Academic Appointments

Honors & Awards

  • Society for Neuroscience Young Investigator Award, Society for Neuroscience (2007)
  • NIH Pioneer Award, National Institutes of Health (2008-2013)
  • Searle Scholar, Searle Foundation (2004-2006)
  • Career Award in the Biomedical Sciences, Burroughes Welcome Fund (2002-2007)
  • McKnight Scholar, McKnight Endowment for Neurosciences (2004-2006)
  • Helen Hay Whitney Fellow, Helen Hay Whitney Foundation (1999-2002)

Professional Education

  • Postdoctoral Fellow, Harvard Medical School, Neuroscience (2003)
  • Ph.D., Stanford University, Neuroscience (1997)
  • B.Sc., Brown University, Neuroscience (1990)

Research & Scholarship

Current Research and Scholarly Interests

Our lab studies the underlying neurobiology of autism and other neuro-developmental disorders. We are particularly interested in understanding how electrical activity and calcium signals control the development of the brain and how this is altered in children with autism spectrum disorders. We are also developing new tools to study and repair the developing brain.


2013-14 Courses

Graduate and Fellowship Programs


Journal Articles

  • SHANK3 and IGF1 restore synaptic deficits in neurons from 22q13 deletion syndrome patients NATURE Shcheglovitov, A., Shcheglovitova, O., Yazawa, M., Portmann, T., Shu, R., Sebastiano, V., Krawisz, A., Froehlich, W., Bernstein, J. A., Hallmayer, J. F., Dolmetsch, R. E. 2013; 503 (7475): 267-?


    Phelan-McDermid syndrome (PMDS) is a complex neurodevelopmental disorder characterized by global developmental delay, severely impaired speech, intellectual disability, and an increased risk of autism spectrum disorders (ASDs). PMDS is caused by heterozygous deletions of chromosome 22q13.3. Among the genes in the deleted region is SHANK3, which encodes a protein in the postsynaptic density (PSD). Rare mutations in SHANK3 have been associated with idiopathic ASDs, non-syndromic intellectual disability, and schizophrenia. Although SHANK3 is considered to be the most likely candidate gene for the neurological abnormalities in PMDS patients, the cellular and molecular phenotypes associated with this syndrome in human neurons are unknown. We generated induced pluripotent stem (iPS) cells from individuals with PMDS and autism and used them to produce functional neurons. We show that PMDS neurons have reduced SHANK3 expression and major defects in excitatory, but not inhibitory, synaptic transmission. Excitatory synaptic transmission in PMDS neurons can be corrected by restoring SHANK3 expression or by treating neurons with insulin-like growth factor 1 (IGF1). IGF1 treatment promotes formation of mature excitatory synapses that lack SHANK3 but contain PSD95 and N-methyl-D-aspartate (NMDA) receptors with fast deactivation kinetics. Our findings provide direct evidence for a disruption in the ratio of cellular excitation and inhibition in PMDS neurons, and point to a molecular pathway that can be recruited to restore it.

    View details for DOI 10.1038/nature12618

    View details for Web of Science ID 000326894200052

    View details for PubMedID 24132240

  • In Vitro Human Corticogenesis NEURON Wang, Y., Dolmetsch, R. 2013; 77 (3): 379-381


    Whether neurons generated in vitro from human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) have in vivo-like properties is unknown. In this issue of Neuron, Espuny-Camacho et al. (2013) show that ESC-/iPSC-derived cortical neurons make specific projections and functional synapses when transplanted into a neonatal mouse brain.

    View details for DOI 10.1016/j.neuron.2013.01.023

    View details for Web of Science ID 000317030800002

    View details for PubMedID 23395367

  • Timothy syndrome is associated with activity-dependent dendritic retraction in rodent and human neurons NATURE NEUROSCIENCE Krey, J. F., Pasca, S. P., Shcheglovitov, A., Yazawa, M., Schwemberger, R., Rasmusson, R., Dolmetsch, R. E. 2013; 16 (2): 201-209


    L-type voltage gated calcium channels have an important role in neuronal development by promoting dendritic growth and arborization. A point mutation in the gene encoding Ca(V)1.2 causes Timothy syndrome, a neurodevelopmental disorder associated with autism spectrum disorders (ASDs). We report that channels with the Timothy syndrome alteration cause activity-dependent dendrite retraction in rat and mouse neurons and in induced pluripotent stem cell (iPSC)-derived neurons from individuals with Timothy syndrome. Dendrite retraction was independent of calcium permeation through the mutant channel, was associated with ectopic activation of RhoA and was inhibited by overexpression of the channel-associated GTPase Gem. These results suggest that Ca(V)1.2 can activate RhoA signaling independently of Ca(2+) and provide insights into the cellular basis of Timothy syndrome and other ASDs.

    View details for DOI 10.1038/nn.3307

    View details for Web of Science ID 000314260200017

    View details for PubMedID 23313911

  • Using iPSC-derived neurons to uncover cellular phenotypes associated with Timothy syndrome NATURE MEDICINE Pasca, S. P., Portmann, T., Voineagu, I., Yazawa, M., Shcheglovitov, A., Pasca, A. M., Cord, B., Palmer, T. D., Chikahisa, S., Nishino, S., Bernstein, J. A., Hallmayer, J., Geschwind, D. H., Dolmetsch, R. E. 2011; 17 (12): 1657-U176


    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.

    View details for DOI 10.1038/nm.2576

    View details for Web of Science ID 000297978000039

    View details for PubMedID 22120178

  • MicroRNA-mediated conversion of human fibroblasts to neurons NATURE Yoo, A. S., Sun, A. X., Li, L., Shcheglovitov, A., Portmann, T., Li, Y., Lee-Messer, C., Dolmetsch, R. E., Tsien, R. W., Crabtree, G. R. 2011; 476 (7359): 228-U123


    Neurogenic transcription factors and evolutionarily conserved signalling pathways have been found to be instrumental in the formation of neurons. However, the instructive role of microRNAs (miRNAs) in neurogenesis remains unexplored. We recently discovered that miR-9* and miR-124 instruct compositional changes of SWI/SNF-like BAF chromatin-remodelling complexes, a process important for neuronal differentiation and function. Nearing mitotic exit of neural progenitors, miR-9* and miR-124 repress the BAF53a subunit of the neural-progenitor (np)BAF chromatin-remodelling complex. After mitotic exit, BAF53a is replaced by BAF53b, and BAF45a by BAF45b and BAF45c, which are then incorporated into neuron-specific (n)BAF complexes essential for post-mitotic functions. Because miR-9/9* and miR-124 also control multiple genes regulating neuronal differentiation and function, we proposed that these miRNAs might contribute to neuronal fates. Here we show that expression of miR-9/9* and miR-124 (miR-9/9*-124) in human fibroblasts induces their conversion into neurons, a process facilitated by NEUROD2. Further addition of neurogenic transcription factors ASCL1 and MYT1L enhances the rate of conversion and the maturation of the converted neurons, whereas expression of these transcription factors alone without miR-9/9*-124 was ineffective. These studies indicate that the genetic circuitry involving miR-9/9*-124 can have an instructive role in neural fate determination.

