Search

The syndromic autism spectrum disorder (ASD) Timothy syndrome (TS) is caused by a point mutation in the alternatively spliced exon 8A of the calcium channel Ca v 1.2. Using mouse brain and human induced pluripotent stem cells (iPSCs), we provide evidence that the TS mutation prevents a normal developmental switch in Ca v 1.2 exon utilization, resulting in persistent expression of gain-of-function mutant channels during neuronal differentiation. In iPSC models, the TS mutation reduces the abundance of SATB2-expressing cortical projection neurons, leading to excess CTIP2+ neurons. We show that expression of TS-Ca v 1.2 channels in the embryonic mouse cortex recapitulates these differentiation defects in a calcium-dependent manner and that in utero Ca v 1.2 gain-and-loss of function reciprocally regulates the abundance of these neuronal populations. Our findings support the idea that disruption of developmentally regulated calcium channel splicing patterns instructively alters differentiation in the developing cortex, providing important in vivo insights into the pathophysiology of a syndromic ASD., Competing Interests: GP, CH, AA, RP, AR, SP, TP No competing interests declared, TP is currently an employee of Neucyte, Inc, RD is currently an employee of Novartis Institutes for Biomedical Research, (© 2019, Panagiotakos et al.)

Store-operated calcium entry (SOCE) regulates a wide variety of essential cellular functions. SOCE is mediated by STIM1 and STIM2, which sense depletion of ER Ca(2+) stores and activate Orai channels in the plasma membrane. Although the amplitude and dynamics of SOCE are considered important determinants of Ca(2+)-dependent responses, the underlying modulatory mechanisms are unclear. In this paper, we identify STIM2β, a highly conserved alternatively spliced isoform of STIM2, which, in contrast to all known STIM isoforms, is a potent inhibitor of SOCE. Although STIM2β does not by itself strongly bind Orai1, it is recruited to Orai1 channels by forming heterodimers with other STIM isoforms. Analysis of STIM2β mutants and Orai1-STIM2β chimeras suggested that it actively inhibits SOCE through a sequence-specific allosteric interaction with Orai1. Our results reveal a previously unrecognized functional flexibility in the STIM protein family by which alternative splicing creates negative and positive regulators of SOCE to shape the amplitude and dynamics of Ca(2+) signals., (© 2015 Rana et al.)

The human α1A voltage-dependent calcium channel (Cav2.1) is a pore-forming essential subunit embedded in the plasma membrane. Its cytoplasmic carboxyl(C)-tail contains a small poly-glutamine (Q) tract, whose length is normally 4∼19 Q, but when expanded up to 20∼33Q, the tract causes an autosomal-dominant neurodegenerative disorder, spinocerebellar ataxia type 6 (SCA6). A recent study has shown that a 75-kDa C-terminal fragment (CTF) containing the polyQ tract remains soluble in normal brains, but becomes insoluble mainly in the cytoplasm with additional localization to the nuclei of human SCA6 Purkinje cells. However, the mechanism by which the CTF aggregation leads to neurodegeneration is completely elusive, particularly whether the CTF exerts more toxicity in the nucleus or in the cytoplasm. We tagged recombinant (r)CTF with either nuclear-localization or nuclear-export signal, created doxycyclin-inducible rat pheochromocytoma (PC12) cell lines, and found that the CTF is more toxic in the cytoplasm than in the nucleus, the observations being more obvious with Q28 (disease range) than with Q13 (normal-length). Surprisingly, the CTF aggregates co-localized both with cAMP response element-binding protein (CREB) and phosphorylated-CREB (p-CREB) in the cytoplasm, and Western blot analysis showed that the quantity of CREB and p-CREB were both decreased in the nucleus when the rCTF formed aggregates in the cytoplasm. In human brains, polyQ aggregates also co-localized with CREB in the cytoplasm of SCA6 Purkinje cells, but not in other conditions. Collectively, the cytoplasmic Cav2.1-CTF aggregates are sufficient to cause cell death, and one of the pathogenic mechanisms may be abnormal CREB trafficking in the cytoplasm and reduced CREB and p-CREB levels in the nuclei.

