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High voltage-gated Ca 2+ 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 α 1 and the auxiliary β, α 2 δ and γ subunits. Four genes code for the α 2 δ isoforms. At the mRNA level, mouse chromaffin cells (MCCs) express predominantly the CACNA2D1 gene coding for the α 2 δ-1 isoform. Here we show that α 2 δ-1 deletion led to ∼60% reduced HVCC Ca 2+ influx with slower inactivation kinetics. Pharmacological dissection showed that HVCC composition remained similar in α 2 δ-1 -/- MCCs compared to wild-type (WT), demonstrating that α 2 δ-1 exerts similar functional effects on all HVCC isoforms. Consistent with reduced HVCC Ca 2+ influx, α 2 δ-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 α 2 δ-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 Ca 2+ -dependent K + currents. Additionally, despite the reduced HVCC Ca 2+ influx, the intracellular Ca 2+ transients and vesicle exocytosis or endocytosis were unaltered in α 2 δ-1 -/- MCCs compared to WT during sustained stimulation. In conclusion, our study shows that α 2 δ-1 genetic deletion reduces Ca 2+ influx in cultured MCCs but leads to a paradoxical increase in catecholamine secretion due to increased excitability. KEY POINTS: Deletion of the α 2 δ-1 high voltage-gated Ca 2+ channel (HVCC) subunit reduces mouse chromaffin cell (MCC) Ca 2+ influx by ∼60% but causes a paradoxical increase in induced excitability. MCC intracellular Ca 2+ transients are unaffected by the reduced HVCC Ca 2+ influx. Deletion of α 2 δ-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 α 2 δ-1 loss-of-function mutations., (© 2024 The Author(s). The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

α 2 δ proteins serve as auxiliary subunits of voltage-gated calcium channels, which are essential components of excitable cells such as skeletal and heart muscles, nerve cells of the brain and the peripheral nervous system, as well as endocrine cells. Over the recent years, α 2 δ proteins have been identified as critical regulators of synaptic functions, including the formation and differentiation of synapses. These functions require signalling mechanisms which are partly independent of calcium channels. Hence, in light of these features it is not surprising that the genes encoding for the four α 2 δ isoforms have recently been linked to neurological and neurodevelopmental disorders including epilepsy, autism spectrum disorders, schizophrenia, and depressive and bipolar disorders. Despite the increasing number of identified disease-associated mutations, the underlying pathophysiological mechanisms are only beginning to emerge. However, a thorough understanding of the pathophysiological role of α 2 δ proteins ideally serves two purposes: first, it will contribute to our understanding of general pathological mechanisms in synaptic disorders. Second, it may support the future development of novel and specific treatments for brain disorders. In this context, it is noteworthy that the antiepileptic and anti-allodynic drugs gabapentin and pregabalin both act via binding to α 2 δ proteins and are among the top sold drugs for treating neuropathic pain. In this book chapter, we will discuss recent developments in our understanding of the functions of α 2 δ proteins, both as calcium channel subunits and as independent regulatory entities. Furthermore, we present and summarize recently identified and likely pathogenic mutations in the genes encoding α 2 δ proteins and discuss potential underlying pathophysiological consequences at the molecular and structural level., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

The α 2 δ auxiliary subunits of voltage-gated calcium channels (VGCC) were traditionally regarded as modulators of biophysical channel properties. In recent years, channel-independent functions of these subunits, such as involvement in synapse formation, have been identified. In the central nervous system, α 2 δ isoforms 1, 2, and 3 are strongly expressed, regulating glutamatergic synapse formation by a presynaptic mechanism. Although the α 2 δ-4 isoform is predominantly found in the retina with very little expression in the brain, it was recently linked to brain functions. In contrast, Cachd1, a novel α 2 δ-like protein, shows strong expression in brain, but its function in neurons is not yet known. Therefore, we aimed to investigate the presynaptic functions of α 2 δ-4 and Cachd1 by expressing individual proteins in cultured hippocampal neurons. Both α 2 δ-4 and Cachd1 are expressed in the presynaptic membrane and could rescue a severe synaptic defect present in triple knockout/knockdown neurons that lacked the α 2 δ-1-3 isoforms (α 2 δ TKO/KD). This observation suggests that presynaptic localization and the regulation of synapse formation in glutamatergic neurons is a general feature of α 2 δ proteins. In contrast to this redundant presynaptic function, α 2 δ-4 and Cachd1 differentially regulate the abundance of presynaptic calcium channels and the amplitude of presynaptic calcium transients. These functional differences may be caused by subtle isoform-specific differences in α 1 -α 2 δ protein-protein interactions, as revealed by structural homology modelling. Taken together, our study identifies both α 2 δ-4 and Cachd1 as presynaptic regulators of synapse formation, differentiation, and calcium channel functions that can at least partially compensate for the loss of α 2 δ-1-3. Moreover, we show that regulating glutamatergic synapse formation and differentiation is a critical and surprisingly redundant function of α 2 δ and Cachd1.