    View details for DOI 10.1038/nature10323

    View details for Web of Science ID 000293731900041

    View details for PubMedID 21753754

  • The Human Brain in a Dish: The Promise of iPSC-Derived Neurons CELL Dolmetsch, R., Geschwind, D. H. 2011; 145 (6): 831-834


    Induced pluripotent stem cell-derived neurons from patients promise to fill an important niche between studies in humans and model organisms in deciphering mechanisms and identifying therapeutic avenues for neurologic and psychiatric diseases. Recent work begins to tap this potential and also highlights challenges that must be overcome to be fully realized.

    View details for DOI 10.1016/j.cell.2011.05.034

    View details for Web of Science ID 000291461600011

    View details for PubMedID 21663789

  • Using induced pluripotent stem cells to investigate cardiac phenotypes in Timothy syndrome NATURE Yazawa, M., Hsueh, B., Jia, X., Pasca, A. M., Bernstein, J. A., Hallmayer, J., Dolmetsch, R. E. 2011; 471 (7337): 230-U120


    Individuals with congenital or acquired prolongation of the QT interval, or long QT syndrome (LQTS), are at risk of life-threatening ventricular arrhythmia. LQTS is commonly genetic in origin but can also be caused or exacerbated by environmental factors. A missense mutation in the L-type calcium channel Ca(V)1.2 leads to LQTS in patients with Timothy syndrome. To explore the effect of the Timothy syndrome mutation on the electrical activity and contraction of human cardiomyocytes, we reprogrammed human skin cells from Timothy syndrome patients to generate induced pluripotent stem cells, and differentiated these cells into cardiomyocytes. Electrophysiological recording and calcium (Ca(2+)) imaging studies of these cells revealed irregular contraction, excess Ca(2+) influx, prolonged action potentials, irregular electrical activity and abnormal calcium transients in ventricular-like cells. We found that roscovitine, a compound that increases the voltage-dependent inactivation of Ca(V)1.2 (refs 6-8), restored the electrical and Ca(2+) signalling properties of cardiomyocytes from Timothy syndrome patients. This study provides new opportunities for studying the molecular and cellular mechanisms of cardiac arrhythmias in humans, and provides a robust assay for developing new drugs to treat these diseases.

    View details for DOI 10.1038/nature09855

    View details for Web of Science ID 000288170200040

    View details for PubMedID 21307850

  • LRRK2 mutant iPSC-derived DA neurons demonstrate increased susceptibility to oxidative stress. Cell stem cell Nguyen, H. N., Byers, B., Cord, B., Shcheglovitov, A., Byrne, J., Gujar, P., Kee, K., Schüle, B., Dolmetsch, R. E., Langston, W., Palmer, T. D., Pera, R. R. 2011; 8 (3): 267-280


    Studies of Parkinson's disease (PD) have been hindered by lack of access to affected human dopaminergic (DA) neurons. Here, we report generation of induced pluripotent stem cells that carry the p.G2019S mutation (G2019S-iPSCs) in the Leucine-Rich Repeat Kinase-2 (LRRK2) gene, the most common PD-related mutation, and their differentiation into DA neurons. The high penetrance of the LRRK2 mutation and its clinical resemblance to sporadic PD suggest that these cells could provide a valuable platform for disease analysis and drug development. We found that DA neurons derived from G2019S-iPSCs showed increased expression of key oxidative stress-response genes and α-synuclein protein. The mutant neurons were also more sensitive to caspase-3 activation and cell death caused by exposure to stress agents, such as hydrogen peroxide, MG-132, and 6-hydroxydopamine, than control DA neurons. This enhanced stress sensitivity is consistent with existing understanding of early PD phenotypes and represents a potential therapeutic target.

    View details for DOI 10.1016/j.stem.2011.01.013

    View details for PubMedID 21362567

  • The CRAC Channel Activator STIM1 Binds and Inhibits L-Type Voltage-Gated Calcium Channels SCIENCE Park, C. Y., Shcheglovitov, A., Dolmetsch, R. 2010; 330 (6000): 101-105


    Voltage- and store-operated calcium (Ca(2+)) channels are the major routes of Ca(2+) 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 Ca(2+) channels, directly suppresses depolarization-induced opening of the voltage-gated Ca(2+) channel Ca(V)1.2. STIM1 binds to the C terminus of Ca(V)1.2 through its Ca(2+) release-activated Ca(2+) 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.

    View details for DOI 10.1126/science.1191027

    View details for Web of Science ID 000282334500045

    View details for PubMedID 20929812

  • PIKfyve regulates Ca(V)1.2 degradation and prevents excitotoxic cell death JOURNAL OF CELL BIOLOGY Tsuruta, F., Green, E. M., Rousset, M., Dolmetsch, R. E. 2009; 187 (2): 279-294


    Voltage-gated Ca(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(V)1.2 channels, resulting in decreased Ca(2+) entry and reduced toxicity. Ca(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(2)) and regulates endosome and lysosome function. Sustained activation of glutamate receptors recruits PIKfyve to Ca(V)1.2 channels, increases cellular levels of PtdIns(3,5)P(2), and promotes targeting of Ca(V)1.2 to lysosomes. Knockdown of PIKfyve prevents Ca(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(2) regulation.

    View details for DOI 10.1083/jcb.200903028

    View details for Web of Science ID 000270914600014

    View details for PubMedID 19841139

  • Induction of protein-protein interactions in live cells using light NATURE BIOTECHNOLOGY Yazawa, M., Sadaghiani, A. M., Hsueh, B., Dolmetsch, R. E. 2009; 27 (10): 941-U105


    Protein-protein interactions are essential for many cellular processes. We have developed a technology called light-activated dimerization (LAD) to artificially induce protein hetero- and homodimerization in live cells using light. Using the FKF1 and GIGANTEA (GI) proteins of Arabidopsis thaliana, we have generated protein tags whose interaction is controlled by blue light. We demonstrated the utility of this system with LAD constructs that can recruit the small G-protein Rac1 to the plasma membrane and induce the local formation of lamellipodia in response to focal illumination. We also generated a light-activated transcription factor by fusing domains of GI and FKF1 to the DNA binding domain of Gal4 and the transactivation domain of VP16, respectively, showing that this technology is easily adapted to other systems. These studies set the stage for the development of light-regulated signaling molecules for controlling receptor activation, synapse formation and other signaling events in organisms.

