High-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology.

Incorporation of noncanonical amino acids (ncAAs) can endow proteins with novel functionalities, such as crosslinking or fluorescence. In ion channels, the function of these variants can be studied with great precision using standard electrophysiology, but this approach is typically labor intensive...

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Autores principales:	Nina Braun, Søren Friis, Christian Ihling, Andrea Sinz, Jacob Andersen, Stephan A Pless
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/be6c4a9da5ab4067909c632cb3b953e3
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spelling	oai:doaj.org-article:be6c4a9da5ab4067909c632cb3b953e32021-12-02T19:54:37ZHigh-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology.1544-91731545-788510.1371/journal.pbio.3001321https://doaj.org/article/be6c4a9da5ab4067909c632cb3b953e32021-09-01T00:00:00Zhttps://doi.org/10.1371/journal.pbio.3001321https://doaj.org/toc/1544-9173https://doaj.org/toc/1545-7885Incorporation of noncanonical amino acids (ncAAs) can endow proteins with novel functionalities, such as crosslinking or fluorescence. In ion channels, the function of these variants can be studied with great precision using standard electrophysiology, but this approach is typically labor intensive and low throughput. Here, we establish a high-throughput protocol to conduct functional and pharmacological investigations of ncAA-containing human acid-sensing ion channel 1a (hASIC1a) variants in transiently transfected mammalian cells. We introduce 3 different photocrosslinking ncAAs into 103 positions and assess the function of the resulting 309 variants with automated patch clamp (APC). We demonstrate that the approach is efficient and versatile, as it is amenable to assessing even complex pharmacological modulation by peptides. The data show that the acidic pocket is a major determinant for current decay, and live-cell crosslinking provides insight into the hASIC1a-psalmotoxin 1 (PcTx1) interaction. Further, we provide evidence that the protocol can be applied to other ion channels, such as P2X2 and GluA2 receptors. We therefore anticipate the approach to enable future APC-based studies of ncAA-containing ion channels in mammalian cells.Nina BraunSøren FriisChristian IhlingAndrea SinzJacob AndersenStephan A PlessPublic Library of Science (PLoS)articleBiology (General)QH301-705.5ENPLoS Biology, Vol 19, Iss 9, p e3001321 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Biology (General) QH301-705.5
spellingShingle	Biology (General) QH301-705.5 Nina Braun Søren Friis Christian Ihling Andrea Sinz Jacob Andersen Stephan A Pless High-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology.
description	Incorporation of noncanonical amino acids (ncAAs) can endow proteins with novel functionalities, such as crosslinking or fluorescence. In ion channels, the function of these variants can be studied with great precision using standard electrophysiology, but this approach is typically labor intensive and low throughput. Here, we establish a high-throughput protocol to conduct functional and pharmacological investigations of ncAA-containing human acid-sensing ion channel 1a (hASIC1a) variants in transiently transfected mammalian cells. We introduce 3 different photocrosslinking ncAAs into 103 positions and assess the function of the resulting 309 variants with automated patch clamp (APC). We demonstrate that the approach is efficient and versatile, as it is amenable to assessing even complex pharmacological modulation by peptides. The data show that the acidic pocket is a major determinant for current decay, and live-cell crosslinking provides insight into the hASIC1a-psalmotoxin 1 (PcTx1) interaction. Further, we provide evidence that the protocol can be applied to other ion channels, such as P2X2 and GluA2 receptors. We therefore anticipate the approach to enable future APC-based studies of ncAA-containing ion channels in mammalian cells.
format	article
author	Nina Braun Søren Friis Christian Ihling Andrea Sinz Jacob Andersen Stephan A Pless
author_facet	Nina Braun Søren Friis Christian Ihling Andrea Sinz Jacob Andersen Stephan A Pless
author_sort	Nina Braun
title	High-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology.
title_short	High-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology.
title_full	High-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology.
title_fullStr	High-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology.
title_full_unstemmed	High-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology.
title_sort	high-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology.
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/be6c4a9da5ab4067909c632cb3b953e3
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High-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology.

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