In nerve cells the genes encoding for α 2 δ subunits of voltage-gated calcium channels have been linked to synaptic functions and neurological disease. Here we show that α 2 δ subunits are essential for the formation and organization of glutamatergic synapses. Using a cellular α 2 δ subunit triple-knockout/knockdown model, we demonstrate a failure in presynaptic differentiation evidenced by defective presynaptic calcium channel clustering and calcium influx, smaller presynaptic active zones, and a strongly reduced accumulation of presynaptic vesicle-associated proteins (synapsin and vGLUT). The presynaptic defect is associated with the downscaling of postsynaptic AMPA receptors and the postsynaptic density. The role of α 2 δ isoforms as synaptic organizers is highly redundant, as each individual α 2 δ isoform can rescue presynaptic calcium channel trafficking and expression of synaptic proteins. Moreover, α 2 δ-2 and α 2 δ-3 with mutated metal ion-dependent adhesion sites can fully rescue presynaptic synapsin expression but only partially calcium channel trafficking, suggesting that the regulatory role of α 2 δ subunits is independent from its role as a calcium channel subunit. Our findings influence the current view on excitatory synapse formation. First, our study suggests that postsynaptic differentiation is secondary to presynaptic differentiation. Second, the dependence of presynaptic differentiation on α 2 δ implicates α 2 δ subunits as potential nucleation points for the organization of synapses. Finally, our results suggest that α 2 δ subunits act as transsynaptic organizers of glutamatergic synapses, thereby aligning the synaptic active zone with the postsynaptic density., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

α 2 δ proteins are membrane-anchored extracellular glycoproteins which are abundantly expressed in the brain and the peripheral nervous system. They serve as regulatory subunits of voltage-gated calcium channels and, particularly in nerve cells, regulate presynaptic and postsynaptic functions independently from their role as channel subunits. α 2 δ proteins are the targets of the widely prescribed anti-epileptic and anti-allodynic drugs gabapentin and pregabalin, particularly for the treatment of neuropathic pain conditions. Recently, the human genes (CACNA2D1-4) encoding for the four known α 2 δ proteins (isoforms α 2 δ-1 to α 2 δ-4) have been linked to a large variety of neurological and neuropsychiatric disorders including epilepsy, autism spectrum disorders, bipolar disorders, schizophrenia, and depressive disorders. Here, we provide an overview of the hitherto identified disease associations of all known α 2 δ genes, hypothesize on the pathophysiological mechanisms considering their known physiological roles, and discuss the most immanent future research questions. Elucidating their specific physiological and pathophysiological mechanisms may open the way for developing entirely novel therapeutic paradigms for treating brain disorders.

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.)

P/Q-type channels are the principal presynaptic calcium channels in brain functioning in neurotransmitter release. They are composed of the pore-forming CaV2.1 α1 subunit and the auxiliary α2δ-2 and β4 subunits. β4 is encoded by CACNB4, and its multiple splice variants serve isoform-specific functions as channel subunits and transcriptional regulators in the nucleus. In two siblings with intellectual disability, psychomotor retardation, blindness, epilepsy, movement disorder and cerebellar atrophy we identified rare homozygous variants in the genes LTBP1, EMILIN1, CACNB4, MINAR1, DHX38 and MYO15 by whole-exome sequencing. In silico tools, animal model, clinical, and genetic data suggest the p.(Leu126Pro) CACNB4 variant to be likely pathogenic. To investigate the functional consequences of the CACNB4 variant, we introduced the corresponding mutation L125P into rat β4b cDNA. Heterologously expressed wild-type β4b associated with GFP-CaV1.2 and accumulated in presynaptic boutons of cultured hippocampal neurons. In contrast, the β4b-L125P mutant failed to incorporate into calcium channel complexes and to cluster presynaptically. When co-expressed with CaV2.1 in tsA201 cells, β4b and β4b-L125P augmented the calcium current amplitudes, however, β4b-L125P failed to stably complex with α1 subunits. These results indicate that p.Leu125Pro disrupts the stable association of β4b with native calcium channel complexes, whereas membrane incorporation, modulation of current density and activation properties of heterologously expressed channels remained intact. Wildtype β4b was specifically targeted to the nuclei of quiescent excitatory cells. Importantly, the p.Leu125Pro mutation abolished nuclear targeting of β4b in cultured myotubes and hippocampal neurons. While binding of β4b to the known interaction partner PPP2R5D (B56δ) was not affected by the mutation, complex formation between β4b-L125P and the neuronal TRAF2 and NCK interacting kinase (TNIK) seemed to be disturbed. In summary, our data suggest that the homozygous CACNB4 p.(Leu126Pro) variant underlies the severe neurological phenotype in the two siblings, most likely by impairing both channel and non-channel functions of β4b., Competing Interests: The authors have declared that no competing interests exist.

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.)

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.