    View details for DOI 10.1038/nbt.1569

    View details for Web of Science ID 000271472500025

    View details for PubMedID 19801976

  • STIM1 and calmodulin interact with Orai1 to induce Ca2+-dependent inactivation of CRAC channels PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Mullins, F. M., Park, C. Y., Dolmetsch, R. E., Lewis, R. S. 2009; 106 (36): 15495-15500


    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.

    View details for DOI 10.1073/pnas.0906781106

    View details for Web of Science ID 000269632400074

    View details for PubMedID 19706428

  • STIM1 Clusters and Activates CRAC Channels via Direct Binding of a Cytosolic Domain to Orai1 CELL Park, C. Y., Hoover, P. J., Mullins, F. M., Bachhawat, P., Covington, E. D., Raunser, S., Walz, T., Garcia, K. C., Dolmetsch, R. E., Lewis, R. S. 2009; 136 (5): 876-890


    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.

    View details for DOI 10.1016/j.cell.2009.02.014

    View details for Web of Science ID 000263930900011

    View details for PubMedID 19249086

  • The tumor suppressor eIF3e mediates calcium-dependent internalization of the L-type calcium channel Ca(v)1.2 NEURON Green, E. M., Barrett, C. F., Bultynck, G., Shamah, S. M., Dolmetsch, R. E. 2007; 55 (4): 615-632


    Voltage-gated calcium channels (VGCCs) convert electrical activity into calcium (Ca2+) signals that regulate cellular excitability, differentiation, and connectivity. The magnitude and kinetics of Ca2+ signals depend on the number of VGCCs at the plasma membrane, but little is known about the regulation of VGCC surface expression. We report that electrical activity causes internalization of the L-type Ca2+ channel (LTC) CaV1.2 and that this is mediated by binding to the tumor suppressor eIF3e/Int6 (eukaryotic initiation factor 3 subunit e). Using total internal reflection microscopy, we identify a population of CaV1.2 containing endosomes whose rapid trafficking is strongly regulated by Ca2+. We define a domain in the II-III loop of CaV1.2 that binds eIF3e and is essential for the activity dependence of both channel internalization and endosomal trafficking. These findings provide a mechanism for activity-dependent internalization and trafficking of CaV1.2 and provide a tantalizing link between Ca2+ homeostasis and a mammalian oncogene.

    View details for DOI 10.1016/j.neuron.2007.07.024

    View details for Web of Science ID 000249159200010

    View details for PubMedID 17698014

  • Molecular mechanisms of autism: a possible role for Ca2+ signaling CURRENT OPINION IN NEUROBIOLOGY Krey, J. F., Dolmetsch, R. E. 2007; 17 (1): 112-119


    Autism spectrum disorders (ASDs) are a group of developmental disorders characterized by social and emotional deficits, language impairments and stereotyped behaviors that manifest in early postnatal life. The molecular mechanisms that underlie ASDs are not known, but several recent developments suggest that some forms of autism are caused by failures in activity-dependent regulation of neural development. Mutations of several voltage-gated and ligand-gated ion channels that regulate neuronal excitability and Ca2+ signaling have been associated with ASDs. In addition, Ca2+-regulated signaling proteins involved in synapse formation and dendritic growth have been implicated in ASDs. These recent advances suggest a set of signaling pathways that might have a role in generating these increasingly prevalent disorders.

    View details for DOI 10.1016/j.conb.2007.01.010

    View details for Web of Science ID 000244771100016

    View details for PubMedID 17275285

  • The C terminus of the L-type voltage-gated calcium channel Ca(v)1.2 encodes a transcription factor CELL Gomez-Ospina, N., Tsuruta, F., Barreto-Chang, O., Hu, L., Dolmetsch, R. 2006; 127 (3): 591-606


    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(V)1.2, 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.

    View details for DOI 10.1016/j.cell.2006.10.017

    View details for Web of Science ID 000241937000022

    View details for PubMedID 17081980

  • Cell signaling. The double life of a transcription factor takes it outside the nucleus. Science Park, C. Y., Dolmetsch, R. 2006; 314 (5796): 64-65

    View details for PubMedID 17023638

  • Calcium oscillations increase the efficiency and specificity of gene expression NATURE Dolmetsch, R. E., XU, K. L., Lewis, R. S. 1998; 392 (6679): 933-936


    Cytosolic calcium ([Ca2+]i) oscillations are a nearly universal mode of signalling in excitable and non-excitable cells. Although Ca2+ is known to mediate a diverse array of cell functions, it is not known whether oscillations contribute to the efficiency or specificity of signalling or are merely an inevitable consequence of the feedback control of [Ca2+]i. We have developed a Ca2+ clamp technique to investigate the roles of oscillation amplitude and frequency in regulating gene expression driven by the proinflammatory transcription factors NF-AT, Oct/OAP and NF-kappaB. Here we report that oscillations reduce the effective Ca2+ threshold for activating transcription factors, thereby increasing signal detection at low levels of stimulation. In addition, specificity is encoded by the oscillation frequency: rapid oscillations stimulate all three transcription factors, whereas infrequent oscillations activate only NF-kappaB. The genes encoding the cytokines interleukin (IL)-2 and IL-8 are also frequency-sensitive in a way that reflects their degree of dependence on NF-AT versus NF-kappaB. Our results provide direct evidence that [Ca2+]i oscillations increase both the efficacy and the information content of Ca2+ signals that lead to gene expression and cell differentiation.

    View details for Web of Science ID 000073359900052

    View details for PubMedID 9582075

  • Differential activation of transcription factors induced by Ca2+ response amplitude and duration NATURE Dolmetsch, R. E., Lewis, R. S., Goodnow, C. C., Healy, J. I. 1997; 386 (6627): 855-858


    An increase in the intracellular calcium ion concentration ([Ca2+]i) controls a diverse range of cell functions, including adhesion, motility, gene expression and proliferation. Calcium signalling patterns can occur as single transients, repetitive oscillations or sustained plateaux, but it is not known whether these patterns are responsible for encoding the specificity of cellular responses. We report here that the amplitude and duration of calcium signals in B lymphocytes controls differential activation of the pro-inflammatory transcriptional regulators NF-kappaB, c-Jun N-terminal kinase (JNK) and NFAT. NF-kappaB and JNK are selectively activated by a large transient [Ca2+]i rise, whereas NFAT is activated by a low, sustained Ca2+ plateau. Differential activation results from differences in the Ca2+ sensitivities and kinetic behaviour of the three pathways. Our results show how downstream effectors can decode information contained in the amplitude and duration of Ca2+ signals, revealing a mechanism by which a multifunctional second messenger such as Ca2+ can achieve specificity in signalling to the nucleus.

