Regulation of presynaptic Ca2+ channel abundance at active zones through a balance of delivery and turnover

  1. Karen L Cunningham
  2. Chad W Sauvola
  3. Sara Tavana
  4. J Troy Littleton  Is a corresponding author
  1. Massachusetts Institute of Technology, United States

Abstract

Voltage-gated Ca2+ channels (VGCCs) mediate Ca2+ influx to trigger neurotransmitter release at specialized presynaptic sites termed active zones (AZs). The abundance of VGCCs at AZs regulates neurotransmitter release probability (Pr), a key presynaptic determinant of synaptic strength. Although biosynthesis, delivery and recycling cooperate to establish AZ VGCC abundance, experimentally isolating these distinct regulatory processes has been difficult. Here we describe how the AZ levels of Cacophony (Cac), the sole VGCC mediating synaptic transmission in Drosophila, are determined. We also analyzed the relationship between Cac, the conserved VGCC regulatory subunit α2δ, and the core AZ scaffold protein Bruchpilot (BRP) in establishing a functional AZ. We find Cac and BRP are independently regulated at growing AZs, as Cac is dispensable for AZ formation and structural maturation, and BRP abundance is not limiting for Cac accumulation. Additionally, AZs stop accumulating Cac after an initial growth phase, whereas BRP levels continue to increase given extended developmental time. AZ Cac is also buffered against moderate increases or decreases in biosynthesis, whereas BRP lacks this buffering. To probe mechanisms that determine AZ Cac abundance, intravital FRAP and Cac photoconversion were used to separately measure delivery and turnover at individual AZs over a multi-day period. Cac delivery occurs broadly across the AZ population, correlates with AZ size, and is rate-limited by α2δ. Although Cac does not undergo significant lateral transfer between neighboring AZs over the course of development, Cac removal from AZs does occur and is promoted by new Cac delivery, generating a cap on Cac accumulation at mature AZs. Together these findings reveal how Cac biosynthesis, synaptic delivery, and recycling set the abundance of VGCCs at individual AZs throughout synapse development and maintenance.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting file; Source Data files have been provided for Figures 1 to 7.

Article and author information

Author details

  1. Karen L Cunningham

    The Picower Institute for Learning and Memory, Massachusetts Institute of Technology, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Chad W Sauvola

    The Picower Institute for Learning and Memory, Massachusetts Institute of Technology, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Sara Tavana

    The Picower Institute for Learning and Memory, Massachusetts Institute of Technology, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. J Troy Littleton

    The Picower Institute for Learning and Memory, Massachusetts Institute of Technology, Cambridge, United States
    For correspondence
    troy@mit.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5576-2887

Funding

National Institute of Mental Health (MH104536)

  • J Troy Littleton

National Institute of Neurological Disorders and Stroke (NS117588)

  • J Troy Littleton

National Institutes of Health (T32GM007287)

  • Karen L Cunningham

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

Reviewing Editor

  1. P Robin Hiesinger, Institute for Biology Free University Berlin, Germany

Version history

  1. Received: March 15, 2022
  2. Preprint posted: March 16, 2022 (view preprint)
  3. Accepted: July 13, 2022
  4. Accepted Manuscript published: July 14, 2022 (version 1)
  5. Version of Record published: August 4, 2022 (version 2)

Copyright

© 2022, Cunningham 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.

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  1. Karen L Cunningham
  2. Chad W Sauvola
  3. Sara Tavana
  4. J Troy Littleton
(2022)
Regulation of presynaptic Ca2+ channel abundance at active zones through a balance of delivery and turnover
eLife 11:e78648.
https://doi.org/10.7554/eLife.78648

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https://doi.org/10.7554/eLife.78648

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