α 2 δ2 modulates the abundance and function of voltage-gated calcium channels. Here we show that α 2 δ2 mRNA is expressed in neonatal and mature hair cells. A functional α 2 δ2-null mouse, the ducky mouse (du), showed elevated auditory brainstem response click and frequency-dependent hearing thresholds. Otoacoustic emissions were not impaired pointing to normal outer hair cell function. Peak Ca 2+ and Ba 2+ currents of mature du/du inner hair cells (IHCs) were reduced by 30-40%, respectively, and gating properties, such as the voltage of half-maximum activation and voltage sensitivity, were altered, indicating that Ca v 1.3 channels normally coassemble with α 2 δ2 at IHC presynapses. The reduction of depolarization-evoked exocytosis in du/du IHCs reflected their reduced Ca 2+ currents. Ca 2+ - and voltage-dependent K + (BK) currents and the expression of the pore-forming BKα protein were normal. Ca v 1.3 and Ca v β2 protein expression was unchanged in du/du IHCs, forming clusters at presynaptic ribbons. However, the close apposition of presynaptic Ca v 1.3 clusters with postsynaptic glutamate receptor GluA4 and PSD-95 clusters was significantly impaired in du/du mice. This implies that, in addition to controlling the expression and gating properties of Ca v 1.3 channels, the largely extracellularly localized α 2 δ2 subunit moreover plays a so far unknown role in mediating trans-synaptic alignment of presynaptic Ca 2+ channels and postsynaptic AMPA receptors., Significance Statement: Inner hair cells possess calcium channels that are essential for transmitting sound information into synaptic transmitter release. Voltage-gated calcium channels can coassemble with auxiliary subunit α 2 δ isoforms 1-4. We found that hair cells of the mouse express the auxiliary subunit α 2 δ2, which is needed for normal hearing thresholds. Using a mouse model with a mutant, nonfunctional α 2 δ2 protein, we showed that the α 2 δ2 protein is necessary for normal calcium currents and exocytosis in inner hair cells. Unexpectedly, the α 2 δ2 protein is moreover required for the optimal spatial alignment of presynaptic calcium channels and postsynaptic glutamate receptor proteins across the synaptic cleft. This suggests that α 2 δ2 plays a novel role in organizing the synapse., (Copyright © 2016 the authors 0270-6474/16/3611024-13$15.00/0.)

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.

In neurons L-type calcium currents function in gene regulation and synaptic plasticity, while excessive calcium influx leads to excitotoxicity and neurodegeneration. The major neuronal Ca(V)1.2 L-type channels are localized in clusters in dendritic shafts and spines. Whereas Ca(V)1.2 clusters remain stable during NMDA-induced synaptic depression, L-type calcium currents are rapidly downregulated during strong excitatory stimulation. Here we used fluorescence recovery after photobleaching (FRAP), live cell-labeling protocols, and single particle tracking (SPT) to analyze the turnover and surface traffic of Ca(V)1.2 in dendrites of mature cultured mouse and rat hippocampal neurons, respectively. FRAP analysis of channels extracellularly tagged with superecliptic pHluorin (Ca(V)1.2-SEP) demonstrated ∼20% recovery within 2 min without reappearance of clusters. Pulse-chase labeling showed that membrane-expressed Ca(V)1.2-HA is not internalized within1 h, while blocking dynamin-dependent endocytosis resulted in increased cluster density after 30 min. Together, these results suggest a turnover rate of clustered Ca(V)1.2s on the hour time scale. Direct recording of the lateral movement in the membrane using SPT demonstrated that dendritic Ca(V)1.2s show highly confined mobility with diffusion coefficients of ∼0.005 μm² s⁻¹. Consistent with the mobile Ca(V)1.2 fraction observed in FRAP, a ∼30% subpopulation of channels reversibly exchanged between confined and diffusive states. Remarkably, high potassium depolarization did not alter the recovery rates in FRAP or the diffusion coefficients in SPT analyses. Thus, an equilibrium of clustered and dynamic Ca(V)1.2s maintains stable calcium channel complexes involved in activity-dependent cell signaling, whereas the minor mobile channel pool in mature neurons allows limited capacity for short-term adaptations.