Calcium current through voltage-gated calcium channels (VGCC) controls gene expression. Here, we describe a novel signalling pathway in which the VGCC Cacnb4 subunit directly couples neuronal excitability to transcription. Electrical activity induces Cacnb4 association to Ppp2r5d, a regulatory subunit of PP2A phosphatase, followed by (i) nuclear translocation of Cacnb4/Ppp2r5d/PP2A, (ii) association with the tyrosine hydroxylase (TH) gene promoter through the nuclear transcription factor thyroid hormone receptor alpha (TRα), and (iii) histone binding through association of Cacnb4 with HP1γ concomitantly with Ser(10) histone H3 dephosphorylation by PP2A. This signalling cascade leads to TH gene repression by Cacnb4 and is controlled by the state of interaction between the SH3 and guanylate kinase (GK) modules of Cacnb4. The human R482X CACNB4 mutation, responsible for a form of juvenile myoclonic epilepsy, prevents association with Ppp2r5 and nuclear targeting of the complex by altering Cacnb4 conformation. These findings demonstrate that an intact VGCC subunit acts as a repressor recruiting platform to control neuronal gene expression.

Synapses are asymmetric cellular adhesions that are critical for nervous system development and function, but the mechanisms that induce their formation are not well understood. We have previously identified thrombospondin as an astrocyte-secreted protein that promotes central nervous system (CNS) synaptogenesis. Here, we identify the neuronal thrombospondin receptor involved in CNS synapse formation as alpha2delta-1, the receptor for the anti-epileptic and analgesic drug gabapentin. We show that the VWF-A domain of alpha2delta-1 interacts with the epidermal growth factor-like repeats common to all thrombospondins. alpha2delta-1 overexpression increases synaptogenesis in vitro and in vivo and is required postsynaptically for thrombospondin- and astrocyte-induced synapse formation in vitro. Gabapentin antagonizes thrombospondin binding to alpha2delta-1 and powerfully inhibits excitatory synapse formation in vitro and in vivo. These findings identify alpha2delta-1 as a receptor involved in excitatory synapse formation and suggest that gabapentin may function therapeutically by blocking new synapse formation.

Ca(2+)-dependent inactivation (CDI) is a key regulator and hallmark of the Ca(2+) release-activated Ca(2+) (CRAC) channel, a prototypic store-operated Ca(2+) channel. Although the roles of the endoplasmic reticulum Ca(2+) sensor STIM1 and the channel subunit Orai1 in CRAC channel activation are becoming well understood, the molecular basis of CDI remains unclear. Recently, we defined a minimal CRAC activation domain (CAD; residues 342-448) that binds directly to Orai1 to activate the channel. Surprisingly, CAD-induced CRAC currents lack fast inactivation, revealing a critical role for STIM1 in this gating process. Through truncations of full-length STIM1, we identified a short domain (residues 470-491) C-terminal to CAD that is required for CDI. This domain contains a cluster of 7 acidic amino acids between residues 475 and 483. Neutralization of aspartate or glutamate pairs in this region either reduced or enhanced CDI, whereas the combined neutralization of six acidic residues eliminated inactivation entirely. Based on bioinformatics predictions of a calmodulin (CaM) binding site on Orai1, we also investigated a role for CaM in CDI. We identified a membrane-proximal N-terminal domain of Orai1 (residues 68-91) that binds CaM in a Ca(2+)-dependent manner and mutations that eliminate CaM binding abrogate CDI. These studies identify novel structural elements of STIM1 and Orai1 that are required for CDI and support a model in which CaM acts in concert with STIM1 and the N terminus of Orai1 to evoke rapid CRAC channel inactivation.