    View details for Web of Science ID A1997WV70600060

    View details for PubMedID 9126747

  • State-dependent signaling by Cav1.2 regulates hair follicle stem cell function. Genes & development Yucel, G., Altindag, B., Gomez-Ospina, N., Rana, A., Panagiotakos, G., Lara, M. F., Dolmetsch, R., Oro, A. E. 2013; 27 (11): 1217-1222


    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. Cav1.2(TS) 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.

    View details for DOI 10.1101/gad.216556.113

    View details for PubMedID 23752588

  • Competition between a-actinin and Ca(2+)-Calmodulin Controls Surface Retention of the L-type Ca(2+) Channel CaV1.2. Neuron Hall, D. D., Dai, S., Tseng, P., Malik, Z., Nguyen, M., Matt, L., Schnizler, K., Shephard, A., Mohapatra, D. P., Tsuruta, F., Dolmetsch, R. E., Christel, C. J., Lee, A., Burette, A., Weinberg, R. J., Hell, J. W. 2013; 78 (3): 483-497


    Regulation of neuronal excitability and cardiac excitation-contraction coupling requires the proper localization of L-type Ca(2+) channels. We show that the actin-binding protein ?-actinin binds to the C-terminal surface targeting motif of ?11.2, the central pore-forming CaV1.2 subunit, in order to foster its surface expression. Disruption of ?-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 ?-actinin from their shared binding site on ?11.2 upon Ca(2+) influx through L-type channels, but not through NMDAR, thereby triggering loss of CaV1.2 from spines. Coexpression of a Ca(2+)-binding-deficient calmodulin mutant does not affect basal CaV1.2 surface expression but inhibits its internalization upon Ca(2+) influx. We conclude that ?-actinin stabilizes CaV1.2 at the plasma membrane and that its displacement by Ca(2+)-calmodulin triggers Ca(2+)-induced endocytosis of CaV1.2, thus providing an important negative feedback mechanism for Ca(2+) influx.

    View details for DOI 10.1016/j.neuron.2013.02.032

    View details for PubMedID 23664615

  • A Promoter in the Coding Region of the Calcium Channel Gene CACNA1C Generates the Transcription Factor CCAT PLOS ONE Gomez-Ospina, N., Panagiotakos, G., Portmann, T., Pasca, S. P., Rabah, D., Budzillo, A., Kinet, J. P., Dolmetsch, R. E. 2013; 8 (4)


    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.

    View details for DOI 10.1371/journal.pone.0060526

    View details for Web of Science ID 000317893400012

    View details for PubMedID 23613729

  • Calcium influx through L-type Ca(V)1.2 Ca2+ channels regulates mandibular development JOURNAL OF CLINICAL INVESTIGATION Ramachandran, K. V., Hennessey, J. A., Barnett, A. S., Yin, X., Stadt, H. A., Foster, E., Shah, R. A., Yazawa, M., Dolmetsch, R. E., Kirby, M. L., Pitt, G. S. 2013; 123 (4): 1638-1646


    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.

    View details for DOI 10.1172/JCI66903

    View details for Web of Science ID 000317021800025

    View details for PubMedID 23549079

  • Timothy syndrome is associated with activity-dependent dendritic retraction in rodent and human neurons. Nature neuroscience Krey, J. F., Pasca, S. P., Shcheglovitov, A., Yazawa, M., Schwemberger, R., Rasmusson, R., Dolmetsch, R. E. 2013; 16 (2): 201-209


    L-type voltage gated calcium channels have an important role in neuronal development by promoting dendritic growth and arborization. A point mutation in the gene encoding Ca(V)1.2 causes Timothy syndrome, a neurodevelopmental disorder associated with autism spectrum disorders (ASDs). We report that channels with the Timothy syndrome alteration cause activity-dependent dendrite retraction in rat and mouse neurons and in induced pluripotent stem cell (iPSC)-derived neurons from individuals with Timothy syndrome. Dendrite retraction was independent of calcium permeation through the mutant channel, was associated with ectopic activation of RhoA and was inhibited by overexpression of the channel-associated GTPase Gem. These results suggest that Ca(V)1.2 can activate RhoA signaling independently of Ca(2+) and provide insights into the cellular basis of Timothy syndrome and other ASDs.

    View details for DOI 10.1038/nn.3307

    View details for PubMedID 23313911

  • Modeling Timothy Syndrome with iPS Cells JOURNAL OF CARDIOVASCULAR TRANSLATIONAL RESEARCH Yazawa, M., Dolmetsch, R. E. 2013; 6 (1): 1-9


    Genetic mutations in ion channel genes that are associated with cardiac arrhythmias have been identified over the past several decades. However, little is known about the pathophysiological processes. An important limitation has been the difficulty of using human cardiomyocytes to study arrhythmias and identify drugs. To circumvent this issue, we have developed a method using human-induced pluripotent stem cells to generate cardiomyocytes from individuals with Timothy syndrome (TS), a genetic disorder characterized by QT prolongation, ventricular tachycardia, and autism. The TS ventricular-like cardiomyocytes exhibit deficits in contraction, electrical signaling, and calcium handling, as revealed by live cell imaging and electrophysiological studies. We tested candidate drugs in TS cardiomyocytes and found that roscovitine could successfully rescue these cellular phenotypes. The use of a human cellular model of cardiac arrhythmias provides a useful new platform not only to study disease mechanisms but also to develop new therapies to treat cardiac arrhythmias.

    View details for DOI 10.1007/s12265-012-9444-x

    View details for Web of Science ID 000313657700001

    View details for PubMedID 23299782

  • Cytoplasmic location of a1A voltage-gated calcium channel C-terminal fragment (Cav2.1-CTF) aggregate is sufficient to cause cell death. PloS one Takahashi, M., Obayashi, M., Ishiguro, T., Sato, N., Niimi, Y., Ozaki, K., Mogushi, K., Mahmut, Y., Tanaka, H., Tsuruta, F., Dolmetsch, R., Yamada, M., Takahashi, H., Kato, T., Mori, O., Eishi, Y., Mizusawa, H., Ishikawa, K. 2013; 8 (3)


    The human ?1A 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.

