Neuronal-Specific Splicing of CaV1.2 L-type Calcium Channels

Summary

Principal Investigator: Kristin Webster
Abstract: DESCRIPTION (provided by applicant): L-type CaV1.2 voltage-gated calcium channels are essential for mediating muscle contraction, hormone release from endocrine cells, and activity-dependent gene expression in neurons. Activity-dependent gene expression is an important mechanism for coupling repeated synaptic input to lasting changes in the neurons. This neuronal-specific function of CaV1.2 channels might arise from brain-specific alternative splicing. The N- terminus of CaV1.2 is encoded by alternative first exons;exon 1a (e1a) is the cardiac isoform and exon1b (e1b) has a broad expression pattern and is the only isoform identified to date in neuronal tissue. Preliminary data suggest that in the presence of the splicin factor Nova-2, e1a is repressed in brain and therefore promotes e1b expression. I hypothesize that e1b is essential for facilitating intracellular calcium signaling through the MAPK pathway to induce activity-dependent gene expression. Preliminary RT-PCR experiments demonstrate aberrant inclusion of e1a-CaV1.2 in Nova-2 -/- brain tissue but not in wild-type (WT). I hypothesize that Nova-2 represses the cardiac isoform (e1a-CaV1.2) in brain. Aim 1 will characterize e1a and e1b expression in specific WT and Nova-2 -/- tissues in the nervous system (cerebellum, cortex, and hippocampus) and cardiac tissue through RT-PCR. Ribonuclease protection assays will be used to quantify expression levels. I hypothesize that the switch in splicing in Nova-2 -/- neurons will lead to a decrease in e1b containing transcripts and an increase in e1a transcripts. Nova-2 cDNA will be transfected into Nova-2 -/- neurons to examine if Nova-2 represses e1a expression. These experiments will determine if Nova-2 is both necessary and sufficient to repress e1a in brain. Aim 2 will address how e1a and e1b differentially affect the neuronal function of CaV1.2, to induce activity dependent gene expression. First, I will analyze the electrophysiological properties (such as current densities and activation/inactivation kinetics) of the e1a/e1b CaV1.2 isoforms transfected into tsA201 cells. I do not expect there to be a difference in channel properties, because I suspect the difference between the isoforms instead lies in the induction of the calcium signaling pathway necessary for activity-dependent gene expression. To address this, I will use a KCl depolarization assay to examine isoform efficiency at coupling membrane depolarization to CREB phosphorylation, a hallmark of activation of the MAPK pathway by L-type channels. I will use siRNA and injection of isoform specific cDNA to manipulate levels of e1a and e1b isoforms in WT neurons to specifically link differences observed in CREB phosphorylation to differences in e1a/e1b expression and not another alternatively spliced exon in CaV1.2 or other transcripts regulated by Nova-2. I hypothesize that the e1b isoform will more efficiently induce CREB phosphorylation. These experiments will advance our understanding of how neuronal specific isoforms of CaV1.2 contribute to essential neuronal functions.
Funding Period: 2012-09-16 - 2014-09-15
more information: NIH RePORT

Detail Information

Research Grants30

  1. Signaling Processes Underlying Cardiovascular Function
    Jeffrey Robbins; Fiscal Year: 2013
    ..These projects are supported by 3 Cores: Core A: The Administrative Core;Core B: The Physiology Core and Core C: The Imaging-Cell Culture Core. (End of Abstract) ..
  2. Prepubertal Stress, Windows of Risk &Sex Bias for Affective Disturbance
    C NEILL NEILL EPPERSON; Fiscal Year: 2013
    ..The Center would provide an intellectual platform with important resources to encourage established investigators, and their mentees, to consider sex and gender as crucial factors in their research. ..
  3. Intellectual and Development Disabilities Research Center
    Marc Yudkoff; Fiscal Year: 2013
    ..5 million from NICHD). The Center includes an excess of 70 Penn faculty at 15 departments at the Schools of Medicine, Veterinary Medicine, Nursing, the Wistar Institute, and the College of Arts and Sciences. ..
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    SCOTT THOMAS GRAFTON; Fiscal Year: 2013
    ..The collaborative effort can be expected to significantly advance our knowledge about mechanisms that support motor cortex plasticity. ..
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    Daniel A Beard; Fiscal Year: 2013
    ..This proposal targets the grand challenge of understanding complex multi-faceted disease phenotypes through experiments and simulations that capture the complex genotype-environment-phenotype relationship. ..
  6. CARDIAC ENERGY METABOLISM IN HEART FAILURE
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    ..In addition, there will be four Cores (Administrative, Large Animal/Histology, Metabolism, and Mitochondria/Mass Spec). ..
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  8. (INTRODUCTION) MECHANISMS OF ACCLIMATIZATION: FETUS AND ADULT
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  9. Role of Sphingolipids in the Pathobiology of Lung Injury
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    ..Together, this PPG addresses critical needs (insights, biomarkers, therapies) in ALI and RILI facilitating development of pharmacogenomic assays and SIP-based therapies for inflammatory lung injury. ..
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  11. Vermont Center on Behavior and Health
    Stephen T Higgins; Fiscal Year: 2013
    ..S. public health. ..
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    Yi Xing; Fiscal Year: 2013
    ..These studies will provide significant insight into how splicing is regulated, and how genetic variations disrupt splicing in human diseases. ..
  13. EARLY EVENTS IN ALZHEIMER PATHOGENESIS
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    ..The synergy between our aims, approaches, and measures will enable us to meet our goal of defining early cellular interactions toward development of rational interventions in AD. ..
  14. A Gene therapeutic approach to stable suppression of HIV-1 replication
    MICHAEL R FARZAN; Fiscal Year: 2013
    ..These studies will establish principles and protocols directly applicable to subsequent human clinical trials. ..
  15. Alzheimer's Disease Research Center
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    ..It will provide an environment and core resources to enhance research, foster professional and community training, and coordinate interdisciplinary research. ..
  16. IPF Fibroblast Phenotype
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    ..A major objective of this Program Project is to inform decisions of the IPF Clinical Network by providing information that can be translated into novel therapeutic strategies for IPF. ..
  17. Plasticity at the Excitatory Synapse
    Richard L Huganir; Fiscal Year: 2013
    ..Understanding these basic mechanisms of synaptic transmission and plasticity will provide insight into normal and abnormal brain function. ..
  18. Strategies for Improved Shock Wave Lithotripsy
    JAMES ALEXANDER MCATEER; Fiscal Year: 2013
    ..and the session can be ended * Determine the mechanism by which cavitation within a vessel causes hemorrhage * Develop numerical models to understand the role of cavitation and non-cavitational mechanisms in causing tissue damage ..
  19. Epigenetic and Transcriptional Dysregulation in Autism Spectrum Disorder
    Daniel H Geschwind; Fiscal Year: 2013
    ..In addition, these data will be made available to the community in a web-based format to be of maximum utility. ..
  20. Proliferation, Specification &Brain Function
    MARGARET ELIZABETH ROSS; Fiscal Year: 2013
    ..This Program tackles this complexity through the combined efforts of 4 Pis using cutting edge approaches to the elucidation of how developmental signals regulate fate and output of these critically important neurons. ..