A new tool to sense pH changes at the neuromuscular junction synaptic cleft

Abstract Synaptic transmission triggers transient acidification of the synaptic cleft. Recently, it has been shown that pH affects the opening of postsynaptic channels and therefore the production of tools that allow to study these behaviors should result of paramount value. We fused α-bungarotoxin,...

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Autores principales:	M. Blaustein, S. Wirth, G. Saldaña, A. P. Piantanida, M. E. Bogetti, M. E. Martin, A. Colman-Lerner, O. D. Uchitel
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2020
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/982233e478104573b49aa6b8c65a3b46
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spelling	oai:doaj.org-article:982233e478104573b49aa6b8c65a3b462021-12-02T16:08:37ZA new tool to sense pH changes at the neuromuscular junction synaptic cleft10.1038/s41598-020-77154-32045-2322https://doaj.org/article/982233e478104573b49aa6b8c65a3b462020-11-01T00:00:00Zhttps://doi.org/10.1038/s41598-020-77154-3https://doaj.org/toc/2045-2322Abstract Synaptic transmission triggers transient acidification of the synaptic cleft. Recently, it has been shown that pH affects the opening of postsynaptic channels and therefore the production of tools that allow to study these behaviors should result of paramount value. We fused α-bungarotoxin, a neurotoxin derived from the snake Bungarus multicinctus that binds irreversibly to the acetylcholine receptor extracellular domain, to the pH sensitive GFP Super Ecliptic pHluorin, and efficiently expressed it in Pichia pastoris. This sensor allows synaptic changes in pH to be measured without the need of incorporating transgenes into animal cells. Here, we show that incubation of the mouse levator auris muscle with a solution containing this recombinant protein is enough to fluorescently label the endplate post synaptic membrane. Furthermore, we could physiologically alter and measure post synaptic pH by evaluating changes in the fluorescent signal of pHluorin molecules bound to acetylcholine receptors. In fact, using this tool we were able to detect a drop in 0.01 to 0.05 pH units in the vicinity of the acetylcholine receptors following vesicle exocytosis triggered by nerve electrical stimulation. Further experiments will allow to learn the precise changes in pH during and after synaptic activation.M. BlausteinS. WirthG. SaldañaA. P. PiantanidaM. E. BogettiM. E. MartinA. Colman-LernerO. D. UchitelNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 10, Iss 1, Pp 1-7 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q M. Blaustein S. Wirth G. Saldaña A. P. Piantanida M. E. Bogetti M. E. Martin A. Colman-Lerner O. D. Uchitel A new tool to sense pH changes at the neuromuscular junction synaptic cleft
description	Abstract Synaptic transmission triggers transient acidification of the synaptic cleft. Recently, it has been shown that pH affects the opening of postsynaptic channels and therefore the production of tools that allow to study these behaviors should result of paramount value. We fused α-bungarotoxin, a neurotoxin derived from the snake Bungarus multicinctus that binds irreversibly to the acetylcholine receptor extracellular domain, to the pH sensitive GFP Super Ecliptic pHluorin, and efficiently expressed it in Pichia pastoris. This sensor allows synaptic changes in pH to be measured without the need of incorporating transgenes into animal cells. Here, we show that incubation of the mouse levator auris muscle with a solution containing this recombinant protein is enough to fluorescently label the endplate post synaptic membrane. Furthermore, we could physiologically alter and measure post synaptic pH by evaluating changes in the fluorescent signal of pHluorin molecules bound to acetylcholine receptors. In fact, using this tool we were able to detect a drop in 0.01 to 0.05 pH units in the vicinity of the acetylcholine receptors following vesicle exocytosis triggered by nerve electrical stimulation. Further experiments will allow to learn the precise changes in pH during and after synaptic activation.
format	article
author	M. Blaustein S. Wirth G. Saldaña A. P. Piantanida M. E. Bogetti M. E. Martin A. Colman-Lerner O. D. Uchitel
author_facet	M. Blaustein S. Wirth G. Saldaña A. P. Piantanida M. E. Bogetti M. E. Martin A. Colman-Lerner O. D. Uchitel
author_sort	M. Blaustein
title	A new tool to sense pH changes at the neuromuscular junction synaptic cleft
title_short	A new tool to sense pH changes at the neuromuscular junction synaptic cleft
title_full	A new tool to sense pH changes at the neuromuscular junction synaptic cleft
title_fullStr	A new tool to sense pH changes at the neuromuscular junction synaptic cleft
title_full_unstemmed	A new tool to sense pH changes at the neuromuscular junction synaptic cleft
title_sort	new tool to sense ph changes at the neuromuscular junction synaptic cleft
publisher	Nature Portfolio
publishDate	2020
url	https://doaj.org/article/982233e478104573b49aa6b8c65a3b46
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A new tool to sense pH changes at the neuromuscular junction synaptic cleft

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