    View details for DOI 10.1371/journal.pone.0050121

    View details for PubMedID 23505410

  • Cacnb4 directly couples electrical activity to gene expression, a process defective in juvenile epilepsy EMBO JOURNAL Tadmouri, A., Kiyonaka, S., Barbado, M., Rousset, M., Fablet, K., Sawamura, S., Bahembera, E., Pernet-Gallay, K., Arnoult, C., Miki, T., Sadoul, K., Gory-Faure, S., Lambrecht, C., Lesage, F., Akiyama, S., Khochbin, S., Baulande, S., Janssens, V., Andrieux, A., Dolmetsch, R., Ronjat, M., Mori, Y., De Waard, M. 2012; 31 (18): 3730-3744


    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 (TR?), and (iii) histone binding through association of Cacnb4 with HP1? 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.

    View details for DOI 10.1038/emboj.2012.226

    View details for Web of Science ID 000308809100010

    View details for PubMedID 22892567

  • Apelin Enhances Directed Cardiac Differentiation of Mouse and Human Embryonic Stem Cells PLOS ONE Wang, I. E., Wang, X., Ge, X., Anderson, J., Ho, M., Ashley, E., Liu, J., Butte, M. J., Yazawa, M., Dolmetsch, R. E., Quertermous, T., Yang, P. C. 2012; 7 (6)


    Apelin is a peptide ligand for an orphan G-protein coupled receptor (APJ receptor) and serves as a critical gradient for migration of mesodermal cells fated to contribute to the myocardial lineage. The present study was designed to establish a robust cardiac differentiation protocol, specifically, to evaluate the effect of apelin on directed differentiation of mouse and human embryonic stem cells (mESCs and hESCs) into cardiac lineage. Different concentrations of apelin (50, 100, 500 nM) were evaluated to determine its differentiation potential. The optimized dose of apelin was then combined with mesodermal differentiation factors, including BMP-4, activin-A, and bFGF, in a developmentally specific temporal sequence to examine the synergistic effects on cardiac differentiation. Cellular, molecular, and physiologic characteristics of the apelin-induced contractile embryoid bodies (EBs) were analyzed. It was found that 100 nM apelin resulted in highest percentage of contractile EB for mESCs while 500 nM had the highest effects on hESCs. Functionally, the contractile frequency of mESCs-derived EBs (mEBs) responded appropriately to increasing concentration of isoprenaline and diltiazem. Positive phenotype of cardiac specific markers was confirmed in the apelin-treated groups. The protocol, consisting of apelin and mesodermal differentiation factors, induced contractility in significantly higher percentage of hESC-derived EBs (hEBs), up-regulated cardiac-specific genes and cell surface markers, and increased the contractile force. In conclusion, we have demonstrated that the treatment of apelin enhanced cardiac differentiation of mouse and human ESCs and exhibited synergistic effects with mesodermal differentiation factors.

    View details for DOI 10.1371/journal.pone.0038328

    View details for Web of Science ID 000305339900024

    View details for PubMedID 22675543

  • Patient-Specific Induced Pluripotent Stem Cells as a Model for Familial Dilated Cardiomyopathy SCIENCE TRANSLATIONAL MEDICINE Sun, N., Yazawa, M., Liu, J., Han, L., Sanchez-Freire, V., Abilez, O. J., Navarrete, E. G., Hu, S., Wang, L., Lee, A., Pavlovic, A., Lin, S., Chen, R., Hajjar, R. J., Snyder, M. P., Dolmetsch, R. E., Butte, M. J., Ashley, E. A., Longaker, M. T., Robbins, R. C., Wu, J. C. 2012; 4 (130)


    Characterized by ventricular dilatation, systolic dysfunction, and progressive heart failure, dilated cardiomyopathy (DCM) is the most common form of cardiomyopathy in patients. DCM is the most common diagnosis leading to heart transplantation and places a significant burden on healthcare worldwide. The advent of induced pluripotent stem cells (iPSCs) offers an exceptional opportunity for creating disease-specific cellular models, investigating underlying mechanisms, and optimizing therapy. Here, we generated cardiomyocytes from iPSCs derived from patients in a DCM family carrying a point mutation (R173W) in the gene encoding sarcomeric protein cardiac troponin T. Compared to control healthy individuals in the same family cohort, cardiomyocytes derived from iPSCs from DCM patients exhibited altered regulation of calcium ion (Ca(2+)), decreased contractility, and abnormal distribution of sarcomeric ?-actinin. When stimulated with a ?-adrenergic agonist, DCM iPSC-derived cardiomyocytes showed characteristics of cellular stress such as reduced beating rates, compromised contraction, and a greater number of cells with abnormal sarcomeric ?-actinin distribution. Treatment with ?-adrenergic blockers or overexpression of sarcoplasmic reticulum Ca(2+) adenosine triphosphatase (Serca2a) improved the function of iPSC-derived cardiomyocytes from DCM patients. Thus, iPSC-derived cardiomyocytes from DCM patients recapitulate to some extent the morphological and functional phenotypes of DCM and may serve as a useful platform for exploring disease mechanisms and for drug screening.

    View details for DOI 10.1126/scitranslmed.3003552

    View details for Web of Science ID 000303045900004

    View details for PubMedID 22517884

  • Using light to control signaling cascades in live neurons CURRENT OPINION IN NEUROBIOLOGY Rana, A., Dolmetsch, R. E. 2010; 20 (5): 617-622


    Understanding the complexity of neuronal biology requires the manipulation of cellular processes with high specificity and spatio-temporal precision. The recent development of synthetic photo-activatable proteins designed using the light-oxygen-voltage and phytochrome domains provides a new set of tools for genetically targeted optical control of cell signaling. Their modular design, functional diversity, precisely controlled activity and in vivo applicability offer many advantages for investigating neuronal function. Although designing these proteins is still a considerable challenge, future advances in rational protein design and a deeper understanding of their photoactivation mechanisms will allow the development of the next generation of optogenetic techniques.

