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FETCH: A platform for high-throughput quantification of gap junction hemichannel docking

Elizabeth Ransey, Kirill Chesnov, View ORCID ProfileNenad Bursac, View ORCID ProfileKafui Dzirasa
doi: https://doi.org/10.1101/2021.06.07.447352
Elizabeth Ransey
1Dept. of Psychiatry and Behavioral Sciences, Duke University Medical Center, Durham, North Carolina 27710, USA
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Kirill Chesnov
2Dept. of Neurobiology, Duke University Medical Center, Durham, North Carolina 27710, USA
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Nenad Bursac
4Dept. of Biomedical Engineering, Duke University, Durham North Carolina 27708, USA
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Kafui Dzirasa
1Dept. of Psychiatry and Behavioral Sciences, Duke University Medical Center, Durham, North Carolina 27710, USA
2Dept. of Neurobiology, Duke University Medical Center, Durham, North Carolina 27710, USA
3Dept. of Neurosurgery, Duke University Medical Center, Durham, North Carolina 27710, USA
4Dept. of Biomedical Engineering, Duke University, Durham North Carolina 27708, USA
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  • For correspondence: kafui.dzirasa@duke.edu
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ABSTRACT

Gap junctions are membrane spanning channels that connect the cytoplasm of apposed cells, allowing for the passage of small molecules and ions. They are formed by the connexin (Cx) family of proteins which assemble into hexameric hemichannels on each cell and dock to create gap junctional channels between two cells. Despite importance of various Cx isoforms in human physiology and disease, available tools for screening and discriminating their interactions such as hemichannel compatibility, docking and permeability are limited. Here, we developed FETCH (flow enabled tracking of connexosomes in HEK cells), a method which utilizes the generation of annular gap junctions (connexosomes) as downstream indicators of hemichannel compatibility for intercellular docking. First, we show that fluorescent connexosomes create a cellular phenotype that is detectable by flow cytometry analysis. We then show that FETCH identifies homotypic and heterotypic docking of many single isoform connexin hemichannels. Finally, we demonstrate that FETCH captures the impact of disease-relevant connexin protein mutations on gap junction formation. Thus, we establish a new flow cytometry-based method that is amenable to the high-throughput classification of gap junction hemichannel docking.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted June 07, 2021.
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FETCH: A platform for high-throughput quantification of gap junction hemichannel docking
Elizabeth Ransey, Kirill Chesnov, Nenad Bursac, Kafui Dzirasa
bioRxiv 2021.06.07.447352; doi: https://doi.org/10.1101/2021.06.07.447352
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FETCH: A platform for high-throughput quantification of gap junction hemichannel docking
Elizabeth Ransey, Kirill Chesnov, Nenad Bursac, Kafui Dzirasa
bioRxiv 2021.06.07.447352; doi: https://doi.org/10.1101/2021.06.07.447352

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