Store-operated Ca(2+) channels activated by the depletion of Ca(2+) from the endoplasmic reticulum (ER) are a major Ca(2+) entry pathway in nonexcitable cells and are essential for T cell activation and adaptive immunity. After store depletion, the ER Ca(2+) sensor STIM1 and the CRAC channel protein Orai1 redistribute to ER-plasma membrane (PM) junctions, but the fundamental issue of how STIM1 activates the CRAC channel at these sites is unresolved. Here, we identify a minimal, highly conserved 107-aa CRAC activation domain (CAD) of STIM1 that binds directly to the N and C termini of Orai1 to open the CRAC channel. Purified CAD forms a tetramer that clusters CRAC channels, but analysis of STIM1 mutants reveals that channel clustering is not sufficient for channel activation. These studies establish a molecular mechanism for store-operated Ca(2+) entry in which the direct binding of STIM1 to Orai1 drives the accumulation and the activation of CRAC channels at ER-PM junctions.

Changes in the concentration of intracellular Ca2+ ([Ca2+]i) in response to various stimuli play a role in regulating numerous cellular processes, including the activation of gene expression. In neurons, the extraordinary diversity of the response to Ca2+ signaling depends on the location, intensity, and duration of the Ca2+ transient. Interestingly, Ca2+-dependent gene transcription appears to be sensitive both to increases in nuclear Ca2+, which occur after relatively intense stimuli, and to highly localized increases in Ca2+ near the sites of Ca2+ influx. Activation of intracellular signaling pathways by specific types of Ca2+ channels depends on localization of specific Ca2+ receptors close to the channel mouth. The dual regulation of signaling pathways by Ca2+ near channels and in the nucleus may permit neurons to precisely tailor transcriptional activation to specific types of electrical or chemical stimuli and at the same time ensure that only robust stimuli that generate nuclear Ca2+ elevations are converted into long-term changes in gene expression.

Monogenic neurodevelopmental disorders provide key insights into the pathogenesis of disease and help us understand how specific genes control the development of the human brain. Timothy syndrome is caused by a missense mutation in the L-type calcium channel Ca(v)1.2 that is associated with developmental delay and autism. We generated cortical neuronal precursor cells and neurons from induced pluripotent stem cells derived from individuals with Timothy syndrome. Cells from these individuals have defects in calcium (Ca(2+)) signaling and activity-dependent gene expression. They also show abnormalities in differentiation, including decreased expression of genes that are expressed in lower cortical layers and in callosal projection neurons. In addition, neurons derived from individuals with Timothy syndrome show abnormal expression of tyrosine hydroxylase and increased production of norepinephrine and dopamine. This phenotype can be reversed by treatment with roscovitine, a cyclin-dependent kinase inhibitor and atypical L-type-channel blocker. These findings provide strong evidence that Ca(v)1.2 regulates the differentiation of cortical neurons in humans and offer new insights into the causes of autism in individuals with Timothy syndrome.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2006.10.017 Byline: Natalia Gomez-Ospina (1), Fuminori Tsuruta (1), Odmara Barreto-Chang (1), Linda Hu (2), Ricardo Dolmetsch (1) Abstract: Voltage-gated calcium channels play a central role in regulating the electrical and biochemical properties of neurons and muscle cells. One of the ways in which calcium channels regulate long-lasting neuronal properties is by activating signaling pathways that control gene expression, but the mechanisms that link calcium channels to the nucleus are not well understood. We report that a C-terminal fragment of Ca.sub.V1.2, an L-type voltage-gated calcium channel (LTC), translocates to the nucleus and regulates transcription. We show that this calcium channel associated transcription regulator (CCAT) binds to a nuclear protein, associates with an endogenous promoter, and regulates the expression of a wide variety of endogenous genes important for neuronal signaling and excitability. The nuclear localization of CCAT is regulated both developmentally and by changes in intracellular calcium. These findings provide evidence that voltage-gated calcium channels can directly activate transcription and suggest a mechanism linking voltage-gated channels to the function and differentiation of excitable cells. Author Affiliation: (1) Department of Neurobiology, Stanford University School of Medicine, 299 Campus Drive, Stanford, CA 94305, USA (2) Division of Neurosciences, Children's Hospital and Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA Article History: Received 17 March 2006; Revised 25 July 2006; Accepted 16 October 2006 Article Note: (miscellaneous) Published: November 2, 2006