    View details for DOI 10.1016/j.conb.2010.08.018

    View details for Web of Science ID 000283481100015

    View details for PubMedID 20850295

  • The carboxy-terminal fragment of alpha(1A) calcium channel preferentially aggregates in the cytoplasm of human spinocerebellar ataxia type 6 Purkinje cells ACTA NEUROPATHOLOGICA Ishiguro, T., Ishikawa, K., Takahashi, M., Obayashi, M., Amino, T., Sato, N., Sakamoto, M., Fujigasaki, H., Tsuruta, F., Dolmetsch, R., Arai, T., Sasaki, H., Nagashima, K., Kato, T., Yamada, M., Takahashi, H., Hashizume, Y., Mizusawa, H. 2010; 119 (4): 447-464


    Spinocerebellar ataxia type 6 (SCA6) is an autosomal dominant neurodegenerative disease caused by a small polyglutamine (polyQ) expansion (control: 4-20Q; SCA6: 20-33Q) in the carboxyl(C)-terminal cytoplasmic domain of the alpha(1A) voltage-dependent calcium channel (Ca(v)2.1). Although a 75-85-kDa Ca(v)2.1 C-terminal fragment (CTF) is toxic in cultured cells, its existence in human brains and its role in SCA6 pathogenesis remains unknown. Here, we investigated whether the small polyQ expansion alters the expression pattern and intracellular distribution of Ca(v)2.1 in human SCA6 brains. New antibodies against the Ca(v)2.1 C-terminus were used in immunoblotting and immunohistochemistry. In the cerebella of six control individuals, the CTF was detected in sucrose- and SDS-soluble cytosolic fractions; in the cerebella of two SCA6 patients, it was additionally detected in SDS-insoluble cytosolic and sucrose-soluble nuclear fractions. In contrast, however, the CTF was not detected either in the nuclear fraction or in the SDS-insoluble cytosolic fraction of SCA6 extracerebellar tissues, indicating that the CTF being insoluble in the cytoplasm or mislocalized to the nucleus only in the SCA6 cerebellum. Immunohistochemistry revealed abundant aggregates in cell bodies and dendrites of SCA6 Purkinje cells (seven patients) but not in controls (n = 6). Recombinant CTF with a small polyQ expansion (rCTF-Q28) aggregated in cultured PC12 cells, but neither rCTF-Q13 (normal-length polyQ) nor full-length Ca(v)2.1 with Q28 did. We conclude that SCA6 pathogenesis may be associated with the CTF, normally found in the cytoplasm, being aggregated in the cytoplasm and additionally distributed in the nucleus.

    View details for DOI 10.1007/s00401-009-0630-0

    View details for Web of Science ID 000275752500005

    View details for PubMedID 20043227

  • Gabapentin Receptor alpha 2 delta-1 Is a Neuronal Thrombospondin Receptor Responsible for Excitatory CNS Synaptogenesis CELL Eroglu, C., Allen, N. J., Susman, M. W., O'Rourke, N. A., Park, C. Y., Oezkan, E., Chakraborty, C., Mulinyawe, S. B., Annis, D. S., Huberman, A. D., Green, E. M., Lawler, J., Dolmetsch, R., Garcia, K. C., Smith, S. J., Luo, Z. D., Rosenthal, A., Mosher, D. F., Barres, B. A. 2009; 139 (2): 380-392


    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.

    View details for DOI 10.1016/j.cell.2009.09.025

    View details for Web of Science ID 000270857500020

    View details for PubMedID 19818485

  • Calcium imaging of cortical neurons using Fura-2 AM. Journal of visualized experiments : JoVE Barreto-Chang, O. L., Dolmetsch, R. E. 2009


    Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium indicator dyes, which are BAPTA-based organic molecules that change their spectral properties in response to the binding of Ca2+ ions. Calcium indicator dyes fall into two categories, ratio-metric dyes like Fura-2 and Indo-1 and single-wavelength dyes like Fluo-4. Ratio-metric dyes change either their excitation or their emission spectra in response to calcium, allowing the concentration of intracellular calcium to be determined from the ratio of fluorescence emission or excitation at distinct wavelengths. The main advantage of using ratio-metric dyes over single wavelength probes is that the ratio signal is independent of the dye concentration, illumination intensity, and optical path length allowing the concentration of intracellular calcium to be determined independently of these artifacts. One of the most common calcium indicators is Fura-2, which has an emission peak at 505 nM and changes its excitation peak from 340 nm to 380 nm in response to calcium binding. Here we describe the use of Fura-2 to measure intracellular calcium elevations in neurons and other excitable cells.

    View details for DOI 10.3791/1067

    View details for PubMedID 19229178

  • L-type channel regulation of gene expression TRANSCRIPTIONAL REGULATION BY NEURONAL ACTIVITY Gornez-Ospina, N., Dolmetsch, R. 2008: 111-123
  • TrpC3 Regulates Hypertrophy-Associated Gene Expression without Affecting Myocyte Beating or Cell Size PLOS ONE Brenner, J. S., Dolmetsch, R. E. 2007; 2 (8)


    Pathological cardiac hypertrophy is associated with an increased risk of heart failure and cardiovascular mortality. Calcium (Ca(2+)) -regulated gene expression is essential for the induction of hypertrophy, but it is not known how myocytes distinguish between the Ca(2+) signals that regulate contraction and those that lead to cardiac hypertrophy. We used in vitro neonatal rat ventricular myocytes to perform an RNA interference (RNAi) screen for ion channels that mediate Ca(2+)-dependent gene expression in response to hypertrophic stimuli. We identified several ion channels that are linked to hypertrophic gene expression, including transient receptor potential C3 (TrpC3). RNAi-mediated knockdown of TrpC3 decreases expression of hypertrophy-associated genes such as the A- and B-type natriuretic peptides (ANP and BNP) in response to numerous hypertrophic stimuli, while TrpC3 overexpression increases BNP expression. Furthermore, stimuli that induce hypertrophy dramatically increase TrpC3 mRNA levels. Importantly, whereas TrpC3-knockdown strongly reduces gene expression associated with hypertrophy, it has a negligible effect on cell size and on myocyte beating. These results suggest that Ca(2+) influx through TrpC3 channels increases transcription of genes associated with hypertrophy but does not regulate the signaling pathways that control cell size or contraction. Thus TrpC3 may represent an important therapeutic target for the treatment of cardiac hypertrophy and heart failure.

    View details for DOI 10.1371/journal.pone.0000802

    View details for Web of Science ID 000207455400019

    View details for PubMedID 17726532

  • Calcium channels light up NATURE CHEMICAL BIOLOGY Green, E., Dolmetsch, R. E. 2007; 3 (7): 369-370

    View details for DOI 10.1038/nchembio0707-369

    View details for Web of Science ID 000247462800008

    View details for PubMedID 17576420

  • Excitation-transcription coupling: signaling by ion channels to the nucleus. Science's STKE : signal transduction knowledge environment Dolmetsch, R. 2003; 2003 (166): PE4-?


    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.