Reduced pancreatic β-cell function or mass is the critical problem in developing diabetes. Insulin release from β-cells depends on Ca 2+ influx through high voltage-gated Ca 2+ channels (HVCCs). Ca 2+ influx also regulates insulin synthesis and insulin granule priming and contributes to β-cell electrical activity. The HVCCs are multisubunit protein complexes composed of a pore-forming α 1 and auxiliary β and α 2 δ subunits. α 2 δ is a key regulator of membrane incorporation and function of HVCCs. Here we show that genetic deletion of α 2 δ-1, the dominant α 2 δ subunit in pancreatic islets, results in glucose intolerance and diabetes without affecting insulin sensitivity. Lack of the α 2 δ-1 subunit reduces the Ca 2+ currents through all HVCC isoforms expressed in β-cells equally in male and female mice. The reduced Ca 2+ influx alters the kinetics and amplitude of the global Ca 2+ response to glucose in pancreatic islets and significantly reduces insulin release in both sexes. The progression of diabetes in males is aggravated by a selective loss of β-cell mass, while a stronger basal insulin release alleviates the diabetes symptoms in most α 2 δ-1 -/- female mice. Together, these findings demonstrate that the loss of the Ca 2+ channel α 2 δ-1 subunit function increases the susceptibility for developing diabetes in a sex-dependent manner., (© 2017 by the American Diabetes Association.)

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.)

L-type voltage-gated Ca(2+) channels (LTCCs) regulate many physiological functions like muscle contraction, hormone secretion, gene expression, and neuronal excitability. Their activity is strictly controlled by various molecular mechanisms. The pore-forming α1-subunit comprises four repeated domains (I-IV), each connected via an intracellular linker. Here we identified a polybasic plasma membrane binding motif, consisting of four arginines, within the I-II linker of all LTCCs. The primary structure of this motif is similar to polybasic clusters known to interact with polyphosphoinositides identified in other ion channels. We used de novo molecular modeling to predict the conformation of this polybasic motif, immunofluorescence microscopy and live cell imaging to investigate the interaction with the plasma membrane, and electrophysiology to study its role for Cav1.2 channel function. According to our models, this polybasic motif of the I-II linker forms a straight α-helix, with the positive charges facing the lipid phosphates of the inner leaflet of the plasma membrane. Membrane binding of the I-II linker could be reversed after phospholipase C activation, causing polyphosphoinositide breakdown, and was accelerated by elevated intracellular Ca(2+) levels. This indicates the involvement of negatively charged phospholipids in the plasma membrane targeting of the linker. Neutralization of four arginine residues eliminated plasma membrane binding. Patch clamp recordings revealed facilitated opening of Cav1.2 channels containing these mutations, weaker inhibition by phospholipase C activation, and reduced expression of channels (as quantified by ON-gating charge) at the plasma membrane. Our data provide new evidence for a membrane binding motif within the I-II linker of LTCC α1-subunits essential for stabilizing normal Ca(2+) channel function., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

In nerve cells the ubiquitous second messenger calcium regulates a variety of vitally important functions including neurotransmitter release, gene regulation, and neuronal plasticity. The entry of calcium into cells is tightly regulated by voltage-gated calcium channels, which consist of a heteromultimeric complex of a pore forming α₁, and the auxiliary β and α₂δ subunits. Four genes (Cacna2d1-4) encode for the extracellular membrane-attached α₂δ subunits (α₂δ-1 to α₂δ-4), out of which three isoforms (α₂δ-1 to -3) are strongly expressed in the central nervous system. Over the years a wealth of studies has demonstrated the classical role of α₂δ subunits in channel trafficking and calcium current modulation. Recent studies in specialized neuronal cell systems propose roles of α₂δ subunits beyond the classical view and implicate α₂δ subunits as important regulators of synapse formation. These findings are supported by the identification of novel human disease mutations associated with α₂δ subunits and by the fact that α₂δ subunits are the target of the anti-epileptic and anti-allodynic drugs gabapentin and pregabalin. Here we review the recently emerging evidence for specific as well as redundant neuronal roles of α₂δ subunits and discuss the mechanisms for establishing and maintaining specificity.

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.

Voltage-gated calcium channels regulate gene expression by controlling calcium entry through the plasma membrane and by direct interactions of channel fragments and auxiliary β subunits with promoters and the epigenetic machinery in the nucleus. Mutations of the calcium channel β(4) subunit gene (CACNB4) cause juvenile myoclonic epilepsy in humans and ataxia and epileptic seizures in mice. Recently a model has been proposed according to which failed nuclear translocation of the truncated β(4) subunit R482X mutation resulted in altered transcriptional regulation and consequently in neurological disease. Here we examined the nuclear targeting properties of the truncated β(4b(1–481)) subunit in tsA-201 cells, skeletal myotubes, and in hippocampal neurons. Contrary to expectation, nuclear targeting of β(4b(1–481)) was not reduced compared with full-length β(4b) in any one of the three cell systems. These findings oppose an essential role of the β(4) distal C-terminus in nuclear targeting and challenge the idea that the nuclear function of calcium channel β(4) subunits is critically involved in the etiology of epilepsy and ataxia in patients and mouse models with mutations in the CACNB4 gene.

