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Flow fluorometry quantification of anion channel VRAC subunit LRRC8A at the membrane of living U937 cells

Assessing the expression of channels on the cell membrane is a necessary step in studying the functioning of ion channels in living cells. We explore, first, if endogenous VRAC can be assayed using flow cytometry and a commercially available antibody against an extracellular loop of the LRRC8A, also...

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Autores principales: Yurinskaya, Valentina, Aksenov, Nikolay, Moshkov, Alexey, Goryachaya, Tatyana, Shemery, Ashley, Vereninov, Alexey
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Taylor & Francis 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7039630/
https://www.ncbi.nlm.nih.gov/pubmed/32075501
http://dx.doi.org/10.1080/19336950.2020.1730535
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author Yurinskaya, Valentina
Aksenov, Nikolay
Moshkov, Alexey
Goryachaya, Tatyana
Shemery, Ashley
Vereninov, Alexey
author_facet Yurinskaya, Valentina
Aksenov, Nikolay
Moshkov, Alexey
Goryachaya, Tatyana
Shemery, Ashley
Vereninov, Alexey
author_sort Yurinskaya, Valentina
collection PubMed
description Assessing the expression of channels on the cell membrane is a necessary step in studying the functioning of ion channels in living cells. We explore, first, if endogenous VRAC can be assayed using flow cytometry and a commercially available antibody against an extracellular loop of the LRRC8A, also known as SWELL1, subunit of the VRAC channel. The second goal is to determine if an increase in the number of VRAC channels at the cell membrane is responsible for an increase in chloride permeability of the membrane in two well-known cases: during staurosporine (STS)-induced apoptosis and after water balance disturbance caused by hypotonic medium. Human suspension lymphoid cells U937 were used as they are suitable for flow fluorometry and because we have recently studied their membrane chloride permeability during apoptosis. We found that surface expression of endogenous LRRC8A subunits can be quantified in living U937 cells using flow fluorometry with the Alomone Lab antibody. Further, we revealed that treatment of cells for 1 hour using STS or a hypotonic solution did not change the number of LRRC8A subunits to the extent that would correspond to changes in the membrane chloride permeability determined by ion content analysis. This indicates that prolonged increase in chloride permeability of the cell membrane during apoptotic cell shrinkage or cell volume regulation under hypotonicity in U937 cells occurs without altering cell surface expression of VRAC.
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spelling pubmed-70396302020-03-03 Flow fluorometry quantification of anion channel VRAC subunit LRRC8A at the membrane of living U937 cells Yurinskaya, Valentina Aksenov, Nikolay Moshkov, Alexey Goryachaya, Tatyana Shemery, Ashley Vereninov, Alexey Channels (Austin) Brief Report Assessing the expression of channels on the cell membrane is a necessary step in studying the functioning of ion channels in living cells. We explore, first, if endogenous VRAC can be assayed using flow cytometry and a commercially available antibody against an extracellular loop of the LRRC8A, also known as SWELL1, subunit of the VRAC channel. The second goal is to determine if an increase in the number of VRAC channels at the cell membrane is responsible for an increase in chloride permeability of the membrane in two well-known cases: during staurosporine (STS)-induced apoptosis and after water balance disturbance caused by hypotonic medium. Human suspension lymphoid cells U937 were used as they are suitable for flow fluorometry and because we have recently studied their membrane chloride permeability during apoptosis. We found that surface expression of endogenous LRRC8A subunits can be quantified in living U937 cells using flow fluorometry with the Alomone Lab antibody. Further, we revealed that treatment of cells for 1 hour using STS or a hypotonic solution did not change the number of LRRC8A subunits to the extent that would correspond to changes in the membrane chloride permeability determined by ion content analysis. This indicates that prolonged increase in chloride permeability of the cell membrane during apoptotic cell shrinkage or cell volume regulation under hypotonicity in U937 cells occurs without altering cell surface expression of VRAC. Taylor & Francis 2020-02-20 /pmc/articles/PMC7039630/ /pubmed/32075501 http://dx.doi.org/10.1080/19336950.2020.1730535 Text en © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. https://creativecommons.org/licenses/by/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Brief Report
Yurinskaya, Valentina
Aksenov, Nikolay
Moshkov, Alexey
Goryachaya, Tatyana
Shemery, Ashley
Vereninov, Alexey
Flow fluorometry quantification of anion channel VRAC subunit LRRC8A at the membrane of living U937 cells
title Flow fluorometry quantification of anion channel VRAC subunit LRRC8A at the membrane of living U937 cells
title_full Flow fluorometry quantification of anion channel VRAC subunit LRRC8A at the membrane of living U937 cells
title_fullStr Flow fluorometry quantification of anion channel VRAC subunit LRRC8A at the membrane of living U937 cells
title_full_unstemmed Flow fluorometry quantification of anion channel VRAC subunit LRRC8A at the membrane of living U937 cells
title_short Flow fluorometry quantification of anion channel VRAC subunit LRRC8A at the membrane of living U937 cells
title_sort flow fluorometry quantification of anion channel vrac subunit lrrc8a at the membrane of living u937 cells
topic Brief Report
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7039630/
https://www.ncbi.nlm.nih.gov/pubmed/32075501
http://dx.doi.org/10.1080/19336950.2020.1730535
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