Characterization of HA-tagged α9 and α10 nAChRs in the mouse cochlea

Abstract Neurons of the medial olivary complex inhibit cochlear hair cells through the activation of α9α10-containing nicotinic acetylcholine receptors (nAChRs). Efforts to study the localization of these proteins have been hampered by the absence of reliable antibodies. To overcome this obstacle, C...

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Autores principales:	Pankhuri Vyas, Megan Beers Wood, Yuanyuan Zhang, Adam C. Goldring, Fatima-Zahra Chakir, Paul Albert Fuchs, Hakim Hiel
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2020
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/2048d1e745d24753b2e1fe2748d7c4d1
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spelling	oai:doaj.org-article:2048d1e745d24753b2e1fe2748d7c4d12021-12-02T16:18:05ZCharacterization of HA-tagged α9 and α10 nAChRs in the mouse cochlea10.1038/s41598-020-78380-52045-2322https://doaj.org/article/2048d1e745d24753b2e1fe2748d7c4d12020-12-01T00:00:00Zhttps://doi.org/10.1038/s41598-020-78380-5https://doaj.org/toc/2045-2322Abstract Neurons of the medial olivary complex inhibit cochlear hair cells through the activation of α9α10-containing nicotinic acetylcholine receptors (nAChRs). Efforts to study the localization of these proteins have been hampered by the absence of reliable antibodies. To overcome this obstacle, CRISPR-Cas9 gene editing was used to generate mice in which a hemagglutinin tag (HA) was attached to the C-terminus of either α9 or α10 proteins. Immunodetection of the HA tag on either subunit in the organ of Corti of adult mice revealed immunopuncta clustered at the synaptic pole of outer hair cells. These puncta were juxtaposed to immunolabeled presynaptic efferent terminals. HA immunopuncta also occurred in inner hair cells of pre-hearing (P7) but not in adult mice. These immunolabeling patterns were similar for both homozygous and heterozygous mice. All HA-tagged genotypes had auditory brainstem responses not significantly different from those of wild type littermates. The activation of efferent neurons in heterozygous mice evoked biphasic postsynaptic currents not significantly different from those of wild type hair cells. However, efferent synaptic responses were significantly smaller and less frequent in the homozygous mice. We show that HA-tagged nAChRs introduced in the mouse by a CRISPR knock-in are regulated and expressed like the native protein, and in the heterozygous condition mediate normal synaptic function. The animals thus generated have clear advantages for localization studies.Pankhuri VyasMegan Beers WoodYuanyuan ZhangAdam C. GoldringFatima-Zahra ChakirPaul Albert FuchsHakim HielNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 10, Iss 1, Pp 1-15 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Pankhuri Vyas Megan Beers Wood Yuanyuan Zhang Adam C. Goldring Fatima-Zahra Chakir Paul Albert Fuchs Hakim Hiel Characterization of HA-tagged α9 and α10 nAChRs in the mouse cochlea
description	Abstract Neurons of the medial olivary complex inhibit cochlear hair cells through the activation of α9α10-containing nicotinic acetylcholine receptors (nAChRs). Efforts to study the localization of these proteins have been hampered by the absence of reliable antibodies. To overcome this obstacle, CRISPR-Cas9 gene editing was used to generate mice in which a hemagglutinin tag (HA) was attached to the C-terminus of either α9 or α10 proteins. Immunodetection of the HA tag on either subunit in the organ of Corti of adult mice revealed immunopuncta clustered at the synaptic pole of outer hair cells. These puncta were juxtaposed to immunolabeled presynaptic efferent terminals. HA immunopuncta also occurred in inner hair cells of pre-hearing (P7) but not in adult mice. These immunolabeling patterns were similar for both homozygous and heterozygous mice. All HA-tagged genotypes had auditory brainstem responses not significantly different from those of wild type littermates. The activation of efferent neurons in heterozygous mice evoked biphasic postsynaptic currents not significantly different from those of wild type hair cells. However, efferent synaptic responses were significantly smaller and less frequent in the homozygous mice. We show that HA-tagged nAChRs introduced in the mouse by a CRISPR knock-in are regulated and expressed like the native protein, and in the heterozygous condition mediate normal synaptic function. The animals thus generated have clear advantages for localization studies.
format	article
author	Pankhuri Vyas Megan Beers Wood Yuanyuan Zhang Adam C. Goldring Fatima-Zahra Chakir Paul Albert Fuchs Hakim Hiel
author_facet	Pankhuri Vyas Megan Beers Wood Yuanyuan Zhang Adam C. Goldring Fatima-Zahra Chakir Paul Albert Fuchs Hakim Hiel
author_sort	Pankhuri Vyas
title	Characterization of HA-tagged α9 and α10 nAChRs in the mouse cochlea
title_short	Characterization of HA-tagged α9 and α10 nAChRs in the mouse cochlea
title_full	Characterization of HA-tagged α9 and α10 nAChRs in the mouse cochlea
title_fullStr	Characterization of HA-tagged α9 and α10 nAChRs in the mouse cochlea
title_full_unstemmed	Characterization of HA-tagged α9 and α10 nAChRs in the mouse cochlea
title_sort	characterization of ha-tagged α9 and α10 nachrs in the mouse cochlea
publisher	Nature Portfolio
publishDate	2020
url	https://doaj.org/article/2048d1e745d24753b2e1fe2748d7c4d1
work_keys_str_mv	AT pankhurivyas characterizationofhataggeda9anda10nachrsinthemousecochlea AT meganbeerswood characterizationofhataggeda9anda10nachrsinthemousecochlea AT yuanyuanzhang characterizationofhataggeda9anda10nachrsinthemousecochlea AT adamcgoldring characterizationofhataggeda9anda10nachrsinthemousecochlea AT fatimazahrachakir characterizationofhataggeda9anda10nachrsinthemousecochlea AT paulalbertfuchs characterizationofhataggeda9anda10nachrsinthemousecochlea AT hakimhiel characterizationofhataggeda9anda10nachrsinthemousecochlea
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Characterization of HA-tagged α9 and α10 nAChRs in the mouse cochlea

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