Mutations in the CACNA1F gene encoding the Cav1.4 Ca (2+) channel are associated with X-linked congenital stationary night blindness type 2 (CSNB2). Despite the increasing knowledge about the functional behavior of mutated channels in heterologous systems, the pathophysiological mechanisms that result in vision impairment remain to be elucidated. This work provides a thorough functional characterization of the novel IT mouse line that harbors the gain-of-function mutation I745T reported in a New Zealand CSNB2 family. (1) Electroretinographic recordings in IT mice permitted a direct comparison with human data. Our data supported the hypothesis that a hyperpolarizing shift in the voltage-dependence of channel activation-as seen in the IT gain-of-function mutant (2)-may reduce the dynamic range of photoreceptor activity. Morphologically, the retinal outer nuclear layer in adult IT mutants was reduced in size and cone outer segments appeared shorter. The organization of the outer plexiform layer was disrupted, and synaptic structures of photoreceptors had a variable, partly immature, appearance. The associated visual deficiency was substantiated in behavioral paradigms. The IT mouse line serves as a specific model for the functional phenotype of human CSNB2 patients with gain-of-function mutations and may help to further understand the dysfunction in CSNB.

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.

Auxiliary beta subunits are critical determinants of membrane expression and gating properties of voltage-gated calcium channels. Mutations in the beta(4) subunit gene cause ataxia and epilepsy. However, the specific function of beta(4) in neurons and its causal relation to neurological diseases are unknown. Here we report the localization of the beta(4) subunit in the nuclei of cerebellar granule and Purkinje cells. beta(4b) was the only beta isoform showing nuclear targeting when expressed in neurons and skeletal myotubes. Its specific nuclear targeting property was mapped to an N-terminal double-arginine motif, which was necessary and sufficient for targeting beta subunits into the nucleus. Spontaneous electrical activity and calcium influx negatively regulated beta(4b) nuclear localization by a CRM-1-dependent nuclear export mechanism. The activity-dependent shuttling of beta(4b) into and out of the nucleus indicates a specific role of this beta subunit in neurons, in communicating the activity of calcium channels to the nucleus.

The Ca(2+) channel alpha(1S) subunit (Ca(V)1.1) is the voltage sensor in skeletal muscle excitation-contraction (EC) coupling. Upon membrane depolarization, this sensor rapidly triggers Ca(2+) release from internal stores and conducts a slowly activating Ca(2+) current. However, this Ca(2+) current is not essential for skeletal muscle EC coupling. Here, we identified a Ca(V)1.1 splice variant with greatly distinct current properties. The variant of the CACNA1S gene lacking exon 29 was expressed at low levels in differentiated human and mouse muscle, and up to 80% in myotubes. To test its biophysical properties, we deleted exon 29 in a green fluorescent protein (GFP)-tagged alpha(1S) subunit and expressed it in dysgenic (alpha(1S)-null) myotubes. GFP-alpha(1S)Delta 29 was correctly targeted into triads and supported skeletal muscle EC coupling. However, the Ca(2+) currents through GFP-alpha(1S)Delta 29 showed a 30-mV left-shifted voltage dependence of activation and a substantially increased open probability, giving rise to an eightfold increased current density. This robust Ca(2+) influx contributed substantially to the depolarization-induced Ca(2+) transient that triggers contraction. Moreover, deletion of exon 29 accelerated current kinetics independent of the auxiliary alpha(2)delta-1 subunit. Thus, characterizing the Ca(V)1.1 Delta 29 splice variant revealed the structural bases underlying the specific gating properties of skeletal muscle Ca(2+) channels, and it suggests the existence of a distinct mode of EC coupling in developing muscle.

Voltage-gated Ca(2+) channels are multi-subunit complexes involved in many key functions of excitable cells. A multitude of studies in heterologous cells demonstrated that coexpression of the pore-forming alpha(1) subunits with auxiliary alpha(2)delta and beta subunits promotes membrane expression and modulates the biophysical channel properties. New null-mutant animal models and shRNA based knockdown experiments in skeletal muscle cells for the first time demonstrated the physiological roles and possible pathological effects of the alpha(2)delta-1 and beta(1a) subunits in a differentiated excitable cell. The alpha(2)delta-1 subunit is the determinant of the typical current properties of skeletal and cardiac muscle Ca(2+) channels. The beta(1a) subunit links the skeletal muscle Ca(2+) channel to the Ca(2+) release channel in the sarcoplasmic reticulum. Whether these specific functions in muscle indicate similar roles of alpha(2)delta and beta subunits as functional modulator and structural organizer, respectively, in neurons is being discussed.

L-type Ca(2+) currents determine the shape of cardiac action potentials (AP) and the magnitude of the myoplasmic Ca(2+) signal, which regulates the contraction force. The auxiliary Ca(2+) channel subunits alpha(2)delta-1 and beta(2) are important regulators of membrane expression and current properties of the cardiac Ca(2+) channel (Ca(V)1.2). However, their role in cardiac excitation-contraction coupling is still elusive. Here we addressed this question by combining siRNA knockdown of the alpha(2)delta-1 subunit in a muscle expression system with simulation of APs and Ca(2+) transients by using a quantitative computer model of ventricular myocytes. Reconstitution of dysgenic muscle cells with Ca(V)1.2 (GFP-alpha(1C)) recapitulates key properties of cardiac excitation-contraction coupling. Concomitant depletion of the alpha(2)delta-1 subunit did not perturb membrane expression or targeting of the pore-forming GFP-alpha(1C) subunit into junctions between the outer membrane and the sarcoplasmic reticulum. However, alpha(2)delta-1 depletion shifted the voltage dependence of Ca(2+) current activation by 9 mV to more positive potentials, and it slowed down activation and inactivation kinetics approximately 2-fold. Computer modeling revealed that the altered voltage dependence and current kinetics exert opposing effects on the function of ventricular myocytes that in total cause a 60% prolongation of the AP and a 2-fold increase of the myoplasmic Ca(2+) concentration during each contraction. Thus, the Ca(2+) channel alpha(2)delta-1 subunit is not essential for normal Ca(2+) channel targeting in muscle but is a key determinant of normal excitation and contraction of cardiac muscle cells, and a reduction of alpha(2)delta-1 function is predicted to severely perturb normal heart function.

