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Charge and lipophilicity are required for effective block of the hair-cell mechano-electrical transducer channel by FM1-43 and its derivatives

Marco Derudas, Molly O’Reilly, Nerissa K. Kirkwood, Emma Kenyon Orcid Logo, Sybil Grimsey, Siân R. Kitcher, Shawna Workman, James Bull Orcid Logo, Simon E. Ward, Corné J. Kros, Guy P. Richardson

Frontiers in Cell and Developmental Biology, Volume: 11

Swansea University Authors: Emma Kenyon Orcid Logo, Shawna Workman, James Bull Orcid Logo

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DOI (Published version): 10.3389/fcell.2023.1247324

Abstract

The styryl dye FM1-43 is widely used to study endocytosis but behaves as a permeant blocker of the mechano-electrical transducer (MET) channel in sensory hair cells, loading rapidly and specifically into the cytoplasm of hair cells in a MET channel-dependent manner. Patch clamp recordings of mouse o...

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Published in: Frontiers in Cell and Developmental Biology
ISSN: 2296-634X
Published: Frontiers Media SA 2023
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URI: https://cronfa.swan.ac.uk/Record/cronfa65141
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Patch clamp recordings of mouse outer hair cells (OHCs) were used to determine how a series of structural modifications of FM1-43 affect MET channel block. Fluorescence microscopy was used to assess how the modifications influence hair-cell loading in mouse cochlear cultures and zebrafish neuromasts. Cochlear cultures were also used to evaluate otoprotective potential of the modified FM1-43 derivatives. Structure-activity relationships reveal that the lipophilic tail and the cationic head group of FM1-43 are both required for MET channel block in mouse cochlear OHCs; neither moiety alone is sufficient. The extent of MET channel block is augmented by increasing the lipophilicity/bulkiness of the tail, by reducing the number of positive charges in the head group from two to one, or by increasing the distance between the two charged head groups. Loading assays with zebrafish neuromasts and mouse cochlear cultures are broadly in accordance with these observations but reveal a loss of hair-cell specific labelling with increasing lipophilicity. Although FM1-43 and many of its derivatives are generally cytotoxic when tested on cochlear cultures in the presence of an equimolar concentration of the ototoxic antibiotic gentamicin (5 µM), at a 10-fold lower concentration (0.5 µM), two of the derivatives protect OHCs from cell death caused by 48 h-exposure to 5 µM gentamicin.</abstract><type>Journal Article</type><journal>Frontiers in Cell and Developmental Biology</journal><volume>11</volume><journalNumber/><paginationStart/><paginationEnd/><publisher>Frontiers Media SA</publisher><placeOfPublication/><isbnPrint/><isbnElectronic/><issnPrint/><issnElectronic>2296-634X</issnElectronic><keywords>FM1-43, mechano-electrical transducer channel, hair cell, zebrafish, hearing, ototoxicity</keywords><publishedDay>10</publishedDay><publishedMonth>10</publishedMonth><publishedYear>2023</publishedYear><publishedDate>2023-10-10</publishedDate><doi>10.3389/fcell.2023.1247324</doi><url>http://dx.doi.org/10.3389/fcell.2023.1247324</url><notes/><college>COLLEGE NANME</college><department>Biomedical Sciences</department><CollegeCode>COLLEGE CODE</CollegeCode><DepartmentCode>BMS</DepartmentCode><institution>Swansea University</institution><apcterm/><funders>This study was funded by Medical Research Council (MRC) program grant MR/K005561/1 to CK, GR, SW, and Anthony Moore. 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spelling v2 65141 2023-11-28 Charge and lipophilicity are required for effective block of the hair-cell mechano-electrical transducer channel by FM1-43 and its derivatives 8f07d20c6cb93623521101c62c4e4eb3 0000-0002-3898-1866 Emma Kenyon Emma Kenyon true false ec1581e2457be9c8cb0ba977328b3a64 Shawna Workman Shawna Workman true false 20742518482c020c80b81b88e5313356 0000-0002-4373-6830 James Bull James Bull true false 2023-11-28 BMS The styryl dye FM1-43 is widely used to study endocytosis but behaves as a permeant blocker of the mechano-electrical transducer (MET) channel in sensory hair cells, loading rapidly and specifically into the cytoplasm of hair cells in a MET channel-dependent manner. Patch clamp recordings of mouse outer hair cells (OHCs) were used to determine how a series of structural modifications of FM1-43 affect MET channel block. Fluorescence microscopy was used to assess how the modifications influence hair-cell loading in mouse cochlear cultures and zebrafish neuromasts. Cochlear cultures were also used to evaluate otoprotective potential of the modified FM1-43 derivatives. Structure-activity relationships reveal that the lipophilic tail and the cationic head group of FM1-43 are both required for MET channel block in mouse cochlear OHCs; neither moiety alone is sufficient. The extent of MET channel block is augmented by increasing the lipophilicity/bulkiness of the tail, by reducing the number of positive charges in the head group from two to one, or by increasing the distance between the two charged head groups. Loading assays with zebrafish neuromasts and mouse cochlear cultures are broadly in accordance with these observations but reveal a loss of hair-cell specific labelling with increasing lipophilicity. Although FM1-43 and many of its derivatives are generally cytotoxic when tested on cochlear cultures in the presence