Ca(v)1 (L-type) channels and calmodulin-dependent protein kinase II (CaMKII) are key regulators of Ca(2+) signaling in neurons. CaMKII directly potentiates the activity of Ca(v)1.2 and Ca(v)1.3 channels, but the underlying molecular mechanisms are incompletely understood. Here, we report that the CaMKII-associated protein densin is required for Ca(2+)-dependent facilitation of Ca(v)1.3 channels. While neither CaMKII nor densin independently affects Ca(v)1.3 properties in transfected HEK293T cells, the two together augment Ca(v)1.3 Ca(2+) currents during repetitive, but not sustained, depolarizing stimuli. Facilitation requires Ca(2+), CaMKII activation, and its association with densin, as well as densin binding to the Ca(v)1.3 alpha(1) subunit C-terminal domain. Ca(v)1.3 channels and densin are targeted to dendritic spines in neurons and form a complex with CaMKII in the brain. Our results demonstrate a novel mechanism for Ca(2+)-dependent facilitation that may intensify postsynaptic Ca(2+) signals during high-frequency stimulation.

In neurons L-type calcium currents contribute to synaptic plasticity and to activity-dependent gene regulation. The subcellular localization of Ca(V)1.2 and its association with upstream and downstream signaling proteins is important for efficient and specific signal transduction. Here we tested the hypothesis that A-kinase anchoring proteins (AKAPs) or PDZ-proteins are responsible for the targeting and anchoring of Ca(V)1.2 in the postsynaptic compartment of glutamatergic neurons. Double-immunofluorescence labeling of hippocampal neurons transfected with external HA epitope-tagged Ca(V)1.2 demonstrated that clusters of membrane-incorporated Ca(V)1.2-HA were colocalized with AKAP79/150 but not with PSD-95 in the spines and shafts of dendrites. To disrupt the interactions with these scaffold proteins, we mutated known binding sequences for AKAP79/150 and PDZ proteins in the C terminus of Ca(V)1.2-HA. Unexpectedly, the distribution pattern, the density, and the fluorescence intensity of clusters were similar for wild-type and mutant Ca(V)1.2-HA, indicating that interactions with AKAP and PDZ proteins are not essential for the correct targeting of Ca(V)1.2. In agreement, brief treatment with NMDA (a chemical LTD paradigm) caused the degradation of PSD-95 and the redistribution of AKAP79/150 and alpha-actinin from dendritic spines into the shaft, without a concurrent loss or redistribution of Ca(V)1.2-HA clusters. Thus, in the postsynaptic compartment of hippocampal neurons Ca(V)1.2 calcium channels form signaling complexes apart from those of glutamate receptors and PSD-95. Their number and distribution in dendritic spines is not altered upon NMDA-induced disruption of the glutamate receptor signaling complex, and targeting and anchoring of Ca(V)1.2 is independent of its interactions with AKAP79/150 and PDZ proteins.

Single-channel analysis revealed the existence of neuronal L-type Ca2+ channels (LTCCs) with fundamentally different gating properties; in addition to LTCCs resembling cardiac channels, LTCCs with anomalous gating were identified in a variety of neurons, including cerebellar granule cells. Anomalous LTCC gating is mainly characterized by long reopenings after repolarization following strong depolarizations or trains of action potentials. To elucidate the unknown molecular nature of anomalous LTCCs, we performed single-channel patch-clamp recordings from cerebellar granule cells of wild-type, Ca(v)1.3-/- and Ca(v)1.2DHP-/- [containing a mutation in the Ca(v)1.2 alpha1 subunit that eliminates dihydropyridine (DHP) sensitivity] mice. Quantitative reverse transcription-PCR revealed that Ca(v)1.2 accounts for 89% and Ca(v)1.3 for 11% of the LTCC transcripts in wild-type cerebellar granule cells, whereas Ca(v)1.1 and Ca(v)1.4 are expressed at insignificant levels. Anomalous LTCCs were observed in neurons of Ca(v)1.3-/- mice with a frequency not different from wild type. In the presence of the DHP agonist (+)-(S)-202-791, the typical prepulse-induced reopenings of anomalous LTCCs after repolarization were shorter in Ca(v)1.2DHP-/- neurons than in Ca(v)1.3-/- neurons. Reopenings in Ca(v)1.2DHP-/- neurons in the presence of the DHP agonist were similar to those in wild-type neurons in the absence of the agonist. These data show that Ca(v)1.2alpha1 subunits are the pore-forming subunits of anomalous LTCCs in mouse cerebellar granule cells. Given the evidence that Ca(v)1.2 channels are specifically involved in sustained Ras-MAPK (mitogen-activated protein kinase)-dependent cAMP response element-binding protein phosphorylation and LTCC-dependent hippocampal long-term potentiation (LTP) (Moosmang et al., 2005), we discuss the hypothesis that anomalous rather than cardiac-type Ca(v)1.2 channels are specifically involved in LTCC-dependent and gene transcription-dependent LTP.