    View details for PubMedID 12538881

  • Time-lapse imaging of a dynamic phosphorylation dependent protein-protein interaction in mammalian cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Spotts, J. M., Dolmetscht, R. E., Greenberg, M. E. 2002; 99 (23): 15142-15147


    The ability to make sensitive measurements of protein-protein interaction kinetics in single neurons is critical for understanding the molecular and cellular basis of neuronal function. We have developed a reporter technology based on the differential induction of Escherichia coli TEM-1 beta-lactamase (Bla) enzymatic activity that can function as a sensor of the interaction state of two target proteins within single neurons in vivo. To modulate Bla enzymatic activity, we first split the enzyme into two separate, complementary protein fragments that we identified by using a functional screening approach based on circular permutation of the Bla enzyme. The split enzyme was then brought together by the phosphorylation-dependent association of the kinase inducible domain of the cAMP response element binding protein (CREB) and the KIX domain of the CREB binding protein. Using an intracellular substrate whose fluorescence spectrum changes after hydrolysis by Bla, we performed time-lapse ratiometric imaging measurements of Bla enzymatic induction after association of the CREB and CREB binding protein interaction domains. This approach permits direct imaging of protein-protein interactions in single cells with high signal discrimination.

    View details for DOI 10.1073/pnas.232565699

    View details for Web of Science ID 000179224800095

    View details for PubMedID 12415118

  • CREB transcriptional activity in neurons is regulated by multiple, calcium-specific phosphorylation events NEURON Kornhauser, J. M., Cowan, C. W., Shaywitz, A. J., Dolmetsch, R. E., Griffith, E. C., Hu, L. S., Haddad, C., Xia, Z. G., Greenberg, M. E. 2002; 34 (2): 221-233


    The transcription factor CREB mediates diverse responses in the nervous system. It is not known how CREB induces specific patterns of gene expression in response to different extracellular stimuli. We find that Ca(2+) influx into neurons induces CREB phosphorylation at Ser133 and two additional sites, Ser142 and Ser143. While CREB Ser133 phosphorylation is induced by many stimuli, phosphorylation at Ser142 and Ser143 is selectively activated by Ca(2+) influx. The triple phosphorylation of CREB is required for effective Ca(2+) stimulation of CREB-dependent transcription, but the phosphorylation of Ser142 and Ser143, in addition to Ser133, disrupts the interaction of CREB with its cofactor CBP. These results suggest that Ca(2+) influx triggers a specific program of gene expression in neurons by selectively regulating CREB phosphorylation.

    View details for Web of Science ID 000174976200008

    View details for PubMedID 11970864

  • Signaling to the nucleus by an L-type calcium channel - Calmodulin complex through the MAP kinase pathway SCIENCE Dolmetsch, R. E., Pajvani, U., Fife, K., Spotts, J. M., Greenberg, M. E. 2001; 294 (5541): 333-339


    Increases in the intracellular concentration of calcium ([Ca2+]i) activate various signaling pathways that lead to the expression of genes that are essential for dendritic development, neuronal survival, and synaptic plasticity. The mode of Ca2+ entry into a neuron plays a key role in determining which signaling pathways are activated and thus specifies the cellular response to Ca2+. Ca2+ influx through L-type voltage-activated channels (LTCs) is particularly effective at activating transcription factors such as CREB and MEF-2. We developed a functional knock-in technique to investigate the features of LTCs that specifically couple them to the signaling pathways that regulate gene expression. We found that an isoleucine-glutamine ("IQ") motif in the carboxyl terminus of the LTC that binds Ca2+-calmodulin (CaM) is critical for conveying the Ca2+ signal to the nucleus. Ca2+-CaM binding to the LTC was necessary for activation of the Ras/mitogen-activated protein kinase (MAPK) pathway, which conveys local Ca2+ signals from the mouth of the LTC to the nucleus. CaM functions as a local Ca2+ sensor at the mouth of the LTC that activates the MAPK pathway and leads to the stimulation of genes that are essential for neuronal survival and plasticity.

    View details for Web of Science ID 000171601400033

    View details for PubMedID 11598293

  • Calcium regulation of neuronal gene expression PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA West, A. E., Chen, W. G., Dalva, M. B., Dolmetsch, R. E., Kornhauser, J. M., Shaywitz, A. J., Takasu, M. A., Tao, X., Greenberg, M. E. 2001; 98 (20): 11024-11031


    Plasticity is a remarkable feature of the brain, allowing neuronal structure and function to accommodate to patterns of electrical activity. One component of these long-term changes is the activity-driven induction of new gene expression, which is required for both the long-lasting long-term potentiation of synaptic transmission associated with learning and memory, and the activity dependent survival events that help to shape and wire the brain during development. We have characterized molecular mechanisms by which neuronal membrane depolarization and subsequent calcium influx into the cytoplasm lead to the induction of new gene transcription. We have identified three points within this cascade of events where the specificity of genes induced by different types of stimuli can be regulated. By using the induction of the gene that encodes brain-derived neurotrophic factor (BDNF) as a model, we have found that the ability of a calcium influx to induce transcription of this gene is regulated by the route of calcium entry into the cell, by the pattern of phosphorylation induced on the transcription factor cAMP-response element (CRE) binding protein (CREB), and by the complement of active transcription factors recruited to the BDNF promoter. These results refine and expand the working model of activity-induced gene induction in the brain, and help to explain how different types of neuronal stimuli can activate distinct transcriptional responses.

    View details for Web of Science ID 000171237100012

    View details for PubMedID 11572963

  • Gene regulation mediated by calcium signals in T lymphocytes NATURE IMMUNOLOGY Feske, S., Giltnane, J., Dolmetsch, R., Staudt, L. M., Rao, A. 2001; 2 (4): 316-324


    Modulation of many signaling pathways in antigen-stimulated T and B cells results in global changes in gene expression. Here we investigate the contribution of calcium signaling to gene expression in T cells using cell lines from two severe-combined immunodeficiency patients with several cytokine deficiencies and diminished activation of the transcription factor NFAT nuclear factor of activated T cells. These T cells show a strong defect in transmembrane calcium influx that is also apparent in their B cells and fibroblasts. DNA microarray analysis of calcium entry-deficient and control T cells shows that Ca2+ signals both activate and repress gene expression and are largely transduced through the phosphatase calcineurin. We demonstrate an elaborate network of signaling pathways downstream of the T cell receptor, explaining the complexity of changes in gene expression during T cell activation.