Background Common genetic variation and rare mutations in genes encoding calcium channel subunits have pleiotropic effects on risk for multiple neuropsychiatric disorders, including autism spectrum disorder (ASD) and schizophrenia. To gain further mechanistic insights by extending previous gene expression data, we constructed co-expression networks in Timothy syndrome (TS), a monogenic condition with high penetrance for ASD, caused by mutations in the L-type calcium channel, Cav1.2. Methods To identify patient-specific alterations in transcriptome organization, we conducted a genome-wide weighted co-expression network analysis (WGCNA) on neural progenitors and neurons from multiple lines of induced pluripotent stem cells (iPSC) derived from normal and TS (G406R in CACNA1C) individuals. We employed transcription factor binding site enrichment analysis to assess whether TS associated co-expression changes reflect calciumdependent co-regulation. Results We identified reproducible developmental and activity-dependent gene co-expression modules conserved in patient and control cell lines. By comparing cell lines from case and control subjects, we also identified co-expression modules reflecting distinct aspects of TS, including intellectual disability and ASD-related phenotypes. Moreover, by integrating co-expression with transcription factor binding analysis, we showed the TS-associated transcriptional changes were predicted to be co-regulated by calcium-dependent transcriptional regulators, including NFAT, MEF2, CREB, and FOXO, thus providing a mechanism by which altered Ca2+ signaling in TS patients leads to the observed molecular dysregulation. Conclusions We applied WGCNA to construct co-expression networks related to neural development and depolarization in iPSC-derived neural cells from TS and control individuals for the first time. These analyses illustrate how a systems biology approach based on gene networks can yield insights into the molecular mechanisms of neural development and function, and provide clues as to the functional impact of the downstream effects of Ca2+ signaling dysregulation on transcription. [ABSTRACT FROM AUTHOR]

The C-terminus of the voltage-gated calcium channel Cav1.2 encodes a transcription factor, the calcium channel associated transcriptional regulator (CCAT), that regulates neurite extension and inhibits Cav1.2 expression. The mechanisms by which CCAT is generated in neurons and myocytes are poorly understood. Here we show that CCAT is produced by activation of a cryptic promoter in exon 46 of CACNA1C, the gene that encodes CaV1.2. Expression of CCAT is independent of Cav1.2 expression in neuroblastoma cells, in mice, and in human neurons derived from induced pluripotent stem cells (iPSCs), providing strong evidence that CCAT is not generated by cleavage of CaV1.2. Analysis of the transcriptional start sites in CACNA1C and immune-blotting for channel proteins indicate that multiple proteins are generated from the 3′ end of the CACNA1C gene. This study provides new insights into the regulation of CACNA1C, and provides an example of how exonic promoters contribute to the complexity of mammalian genomes. [ABSTRACT FROM AUTHOR]

The human α1A voltage-dependent calcium channel (Cav2.1) is a pore-forming essential subunit embedded in the plasma membrane. Its cytoplasmic carboxyl(C)-tail contains a small poly-glutamine (Q) tract, whose length is normally 4∼19 Q, but when expanded up to 20∼33Q, the tract causes an autosomal-dominant neurodegenerative disorder, spinocerebellar ataxia type 6 (SCA6). A recent study has shown that a 75-kDa C-terminal fragment (CTF) containing the polyQ tract remains soluble in normal brains, but becomes insoluble mainly in the cytoplasm with additional localization to the nuclei of human SCA6 Purkinje cells. However, the mechanism by which the CTF aggregation leads to neurodegeneration is completely elusive, particularly whether the CTF exerts more toxicity in the nucleus or in the cytoplasm. We tagged recombinant (r)CTF with either nuclear-localization or nuclear-export signal, created doxycyclin-inducible rat pheochromocytoma (PC12) cell lines, and found that the CTF is more toxic in the cytoplasm than in the nucleus, the observations being more obvious with Q28 (disease range) than with Q13 (normal-length). Surprisingly, the CTF aggregates co-localized both with cAMP response element-binding protein (CREB) and phosphorylated-CREB (p-CREB) in the cytoplasm, and Western blot analysis showed that the quantity of CREB and p-CREB were both decreased in the nucleus when the rCTF formed aggregates in the cytoplasm. In human brains, polyQ aggregates also co-localized with CREB in the cytoplasm of SCA6 Purkinje cells, but not in other conditions. Collectively, the cytoplasmic Cav2.1-CTF aggregates are sufficient to cause cell death, and one of the pathogenic mechanisms may be abnormal CREB trafficking in the cytoplasm and reduced CREB and p-CREB levels in the nuclei. [ABSTRACT FROM AUTHOR]

