β2-subunit alternative splicing stabilizes Cav2.3 Ca2+ channel activity during continuous midbrain dopamine neuron-like activity

Abstract

In dopaminergic (DA) substantia nigra (SN) neurons Cav2.3 R-type Ca2+-currents contribute to somatodendritic Ca2+-oscillations. This activity may contribute to the selective degeneration of these neurons in Parkinson's disease (PD) since Cav2.3-knockout is neuroprotective in a PD mouse model. Here we show that in tsA-201-cells the membrane-anchored β2-splice variants β2a and β2e are required to stabilize Cav2.3 gating properties allowing sustained Cav2.3 availability during simulated pacemaking and enhanced Ca2+-currents during bursts. We confirmed the expression of β2a- and β2e-subunit transcripts in the mouse SN and in identified SN DA neurons. Patch-clamp recordings of mouse DA midbrain neurons in culture and SN DA neurons in brain slices revealed SNX-482-sensitive R-type Ca2+-currents with voltage-dependent gating properties that suggest modulation by β2a- and/or β2e-subunits. Thus, β-subunit alternative splicing may prevent a fraction of Cav2.3 channels from inactivation in continuously active, highly vulnerable SN DA neurons, thereby also supporting Ca2+ signals contributing to the (patho)physiological role of Cav2.3 channels in PD.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files. Raw data have been provided for mean population data shown in Figures and Tables.

The following previously published data sets were used

Article and author information

Author details

  1. Anita Siller

    Department of Pharmacology and Toxicology, University of Innsbruck, Innsbruck, Austria
    Competing interests
    The authors declare that no competing interests exist.
  2. Nadja T Hofer

    Department of Pharmacology and Toxicology, University of Innsbruck, Innsbruck, Austria
    Competing interests
    The authors declare that no competing interests exist.
  3. Giulia Tomagra

    Department of Drug Science, University of Torino, Torino, Italy
    Competing interests
    The authors declare that no competing interests exist.
  4. Nicole Wiederspohn

    Institute of Applied Physiology, University of Ulm, Ulm, Germany
    Competing interests
    The authors declare that no competing interests exist.
  5. Simon Hess

    Institute for Zoology, Biocenter, University of Cologne, Cologne, Germany
    Competing interests
    The authors declare that no competing interests exist.
  6. Julia Benkert

    Institute of Applied Physiology, University of Ulm, Ulm, Germany
    Competing interests
    The authors declare that no competing interests exist.
  7. Aisylu Gaifullina

    Institute of Applied Physiology, University of Ulm, Ulm, Germany
    Competing interests
    The authors declare that no competing interests exist.
  8. Desiree Spaich

    Institute of Applied Physiology, University of Ulm, Ulm, Germany
    Competing interests
    The authors declare that no competing interests exist.
  9. Johanna Duda

    Institute of Applied Physiology, University of Ulm, Ulm, Germany
    Competing interests
    The authors declare that no competing interests exist.
  10. Christina Poetschke

    Institute of Applied Physiology, University of Ulm, Ulm, Germany
    Competing interests
    The authors declare that no competing interests exist.
  11. Kristina Vilusic

    Department of Pharmacology and Toxicology, University of Innsbruck, Innsbruck, Austria
    Competing interests
    The authors declare that no competing interests exist.
  12. Eva Maria Fritz

    Department of Pharmacology and Toxicology, University of Innsbruck, Innsbruck, Austria
    Competing interests
    The authors declare that no competing interests exist.
  13. Toni Schneider

    Institute of Neurophysiology, University of Cologne, Cologne, Germany
    Competing interests
    The authors declare that no competing interests exist.
  14. Peter Kloppenburg

    Institute for Zoology, Biocenter, University of Cologne, Cologne, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4554-404X
  15. Birgit Liss

    Institute of Applied Physiology, University of Ulm, Ulm, Germany
    Competing interests
    The authors declare that no competing interests exist.
  16. Valentina Carabelli

    Department of Drug Science, University of Torino, Torino, Italy
    Competing interests
    The authors declare that no competing interests exist.
  17. Emilio Carbone

    Department of Drug Science, University of Torino, Torino, Italy
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2239-6280
  18. Nadine Jasmin Ortner

    Department of Pharmacology and Toxicology, University of Innsbruck, Innsbruck, Austria
    For correspondence
    nadine.ortner@uibk.ac.at
    Competing interests
    The authors declare that no competing interests exist.
  19. Jörg Striessnig

    Department of Pharmacology and Toxicology, University of Innsbruck, Innsbruck, Austria
    For correspondence
    joerg.striessnig@uibk.ac.at
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9406-7120

Funding

Austrian Science Fund (P27809,P35722,CavX-DOC 30 doc.fund)

  • Jörg Striessnig

Tyrolean Science Fund (UNI-0404/2345)