Auxiliary channel subunits regulate membrane expression and modulate current properties of voltage-activated Ca(2+) channels and thus are involved in numerous important cell functions, including muscle contraction. Whereas the importance of the alpha(1S), beta(1a), and gamma Ca(2+) channel subunits in skeletal muscle has been determined by using null-mutant mice, the role of the alpha(2)delta-1 subunit in skeletal muscle is still elusive. We addressed this question by small interfering RNA silencing of alpha(2)delta-1 in reconstituted dysgenic (alpha(1S)-null) myotubes and in BC3H1 skeletal muscle cells. Immunofluorescence labeling of the alpha(1S) and alpha(2)delta-1 subunits and whole cell patch clamp recordings demonstrated that triad targeting and functional expression of the skeletal muscle Ca(2+) channel were not compromised by the depletion of the alpha(2)delta-1 subunit. The amplitudes and voltage dependences of L-type Ca(2+) currents and of the depolarization-induced Ca(2+) transients were identical in control and in alpha(2)delta-1-depleted muscle cells. However, alpha(2)delta-1 depletion significantly accelerated the current kinetics, most likely by the conversion of slowly activating into fast activating Ca(2+) channels. Reverse transcription-PCR analysis indicated that alpha(2)delta-1 is the exclusive isoform expressed in differentiated BC3H1 cells and that depletion of alpha(2)delta-1 was not compensated by the up-regulation of any other alpha(2)delta isoform. Thus, in skeletal muscle the Ca(2+) channel alpha(2)delta-1 subunit functions as a major determinant of the characteristic slow L-type Ca(2+) current kinetics. However, this subunit is not essential for targeting of Ca(2+) channels or for their primary physiological role in activating skeletal muscle excitation-contraction coupling.

L-type voltage-gated CaV1.2 calcium channels (CaV1.2) are key regulators of neuronal excitability, synaptic plasticity, and excitation-transcription coupling. Surface-exposed CaV1.2 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 CaV1.2 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 CaV1.2 are completely unexplored. Here, we examined the dynamic states of CaV1.2 depending on phosphorylation on Ser-1700 and Ser-1928 at the channel C terminus. Phosphorylation at these sites is strongly involved in CaV1.2-mediated nuclear factor of activated T cells (NFAT) signaling, long-term potentiation, and responsiveness to adrenergic stimulation. We engineered CaV1.2 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 CaV1.2 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 CaV1.2 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 CaV1.2 at signaling complexes.

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]

In nerve cells the genes encoding for α2δ subunits of voltage-gated calcium channels have been linked to synaptic functions and neurological disease. Here we show that α2δ subunits are essential for the formation and organization of glutamatergic synapses. Using a cellular α2δ subunit triple-knockout/knockdown model, we demonstrate a failure in presynaptic differentiation evidenced by defective presynaptic calcium channel clustering and calcium influx, smaller presynaptic active zones, and a strongly reduced accumulation of presynaptic vesicle-associated proteins (synapsin and vGLUT). The presynaptic defect is associated with the downscaling of postsynaptic AMPA receptors and the postsynaptic density. The role of α2δ isoforms as synaptic organizers is highly redundant, as each individual α2δ isoform can rescue presynaptic calcium channel trafficking and expression of synaptic proteins. Moreover, α2&#948-2 and α2&#948-3 with mutated metal iondependent adhesion sites can fully rescue presynaptic synapsin expression but only partially calcium channel trafficking, suggesting that the regulatory role of α2δ subunits is independent from its role as a calcium channel subunit. Our findings influence the current view on excitatory synapse formation. First, our study suggests that postsynaptic differentiation is secondary to presynaptic differentiation. Second, the dependence of presynaptic differentiation on α2δ implicates α2δ subunits as potential nucleation points for the organization of synapses. Finally, our results suggest that α2δ subunits act as transsynaptic organizers of glutamatergic synapses, thereby aligning the synaptic active zone with the postsynaptic density. [ABSTRACT FROM AUTHOR]