Ca2+-dependent inactivation (CDI) is a key regulator and hallmark of the Ca2+ release-activated Ca2+ (CRAC) channel, a prototypic store-operated Ca2+ channel. Although the roles of the endoplasmic reticulum Ca2+ sensor STIM1 and the channel subunit Orail in CRAC channel activation are becoming well understood, the molecular basis of CDI remains unclear. Recently, we defined a minimal CRAC activation domain (CAD; residues 342-448) that binds directly to Orail to activate the channel. Surprisingly, CADinduced CRAC currents lack fast inactivation, revealing a critical role for STIM1 in this gating process. Through truncations of full-length STIM1, we identified a short domain (residues 470-491) C-terminal to CAD that is required for CDI. This domain contains a cluster of 7 acidic amino acids between residues 475 and 483. Neutralization of aspartate or glutamate pairs in this region either reduced or enhanced CDI, whereas the combined neutralization of six acidic residues eliminated inactivation entirely. Based on bioinformatics predictions of a calmodulin (CaM) binding site on Orail, we also investigated a role for CaM in CDI. We identified a membrane-proximal N-terminal domain of Orail (residues 68-91) that binds CaM in a Ca2+-dependent manner and mutations that eliminate CaM binding abrogate CDI. These studies identify novel structural elements of STIM1 and Orail that are required for CDI and support a model in which CaM acts in concert with STIM1 and the N terminus of Orail to evoke rapid CRAC channel inactivation. [ABSTRACT FROM AUTHOR]

Reports the development of a functional knock-in technique in investigations of the features of L-type voltage-activated channels. Creation of mutant channels for pharmacological blockers; Selective signalling of L-type calcium channels to the nucleus.

Examines the roles of calcium ion (Ca2+) influx and depletion of intracellular Ca2+ stores in the oscillation mechanism using single-cell Ca2+ imaging techniques. Dependence of Ca2+ entry across membranes by stimulation through the antigen receptor of T cells; Model for Ca2+ concentration oscillation in T cells.

Voltage- and store-operated calcium (Ca2+) channels are the major routes of Ca2+ entry in mammalian cells, but little is known about how cells coordinate the activity of these channels to generate coherent calcium signals. We found that STIM1 (stromal interaction molecule 1), the main activator of store-operated Ca2+ channels, directly suppresses depolarization-induced opening of the voltage-gated Ca2+ channel Cav1.2. STIAA1 binds to the C terminus of Cav1.2 through its Ca2+ release-activated Ca2+ activation domain, acutely inhibits gating, and causes long-term internalization of the channel from the membrane. This establishes a previously unknown function for STIM1 and provides a molecular mechanism to explain the reciprocal regulation of these two channels in cells. [ABSTRACT FROM AUTHOR]

The signals regulating stem cell activation during tissue regeneration remain poorly understood. We investigated the baldness associated with mutations in the voltage-gated calcium channel (VGCC) Cav1.2 underlying Timothy syndrome (TS). While hair follicle stem cells express Cav1.2, they lack detectable voltage-dependent calcium currents. Cav1.2TS acts in a dominant-negative manner to markedly delay anagen, while L-type channel blockers act through Cav1.2 to induce anagen and overcome the TS phenotype. Cav1.2 regulates production of the bulge-derived BMP inhibitor follistatin-like1 (Fstl1), derepressing stem cell quiescence. Our findings show how channels act in nonexcitable tissues to regulate stem cells and may lead to novel therapeutics for tissue regeneration. [ABSTRACT FROM AUTHOR]