  • Nadine Jasmin Ortner

Italian Miur (2015FNWP34)

  • Emilio Carbone

Compagnia di San Paolo (CSTO165284)

  • Emilio Carbone

Austrian Science Fund (P35087)

  • Nadine Jasmin Ortner

Hamburg Institute for Advanced Study (Research Fellowship)

  • Birgit Liss

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: All animal experiments and procedures were performed in strict accordance with the European Community's Council Directive 2010/63/UE and approved by the Italian Ministry of Health and the Local Organism responsible for animal welfare at the University of Torino (authorization DGSAF 0011710-P-26/07/2017) and the local authorities at the University of Ulm (Regierungspräsidium Tübingen, Ref: 35/9185.81-3; Reg. Nr. o.147) and University of Cologne (LANUV NRW, Recklinghausen, Germany (84-02.05.20.12.254).

Copyright

© 2022, Siller et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Anita Siller
  2. Nadja T Hofer
  3. Giulia Tomagra
  4. Nicole Wiederspohn
  5. Simon Hess
  6. Julia Benkert
  7. Aisylu Gaifullina
  8. Desiree Spaich
  9. Johanna Duda
  10. Christina Poetschke
  11. Kristina Vilusic
  12. Eva Maria Fritz
  13. Toni Schneider
  14. Peter Kloppenburg
  15. Birgit Liss
  16. Valentina Carabelli
  17. Emilio Carbone
  18. Nadine Jasmin Ortner
  19. Jörg Striessnig
(2022)
β2-subunit alternative splicing stabilizes Cav2.3 Ca2+ channel activity during continuous midbrain dopamine neuron-like activity
eLife 11:e67464.
https://doi.org/10.7554/eLife.67464

Share this article

https://doi.org/10.7554/eLife.67464

Further reading

    1. Neuroscience
    Jian Dong, Mian Chen ... Matthijs Verhage
    Research Article

    Dense core vesicles (DCVs) transport and release various neuropeptides and neurotrophins that control diverse brain functions, but the DCV secretory pathway remains poorly understood. Here, we tested a prediction emerging from invertebrate studies about the crucial role of the intracellular trafficking GTPase Rab10, by assessing DCV exocytosis at single-cell resolution upon acute Rab10 depletion in mature mouse hippocampal neurons, to circumvent potential confounding effects of Rab10’s established role in neurite outgrowth. We observed a significant inhibition of DCV exocytosis in Rab10-depleted neurons, whereas synaptic vesicle exocytosis was unaffected. However, rather than a direct involvement in DCV trafficking, this effect was attributed to two ER-dependent processes, ER-regulated intracellular Ca2+ dynamics, and protein synthesis. Gene Ontology analysis of differentially expressed proteins upon Rab10 depletion identified substantial alterations in synaptic and ER/ribosomal proteins, including the Ca2+ pump SERCA2. In addition, ER morphology and dynamics were altered, ER Ca2+ levels were depleted, and Ca2+ homeostasis was impaired in Rab10-depleted neurons. However, Ca2+ entry using a Ca2+ ionophore still triggered less DCV exocytosis. Instead, leucine supplementation, which enhances protein synthesis, largely rescued DCV exocytosis deficiency. We conclude that Rab10 is required for neuropeptide release by maintaining Ca2+ dynamics and regulating protein synthesis. Furthermore, DCV exocytosis appeared more dependent on (acute) protein synthesis than synaptic vesicle exocytosis.

    1. Neuroscience
    Brian C Ruyle, Sarah Masud ... Jose A Morón
    Research Article

    Millions of Americans suffering from Opioid Use Disorders face a high risk of fatal overdose due to opioid-induced respiratory depression (OIRD). Fentanyl, a powerful synthetic opioid, is a major contributor to the rising rates of overdose deaths. Reversing fentanyl overdoses has proved challenging due to its high potency and the rapid onset of OIRD. We assessed the contributions of central and peripheral mu opioid receptors (MORs) in mediating fentanyl-induced physiological responses. The peripherally restricted MOR antagonist naloxone methiodide (NLXM) both prevented and reversed OIRD to a degree comparable to that of naloxone (NLX), indicating substantial involvement of peripheral MORs to OIRD. Interestingly, NLXM-mediated OIRD reversal did not produce aversive behaviors observed after NLX. We show that neurons in the nucleus of the solitary tract (nTS), the first central synapse of peripheral afferents, exhibit a biphasic activity profile following fentanyl exposure. NLXM pretreatment attenuates this activity, suggesting that these responses are mediated by peripheral MORs. Together, these findings establish a critical role for peripheral MORs, including ascending inputs to the nTS, as sites of dysfunction during OIRD. Furthermore, selective peripheral MOR antagonism could be a promising therapeutic strategy for managing OIRD by sparing CNS-driven acute opioid-associated withdrawal and aversion observed after NLX.