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Protein Kinase C and Calmodulin Serve As Calcium Sensors for Calcium-Stimulated Endocytosis at Synapses.

Authors
  • Jin, Ying-Hui1, 2
  • Wu, Xin-Sheng1
  • Shi, Bo1, 3
  • Zhang, Zhen1
  • Guo, Xiaoli1
  • Gan, Lin4
  • Chen, Zhongqing2
  • Wu, Ling-Gang5
  • 1 National Institute of Neurological Disorders and Stroke, Bethesda, Maryland 20892.
  • 2 Department of Critical Care Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, China 510515. , (China)
  • 3 Biological Sciences Graduate Program, College of Computer, Mathematical, and Natural Sciences, University of Maryland, College Park, Maryland 20740, and.
  • 4 Flaum Eye Institute, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642.
  • 5 National Institute of Neurological Disorders and Stroke, Bethesda, Maryland 20892, [email protected]
Type
Published Article
Journal
Journal of Neuroscience
Publisher
Society for Neuroscience
Publication Date
Nov 27, 2019
Volume
39
Issue
48
Pages
9478–9490
Identifiers
DOI: 10.1523/JNEUROSCI.0182-19.2019
PMID: 31628181
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

Calcium influx triggers and facilitates endocytosis, which recycles vesicles and thus sustains synaptic transmission. Despite decades of studies, the underlying calcium sensor remained not well understood. Here, we examined two calcium binding proteins, protein kinase C (PKC) and calmodulin. Whether PKC is involved in endocytosis was unclear; whether calmodulin acts as a calcium sensor for endocytosis was neither clear, although calmodulin involvement in endocytosis had been suggested. We generated PKC (α or β-isoform) and calmodulin (calmodulin 2 gene) knock-out mice of either sex and measured endocytosis with capacitance measurements, pHluorin imaging and electron microscopy. We found that these knock-outs inhibited slow (∼10-30 s) and rapid (<∼3 s) endocytosis at large calyx-type calyces, and inhibited slow endocytosis and bulk endocytosis (forming large endosome-like structures) at small conventional hippocampal synapses, suggesting the involvement of PKC and calmodulin in three most common forms of endocytosis-the slow, rapid and bulk endocytosis. Inhibition of slow endocytosis in PKC or calmodulin 2 knock-out hippocampal synapses was rescued by overexpressing wild-type PKC or calmodulin, but not calcium-binding-deficient PKC or calmodulin mutant, respectively, suggesting that calcium stimulates endocytosis by binding with its calcium sensor PKC and calmodulin. PKC and calmodulin 2 knock-out inhibited calcium-dependent vesicle mobilization to the readily releasable pool, suggesting that PKC and calmodulin may mediate calcium-dependent facilitation of vesicle mobilization. These findings shed light on the molecular signaling link among calcium, endocytosis and vesicle mobilization that are crucial in maintaining synaptic transmission and neuronal network activity.SIGNIFICANCE STATEMENT Vesicle fusion releases neurotransmitters to mediate synaptic transmission. To sustain synaptic transmission, fused vesicles must be retrieved via endocytosis. Accumulating evidence suggests that calcium influx triggers synaptic vesicle endocytosis. However, how calcium triggers endocytosis is not well understood. Using genetic tools together with capacitance measurements, optical imaging and electron microscopy, we identified two calcium sensors, including protein kinase C (α and β isoforms) and calmodulin, for the most commonly observed forms of endocytosis: slow, rapid, and bulk. We also found that these two proteins are involved in calcium-dependent vesicle mobilization to the readily releasable pool. These results provide the molecular signaling link among calcium, endocytosis, and vesicle mobilization that are essential in sustaining synaptic transmission and neuronal network activity. Copyright © 2019 the authors.

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