α2δ proteins are membrane-anchored extracellular glycoproteins which are abundantly expressed in the brain and the peripheral nervous system. They serve as regulatory subunits of voltage-gated calcium channels and, particularly in nerve cells, regulate presynaptic and postsynaptic functions independently from their role as channel subunits. α2δ proteins are the targets of the widely prescribed anti-epileptic and anti-allodynic drugs gabapentin and pregabalin, particularly for the treatment of neuropathic pain conditions. Recently, the human genes (CACNA2D1–4) encoding for the four known α2δ proteins (isoforms α2δ-1 to α2δ-4) have been linked to a large variety of neurological and neuropsychiatric disorders including epilepsy, autism spectrum disorders, bipolar disorders, schizophrenia, and depressive disorders. Here, we provide an overview of the hitherto identified disease associations of all known α2δ genes, hypothesize on the pathophysiological mechanisms considering their known physiological roles, and discuss the most immanent future research questions. Elucidating their specific physiological and pathophysiological mechanisms may open the way for developing entirely novel therapeutic paradigms for treating brain disorders. [ABSTRACT FROM AUTHOR]

Presynaptic α2δ 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 α2δ isoforms and their role in GABAergic synapses. Using primary neuronal cultures of embryonic mice of both sexes, we here report that presynaptic overexpression of α2δ-2 in GABAergic synapses strongly increases clustering of postsynaptic GABAARs. Strikingly, presynaptic α2δ-2 exerts the same effect in glutamatergic synapses, leading to a mismatched localization of GABAARs. This mismatching is caused by an aberrant wiring of glutamatergic presynaptic boutons with GABAergic postsynaptic positions. The trans-synaptic effect of α2δ is independent of the prototypical cell-adhesion molecules a-neurexins (α-Nrxns); however, α-Nrxns together with α2δ can modulate postsynaptic GABAAR abundance. Finally, exclusion of the alternatively spliced exon 23 of α2δ is essential for the trans-synaptic mechanism. The novel function of α2δ identified here may explain how abnormal α2δ subunit expression can cause excitatory-inhibitory imbalance often associated with neuropsychiatric disorders. [ABSTRACT FROM AUTHOR]

Reduced pancreatic β-cell function or mass is the critical problem in developing diabetes. Insulin release from β-cells depends on Ca2+ influx through high voltage-gated Ca2+ channels (HVCCs). Ca2+ influx also regulates insulin synthesis and insulin granule priming and contributes to β-cell electrical activity. The HVCCs are multisubunit protein complexes composed of a pore-forming α1 and auxiliary β and α2δ subunits. α2δ is a key regulator of membrane incorporation and function of HVCCs. Here we show that genetic deletion of α2δ-1, the dominant α2δ subunit in pancreatic islets, results in glucose intolerance and diabetes without affecting insulin sensitivity. Lack of the α2δ-1 subunit reduces the Ca2+ currents through all HVCC isoforms expressed in β-cells equally in male and female mice. The reduced Ca2+ influx alters the kinetics and amplitude of the global Ca2+ response to glucose in pancreatic islets and significantly reduces insulin release in both sexes. The progression of diabetes in males is aggravated by a selective loss of β-cell mass, while a stronger basal insulin release alleviates the diabetes symptoms in most α2δ-1-/- female mice. Together, these findings demonstrate that the loss of the Ca2+ channel α2δ-1 subunit function increases the susceptibility for developing diabetes in a sex-dependent manner. [ABSTRACT FROM AUTHOR]

The auxiliary subunit α2δ2 modulates the abundance and function of voltage-gated calcium channels. Here we show that α2 δ2 mRNA is expressed in neonatal and mature hair cells. A functional α2 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 Ca2+ and Ba2+ 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 Cav1.3 channels normally coassemble with α2 δ2 at IHC presynapses. The reduction of depolarization-evoked exocytosis in du/du IHCs reflected their reduced Ca2+ currents. Ca2+- and voltage-dependent K+ (BK) currents and the expression of the pore-forming BKα protein were normal. Cav1.3 and Cavβ2 protein expression was unchanged in du/du IHCs, forming clusters at presynaptic ribbons. However, the close apposition of presynaptic Cav1.3 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 Cav1.3 channels, the largely extracellularly localized α2 δ2 subunit moreover plays a so far unknown role in mediating trans-synaptic alignment of presynaptic Ca2+ channels and postsynaptic AMPA receptors. [ABSTRACT FROM AUTHOR]

Skeletal muscle excitation-contraction (EC) coupling is independent of calcium influx. In fact, alternative splicing of the voltage-gated calcium channel CaV1.1 actively suppresses calcium currents in mature muscle. Whether this is necessary for normal development and function of muscle is not known. However, splicing defects that cause aberrant expression of the calcium-conducting developmental CaV1.1e splice variant correlate with muscle weakness in myotonic dystrophy. Here, we deleted CaV1.1 (Cacna1s) exon 29 in mice. These mice displayed normal overall motor performance, although grip force and voluntary running were reduced. Continued expression of the developmental CaV1.1e splice variant in adult mice caused increased calcium influx during EC coupling, altered calcium homeostasis, and spontaneous calcium sparklets in isolated muscle fibers. Contractile force was reduced and endurance enhanced. Key regulators of fiber type specification were dysregulated and the fiber type composition was shifted toward slower fibers. However, oxidative enzyme activity and mitochondrial content declined. These findings indicate that limiting calcium influx during skeletal muscle EC coupling is important for the secondary function of the calcium signal in the activity-dependent regulation of fiber type composition and to prevent muscle disease. [ABSTRACT FROM AUTHOR]

