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Genome Editing Reveals Idiosyncrasy of CNGA2 Ion Channel-Directed Antibody Immunoreactivity Toward Oxytocin

Presynaptic cGMP-gated ion (CNG) channels positively or negatively modulate neurotransmitter secretion as well as the strength of synaptic transmission. Zebrafish cGMP-gated ion channel, CNGA2a (a.k.a. CNGA5), was previously reported to be specifically enriched in synaptic terminals of zebrafish oxy...

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Autores principales: Blechman, Janna, Anbalagan, Savani, Matthews, Gary G., Levkowitz, Gil
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6158348/
https://www.ncbi.nlm.nih.gov/pubmed/30294598
http://dx.doi.org/10.3389/fcell.2018.00117
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author Blechman, Janna
Anbalagan, Savani
Matthews, Gary G.
Levkowitz, Gil
author_facet Blechman, Janna
Anbalagan, Savani
Matthews, Gary G.
Levkowitz, Gil
author_sort Blechman, Janna
collection PubMed
description Presynaptic cGMP-gated ion (CNG) channels positively or negatively modulate neurotransmitter secretion as well as the strength of synaptic transmission. Zebrafish cGMP-gated ion channel, CNGA2a (a.k.a. CNGA5), was previously reported to be specifically enriched in synaptic terminals of zebrafish oxytocin (OXT) neurons. This conclusion was based on immunoreactivity of a monoclonal antibody (mAb) clone L55/54, which was directed against the carboxy terminal tail of the CNGA2a. To study the role of CNGA2a in oxytocin neurons function, we generated zebrafish mutants of cnga2a, cnga2b and oxt genes using clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing. We show that mAb L55/54 specifically recognizes CNGA2a protein when expressed in heterologous cell culture system. Surprisingly, anti-CNGA2a immunoreactivity was not eliminated following knockout of either cnga2a, cnga2b or both. However, knockout of oxt resulted in total loss of anti-CNGA2a mAb immunoreactivity despite the lack of sequence and structural similarities between OXT and CNGA2a proteins. Our results provide a noteworthy lesson of differences in antibody immunoreactivity, which could only be revealed using specific genetic tools.
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spelling pubmed-61583482018-10-05 Genome Editing Reveals Idiosyncrasy of CNGA2 Ion Channel-Directed Antibody Immunoreactivity Toward Oxytocin Blechman, Janna Anbalagan, Savani Matthews, Gary G. Levkowitz, Gil Front Cell Dev Biol Cell and Developmental Biology Presynaptic cGMP-gated ion (CNG) channels positively or negatively modulate neurotransmitter secretion as well as the strength of synaptic transmission. Zebrafish cGMP-gated ion channel, CNGA2a (a.k.a. CNGA5), was previously reported to be specifically enriched in synaptic terminals of zebrafish oxytocin (OXT) neurons. This conclusion was based on immunoreactivity of a monoclonal antibody (mAb) clone L55/54, which was directed against the carboxy terminal tail of the CNGA2a. To study the role of CNGA2a in oxytocin neurons function, we generated zebrafish mutants of cnga2a, cnga2b and oxt genes using clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing. We show that mAb L55/54 specifically recognizes CNGA2a protein when expressed in heterologous cell culture system. Surprisingly, anti-CNGA2a immunoreactivity was not eliminated following knockout of either cnga2a, cnga2b or both. However, knockout of oxt resulted in total loss of anti-CNGA2a mAb immunoreactivity despite the lack of sequence and structural similarities between OXT and CNGA2a proteins. Our results provide a noteworthy lesson of differences in antibody immunoreactivity, which could only be revealed using specific genetic tools. Frontiers Media S.A. 2018-09-20 /pmc/articles/PMC6158348/ /pubmed/30294598 http://dx.doi.org/10.3389/fcell.2018.00117 Text en Copyright © 2018 Blechman, Anbalagan, Matthews and Levkowitz. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Cell and Developmental Biology
Blechman, Janna
Anbalagan, Savani
Matthews, Gary G.
Levkowitz, Gil
Genome Editing Reveals Idiosyncrasy of CNGA2 Ion Channel-Directed Antibody Immunoreactivity Toward Oxytocin
title Genome Editing Reveals Idiosyncrasy of CNGA2 Ion Channel-Directed Antibody Immunoreactivity Toward Oxytocin
title_full Genome Editing Reveals Idiosyncrasy of CNGA2 Ion Channel-Directed Antibody Immunoreactivity Toward Oxytocin
title_fullStr Genome Editing Reveals Idiosyncrasy of CNGA2 Ion Channel-Directed Antibody Immunoreactivity Toward Oxytocin
title_full_unstemmed Genome Editing Reveals Idiosyncrasy of CNGA2 Ion Channel-Directed Antibody Immunoreactivity Toward Oxytocin
title_short Genome Editing Reveals Idiosyncrasy of CNGA2 Ion Channel-Directed Antibody Immunoreactivity Toward Oxytocin
title_sort genome editing reveals idiosyncrasy of cnga2 ion channel-directed antibody immunoreactivity toward oxytocin
topic Cell and Developmental Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6158348/
https://www.ncbi.nlm.nih.gov/pubmed/30294598
http://dx.doi.org/10.3389/fcell.2018.00117
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