    View details for Web of Science ID 000167982900012

    View details for PubMedID 11276202

  • A fluorometric method for estimating the calcium content of internal stores CELL CALCIUM Bergling, S., Dolmetsch, R., Lewis, R. S., Keizer, J. 1998; 23 (4): 251-259


    The concentration of Ca2+ in intracellular stores is an important factor in many aspects of Ca2+ signaling, including the generation of Ca2+ spikes, oscillations and waves, control of mitochondrial respiration, and activation of store-operated Ca2+ channels. Here we describe a consistent method for estimating the content of stores, based on the release of stored Ca2+ by thapsigargin (TG) or ionomycin (IO). Once released from stores, Ca2+ elevates [Ca2+]i transiently before it is pumped across the plasma membrane. If the dependence of the pump rate on [Ca2+]i is known, then the kinetics and amplitude of the Ca2+ transient allows the total amount of releasable Ca2+ to be estimated. We develop this quantitative approach and validate its use in human T cells, in which the Ca2+ clearance rate is an approximately linear function of [Ca2+]i. Our results support the assumption that the ER Ca2+ leak in resting T cells is unregulated, i.e. its rate is proportional to luminal [Ca2+]. The characteristic time constant for basal Ca2+ release is 110-140 s, comparable to that for activation of Ca2+ release-activated Ca2+ (CRAC) channels by TG and consistent with the dependence of ICRAC on store depletion. This method for estimating store content may be useful for quantifying the overlap between functionally distinct stores and for defining the relation between store content and cellular responses.

    View details for Web of Science ID 000073862300007

    View details for PubMedID 9681188

  • Quantitative and qualitative control of antigen receptor signalling in tolerant B lymphocytes IMMUNOLOGICAL TOLERANCE Healy, J. I., Dolmetsch, R. E., Lewis, R. S., Goodnow, C. C. 1998; 215: 137-144


    Lymphocyte antigen receptors, such as the B cell antigen receptor (BCR), have the ability to promote or inhibit immune responses. This functional plasticity is exemplified by BCR-induced mitosis in naïve but not tolerant B cells and is correlated with biochemical differences in the signals triggered by foreign and self antigens. Acute stimulation of naïve B cells with foreign antigen induces a biphasic Ca2+ flux, and activates nuclear signalling through NF-AT, NF-kappa B, JNK and ERK. In tolerant B lymphocytes, by contrast, self antigen triggers only a low Ca2+ plateau, NF-AT and ERK. After removal from self antigen, the BCRs on tolerant B cells reacquire the ability to stimulate a biphasic Ca2+ flux and to promote proliferation. The differences in nuclear signalling between naïve and tolerant cells is brought about in part by differences in the magnitude of the Ca2+ signal. A low, sustained Ca2+ signal, such as that seen in tolerant B cells, activates NF-AT, whereas, a high but transient Ca2+ spike, which resembles that triggered in naïve B cells, activates NF-kappa B and JNK. These findings demonstrate that the quantitative differences in Ca2+ signalling between naïve and tolerant B cells are reversible and contribute to the differential triggering of nuclear signals. The activation of selected transcription factors may in turn account for the different functional responses triggered in naïve and tolerant lymphocytes.

    View details for Web of Science ID 000076284400020

    View details for PubMedID 9760576

  • Different nuclear signals are activated by the B cell receptor during positive versus negative signaling IMMUNITY Healy, J. I., Dolmetsch, R. E., Timmerman, L. A., Cyster, J. G., Thomas, M. L., Crabtree, G. R., Lewis, R. S., Goodnow, C. C. 1997; 6 (4): 419-428


    It is not known how immunogenic versus tolerogenic cellular responses are signaled by receptors such as the B cell antigen receptor (BCR). Here we compare BCR signaling in naive cells that respond positively to foreign antigen and self-tolerant cells that respond negatively to self-antigen. In naive cells, foreign antigen triggered a large biphasic calcium response and activated nuclear signals through NF-AT, NF-kappa B, JNK, and ERK/pp90rsk. In tolerant B cells, self-antigen stimulated low calcium oscillations and activated NF-AT and ERK/pp90rsk but not NF-kappa B or JNK. Self-reactive B cells lacking the phosphatase CD45 did not exhibit calcium oscillations or ERK/pp90rsk activation, nor did they repond negatively to self-antigen. These data reveal striking biochemical differences in BCR signaling to the nucleus during positive selection by foreign antigens and negative selection by self-antigens.

    View details for Web of Science ID A1997XC61900007

    View details for PubMedID 9133421

  • Function follows form: The role of store-operated calcium channels in T-cell activation CELLULAR PHYSIOLOGY AND BIOCHEMISTRY Fanger, C. M., Zweifach, A., Dolmetsch, R. E., Hoth, M., Lewis, R. S. 1997; 7 (3-4): 203-218


    Stimulation through the antigen receptor (TCR) of T lymphocytes triggers cytosolic calcium ([Ca2+]i) oscillations that are critically dependent on Ca2+ entry across the plasma membrane. We have investigated the roles of Ca2+ influx and depletion of intracellular Ca2+ stores in the oscillation mechanism, using single-cell Ca2+ imaging techniques and agents that deplete the stores. Thapsigargin (TG; 5-25 nM), cyclopiazonic acid (CPA; 5-20 microM), and tert-butylhydroquinone (tBHQ; 80-200 microM), inhibitors of endoplasmic reticulum Ca(2+)-ATPases, as well as the Ca2+ ionophore ionomycin (5-40 nM), elicit [Ca2+]i oscillations in human T cells. The oscillation frequency is approximately 5 mHz (for ATPase inhibitors) to approximately 10 mHz (for ionomycin) at 22-24 degrees C. The [Ca2+]i oscillations resemble those evoked by TCR ligation in terms of their shape, amplitude, and an absolute dependence on Ca2+ influx. Ca(2+)-ATPase inhibitors and ionomycin induce oscillations only within a narrow range of drug concentrations that are expected to cause partial depletion of intracellular stores. Ca(2+)-induced Ca2+ release does not appear to be significantly involved, as rapid removal of extracellular Ca2+ elicits the same rate of [Ca2+]i decline during the rising and falling phases of the oscillation cycle. Both transmembrane Ca2+ influx and the content of ionomycin-releasable Ca2+ pools fluctuate in oscillating cells. From these data, we propose a model in which [Ca2+]i oscillations in T cells result from the interaction between intracellular Ca2+ stores and depletion-activated Ca2+ channels in the plasma membrane.

    View details for Web of Science ID A1994NC00500001

    View details for PubMedID 8195779

Conference Proceedings

  • Positive and negative regulation of depletion-activated calcium channels by calcium Lewis, R. S., Dolmetsch, R. E., Zweifach, A. ROCKEFELLER UNIV PRESS. 1996: 241-254

    View details for Web of Science ID A1996BF84C00019

    View details for PubMedID 8809948

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