Background: It is well established that reactive astrocytes express L-type calcium channels (LTCC), but their functional role is completely unknown. We have recently shown that reactive astrocytes highly express the CaV1.2 α1-subunit around β-amyloid (Aβ) plaques in an Alzheimer mouse model. The aim of the present study was to explore whether Aβ peptides may regulate the mRNA expression of all LTCC subunits in primary mouse astrocytes in culture. Methods: Confluent primary astrocytes were incubated with 10 µg/ml of human or murine Aβ or the toxic fragment Aβ25-35 for 3 days or for 3 weeks. The LTCC subunits were determined by quantitative RT-PCR. Results: Our data show that murine Aβ42 slightly but significantly increased CaV1.2 and CaV1.3 expression when incubated for 3 days. This acute treatment with murine Aβ enhanced β2 and β3 mRNA levels but decreased α2δ-2 mRNA expression. When astrocytes were incubated for 3 weeks, the levels of CaV1.2 α1 were significantly decreased by the murine Aβ and the toxic fragment. As a control, the protein kinase C-ε activator DCP-LA displayed a decrease in CaV2.1 expression. Conclusion: In conclusion, our data show that Aβ can differentially regulate LTCC expression in primary mouse astrocytes depending on incubation time. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy (DMD), induced by mutations in the gene encoding for the cytoskeletal protein dystrophin, is an inherited disease characterized by progressive muscle weakness. Besides the relatively well characterized skeletal muscle degenerative processes, DMD is also associated with cardiac complications. These include cardiomyopathy development and cardiac arrhythmias. The current understanding of the pathomechanisms in the heart is very limited, but recent research indicates that dysfunctional ion channels in dystrophic cardiomyocytes play a role. The aim of the present study was to characterize abnormalities in L-type calcium channel function in adult dystrophic ventricular cardiomyocytes. By using the whole cell patchclamp technique, the properties of currents through calcium channels in ventricular cardiomyocytes isolated from the hearts of normal and dystrophic adult mice were compared. Besides the commonly used dystrophin-deficient mdx mouse model for human DMD, we also used mdx-utr mice, which are both dystrophin- and utrophin-deficient. We found that calcium channel currents were significantly increased, and channel inactivation was reduced in dystrophic cardiomyocytes. Both effects enhance the calcium influx during an action potential (AP). Whereas the AP in dystrophic mouse cardiomyocytes was nearly normal, implementation of the enhanced dystrophic calcium conductance in a computer model of a human ventricular cardiomyocyte considerably prolonged the AP. Finally, the described dystrophic calcium channel abnormalities entailed alterations in the electrocardiograms of dystrophic mice. We conclude that gain of function in cardiac L-type calcium channels may disturb the electrophysiology of the dystrophic heart and thereby cause arrhythmias. [ABSTRACT FROM AUTHOR]

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 variableNterminus (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 (β4bβ4a>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. [ABSTRACT FROM AUTHOR]

Increased activity of L-type Ca2+ channels has been implicated in the pathogenesis of dementia and Alzheimer's disease (AD). Previously we detected CaV1.2 α1-subunit-positive expression in reactive astrocytes surrounding the plaques of 12 month-old transgenic mice overexpressing hAβPP751 with the London (V717I) and Swedish (K670M/N671L) mutations. Here we examined whether increased CaV1.2 α1-subunit expression precedes plaque formation or is specifically associated with the increased amyloid-β (Aβ) load in the plaques. Quantitative RT-PCR expression profiling of all high voltage-gated Ca2+ channel subunits (α1, β, and α2δ) revealed no difference in the hippocampi of 2, 4, and 11 month-old wild type (wt) and transgenic (tg) mice. Immunohistochemistry demonstrated that expression of CaV1.2 α1-subunit, but not of the auxiliary β4 Ca2+ channel subunit, specifically associated with Aβ-positive plaques in brains of 11 month tg mice. No difference in CaV1.2 α1-subunit labeling was found in 2 and 4 month-old wt and tg mice prior to plaque formation. The CaV1.2 α1-subunit-positive cells in 11 month-old tg mice also labeled with GFAP, but not with the microglia marker Iba1. In contrast, GFAP-positive cells induced by injection of quinolinic acid did not reveal any CaV1.2 α1-subunit immunoreactivity. Together these results indicate that the expression of CaV1.2 α1-subunits in reactive astrocytes in the tg AD mouse model is related to the increased amyloid-β load in the plaques rather than caused by effects on gene regulation or mechanisms preceding the manifestation of AD as seen by plaque formation. [ABSTRACT FROM AUTHOR]

The article presents a study examining the turnover and surface traffic of Cav1.2 calcium channels in the dendrites of mouse and rat hippocampal neurons through fluorescence recovery after photobleaching (FRAP) technique. The study found a major immobile and a minor mobile population of membrane-expressed Cav1.2 calcium channels in the dendrities. The study also recommends a model for the existence of the channels inside and outside of clusters in a dynamic equilibrium.