Summary: Regulation of neuronal excitability and cardiac excitation-contraction coupling requires the proper localization of L-type Ca2+ channels. We show that the actin-binding protein α-actinin binds to the C-terminal surface targeting motif of α11.2, the central pore-forming CaV1.2 subunit, in order to foster its surface expression. Disruption of α-actinin function by dominant-negative or small hairpin RNA constructs reduces CaV1.2 surface localization in human embryonic kidney 293 and neuronal cultures and dendritic spine localization in neurons. We demonstrate that calmodulin displaces α-actinin from their shared binding site on α11.2 upon Ca2+ influx through L-type channels, but not through NMDAR, thereby triggering loss of CaV1.2 from spines. Coexpression of a Ca2+-binding-deficient calmodulin mutant does not affect basal CaV1.2 surface expression but inhibits its internalization upon Ca2+ influx. We conclude that α-actinin stabilizes CaV1.2 at the plasma membrane and that its displacement by Ca2+-calmodulin triggers Ca2+-induced endocytosis of CaV1.2, thus providing an important negative feedback mechanism for Ca2+ influx. [ABSTRACT FROM AUTHOR]

The identification of a gain-of-function mutation in CACNA1C as the cause of Timothy Syndrome (TS), a rare disorder characterized by cardiac arrhythmias and syndactyly, highlighted unexpected roles for the L-type voltage-gated Ca2+ channel Cav1.2 in nonexcitable cells. How abnormal Ca2+ influx through Cav1.2 underlies phenotypes such as the accompanying syndactyly or craniofacial abnormalities in the majority of affected individuals is not readily explained by established Cav1.2 roles. Here, we show that Cav1.2 is expressed in the first and second pharyngeal arches within the subset of cells that give rise to jaw primordia. Gain-of-function and loss-of-function studies in mouse, in concert with knockdown/rescue and pharmacological approaches in zebrafish, demonstrated that Ca2+ influx through Cav1.2 regulates jaw development. Cranial neural crest migration was unaffected by Cav1.2 knockdown, suggesting a role for Cav1.2 later in development. Focusing on the mandible, we observed that cellular hypertrophy and hyperplasia depended upon Ca2+ signals through Cav1.2, including those that activated the calcineurin signaling pathway. Together, these results provide new insights into the role of voltage-gated Ca2+ channels in nonexcitable cells during development. [ABSTRACT FROM AUTHOR]

Voltage-gated Ca[sup 2+] channels (VGCCs) play a key role in neuronal signaling but can also contribute to cellular dysfunction and death under pathological conditions such as stroke and neurodegenerative diseases. We report that activation of N-methyl-D-aspartic acid receptors causes internalization and degradation of Ca[sub v]1.2 channels, resulting in decreased Ca[sup 2+] entry and reduced toxicity. Ca[sub v]1.2 internalization and degradation requires binding to phosphatidylinositol 3-phosphate 5-kinase (PIKfyve), a lipid kinase which generates phosphatidylinositol (3,5)-bisphosphate (PtdIns(3,5)P[sub 2]) and regulates endosome and lysosome function. Sustained activation of glutamate receptors recruits PIKfyve to Ca[sub v]1.2 channels, increases cellular levels of PtdIns(3,5)P[sub 2], and promotes targeting of Ca[sub v]1.2 to lysosomes. Knockdown of PIKfyve prevents Ca[sub v]1.2 degradation and increases neuronal susceptibility to excitotoxicity. These experiments identify a novel mechanism by which neurons are protected from excitotoxicity and provide a possible explanation for neuronal death in diseases caused by mutations that affect PtdIns(3,5)P[sub 2] regulation. [ABSTRACT FROM AUTHOR]