In neurons L-type calcium currents contribute to synaptic plasticity and to activity-dependent gene regulation. The subcellular localization of CaV1.2 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 CaV1.2 in the postsynaptic compartment of glutamatergic neurons. Double-immunofluorescence labeling of hippocampal neurons transfected with external HA epitope-tagged CaV1.2 demonstrated that clusters of membrane-incorporated CaV1.2-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 CaV1.2-HA. Unexpectedly, the distribution pattern, the density, and the fluorescence intensity of clusters were similar for wild-type and mutant CaV1.2-HA, indicating that interactions with AKAP and PDZ proteins are not essential for the correct targeting of CaV1.2. In agreement, brief treatment with NMDA (a chemical LTD paradigm) caused the degradation of PSD-95 and the redistribution of AKAP79/150 and α-actinin from dendritic spines into the shaft, without a concurrent loss or redistribution of CaV1.2-HA clusters. Thus, in the postsynaptic compartment of hippocampal neurons CaV1.2 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 CaV1.2 is independent of its interactions with AKAP79/150 and PDZ proteins. [ABSTRACT FROM AUTHOR]

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, Cav1.3-/- and Cav1.2DHP-/- [containing a mutation in the Cav1.2 α1 subunit that eliminates dihydropyridine (DHP) sensitivity] mice. Quantitative reverse transcription-PCR revealed that Cav1.2 accounts for 89% and Cav1.3 for 11% of the LTCC transcripts in wild-type cerebellar granule cells, whereas Cav1.1 and Cav1.4 are expressed at insignificant levels. Anomalous LTCCs were observed in neurons of Cav1.3-/- 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 Cav1.2DHP-/- neurons than in Cav1.3-/- neurons. Reopenings in Cav1.2DHP-/- 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 Cav1.2α1 subunits are the pore-forming subunits of anomalous LTCCs in mouse cerebellar granule cells. Given the evidence that Cav1.2 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 Cav1.2 channels are specifically involved in LTCC-dependent and gene transcription-dependent LTP. [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]

L-type voltage-gated CaV1.2 calcium channels (CaV1.2) are key regulators of neuronal excitability, synaptic plasticity, and excitation-transcription coupling. Surface-exposed CaV1.2 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 CaV1.2 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 CaV1.2 are completely unexplored. Here, we examined the dynamic states of CaV1.2 depending on phosphorylation on Ser- 1700 and Ser-1928 at the channel C terminus. Phosphorylation at these sites is strongly involved in CaV1.2-mediated nuclear factor of activated T cells (NFAT) signaling, long-term potentiation, and responsiveness to adrenergic stimulation. We engineered CaV1.2 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 CaV1.2 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 CaV1.2 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 CaV1.2 at signaling complexes. [ABSTRACT FROM AUTHOR]

L-type voltage-gated Ca2+ channels (LTCCs) regulate many physiological functions like muscle contraction, hormone secretion, gene expression, and neuronal excitability. Their activity is strictly controlled by various molecular mechanisms. The poreforming α1-subunit comprises four repeated domains (I-IV), each connected via an intracellular linker. Here we identified a polybasic plasma membrane binding motif, consisting of four arginines, within the I-II linker of all LTCCs. The primary structure of this motif is similar to polybasic clusters known to interact with polyphosphoinositides identified in other ion channels. We used de novo molecular modeling to predict the conformation of this polybasic motif, immunofluorescence microscopy and live cell imaging to investigate the interaction with the plasma membrane, and electrophysiology to study its role for Cav1.2 channel function. According to our models, this polybasic motif of the I-II linker forms a straight α-helix, with the positive charges facing the lipid phosphates of the inner leaflet of the plasma membrane. Membrane binding of the I-II linker could be reversed after phospholipase C activation, causing polyphosphoinositide breakdown, and was accelerated by elevated intracellular Ca2+ levels. This indicates the involvement of negatively charged phospholipids in the plasma membrane targeting of the linker. Neutralization of four arginine residues eliminated plasma membrane binding. Patchclamp recordings revealed facilitated opening of Cav1.2 channels containing these mutations, weaker inhibition by phospholipase Cactivation, and reduced expression of channels (as quantified by ON-gating charge) at the plasma membrane. Our data provide new evidence for a membrane binding motif within the I-II linker of LTCC α1-subunits essential for stabilizing normal Ca2+ channel function. [ABSTRACT FROM AUTHOR]

Abstract: Neurotransmitter release and spontaneous action potentials during cochlear inner hair cell (IHC) development depend on the activity of Cav1.3 voltage-gated L-type Ca2+ channels. Their voltage- and Ca2+-dependent inactivation kinetics are slower than in other tissues but the underlying molecular mechanisms are not yet understood. We found that Rab3-interacting molecule-2α (RIM2α) mRNA is expressed in immature cochlear IHCs and the protein co-localizes with Cav1.3 in the same presynaptic compartment of IHCs. Expression of RIM proteins in tsA-201 cells revealed binding to the β-subunit of the channel complex and RIM-induced slowing of both Ca2+- and voltage-dependent inactivation of Cav1.3 channels. By inhibiting inactivation, RIM induced a non-inactivating current component typical for IHC Cav1.3 currents which should allow these channels to carry a substantial window current during prolonged depolarizations. These data suggest that RIM2 contributes to the stabilization of Cav1.3 gating kinetics in immature IHCs. [Copyright &y& Elsevier]