of an equimolar concentration of the ototoxic antibiotic gentamicin (5 µM), at a 10-fold lower concentration (0.5 µM), two of the derivatives protect OHCs from cell death caused by 48 h-exposure to 5 µM gentamicin. Journal Article Frontiers in Cell and Developmental Biology 11 Frontiers Media SA 2296-634X FM1-43, mechano-electrical transducer channel, hair cell, zebrafish, hearing, ototoxicity 10 10 2023 2023-10-10 10.3389/fcell.2023.1247324 http://dx.doi.org/10.3389/fcell.2023.1247324 COLLEGE NANME Biomedical Sciences COLLEGE CODE BMS Swansea University This study was funded by Medical Research Council (MRC) program grant MR/K005561/1 to CK, GR, SW, and Anthony Moore. SK was funded by a PhD studentship from RNID (S30). GR was the recipient of a Royal Society Wolfson Merit Award. SW was funded by a Swansea Paid Internship Network (SPIN) grant from Swansea University. 2024-01-03T15:25:29.6479087 2023-11-28T16:02:26.4872128 Faculty of Science and Engineering School of Biosciences, Geography and Physics - Biosciences Marco Derudas 1 Molly O’Reilly 2 Nerissa K. Kirkwood 3 Emma Kenyon 0000-0002-3898-1866 4 Sybil Grimsey 5 Siân R. Kitcher 6 Shawna Workman 7 James Bull 0000-0002-4373-6830 8 Simon E. Ward 9 Corné J. Kros 10 Guy P. Richardson 11 65141__29141__ed7238dac4bb4eb0babce57fab85a1ec.pdf fcell-11-1247324.pdf 2023-11-28T16:04:17.1218955 Output 4036506 application/pdf Version of Record true © 2023 Derudas, O’Reilly, Kirkwood, Kenyon, Grimsey, Kitcher, Workman, Bull, Ward, Kros and Richardson. Distributed under the terms of a Creative Commons Attribution 4.0 International License (CC BY 4.0). true eng https:// creativecommons.org/licenses/by/ 4.0/
title Charge and lipophilicity are required for effective block of the hair-cell mechano-electrical transducer channel by FM1-43 and its derivatives
spellingShingle Charge and lipophilicity are required for effective block of the hair-cell mechano-electrical transducer channel by FM1-43 and its derivatives
Emma Kenyon
Shawna Workman
James Bull
title_short Charge and lipophilicity are required for effective block of the hair-cell mechano-electrical transducer channel by FM1-43 and its derivatives
title_full Charge and lipophilicity are required for effective block of the hair-cell mechano-electrical transducer channel by FM1-43 and its derivatives
title_fullStr Charge and lipophilicity are required for effective block of the hair-cell mechano-electrical transducer channel by FM1-43 and its derivatives
title_full_unstemmed Charge and lipophilicity are required for effective block of the hair-cell mechano-electrical transducer channel by FM1-43 and its derivatives
title_sort Charge and lipophilicity are required for effective block of the hair-cell mechano-electrical transducer channel by FM1-43 and its derivatives
author_id_str_mv 8f07d20c6cb93623521101c62c4e4eb3
ec1581e2457be9c8cb0ba977328b3a64
20742518482c020c80b81b88e5313356
author_id_fullname_str_mv 8f07d20c6cb93623521101c62c4e4eb3_***_Emma Kenyon
ec1581e2457be9c8cb0ba977328b3a64_***_Shawna Workman
20742518482c020c80b81b88e5313356_***_James Bull
author Emma Kenyon
Shawna Workman
James Bull
author2 Marco Derudas
Molly O’Reilly
Nerissa K. Kirkwood
Emma Kenyon
Sybil Grimsey
Siân R. Kitcher
Shawna Workman
James Bull
Simon E. Ward
Corné J. Kros
Guy P. Richardson
format Journal article
container_title Frontiers in Cell and Developmental Biology
container_volume 11
publishDate 2023
institution Swansea University
issn 2296-634X
doi_str_mv 10.3389/fcell.2023.1247324
publisher Frontiers Media SA
college_str Faculty of Science and Engineering
hierarchytype
hierarchy_top_id facultyofscienceandengineering
hierarchy_top_title Faculty of Science and Engineering
hierarchy_parent_id facultyofscienceandengineering
hierarchy_parent_title Faculty of Science and Engineering
department_str School of Biosciences, Geography and Physics - Biosciences{{{_:::_}}}Faculty of Science and Engineering{{{_:::_}}}School of Biosciences, Geography and Physics - Biosciences
url http://dx.doi.org/10.3389/fcell.2023.1247324
document_store_str 1
active_str 0
description The styryl dye FM1-43 is widely used to study endocytosis but behaves as a permeant blocker of the mechano-electrical transducer (MET) channel in sensory hair cells, loading rapidly and specifically into the cytoplasm of hair cells in a MET channel-dependent manner. Patch clamp recordings of mouse outer hair cells (OHCs) were used to determine how a series of structural modifications of FM1-43 affect MET channel block. Fluorescence microscopy was used to assess how the modifications influence hair-cell loading in mouse cochlear cultures and zebrafish neuromasts. Cochlear cultures were also used to evaluate otoprotective potential of the modified FM1-43 derivatives. Structure-activity relationships reveal that the lipophilic tail and the cationic head group of FM1-43 are both required for MET channel block in mouse cochlear OHCs; neither moiety alone is sufficient. The extent of MET channel block is augmented by increasing the lipophilicity/bulkiness of the tail, by reducing the number of positive charges in the head group from two to one, or by increasing the distance between the two charged head groups. Loading assays with zebrafish neuromasts and mouse cochlear cultures are broadly in accordance with these observations but reveal a loss of hair-cell specific labelling with increasing lipophilicity. Although FM1-43 and many of its derivatives are generally cytotoxic when tested on cochlear cultures in the presence of an equimolar concentration of the ototoxic antibiotic gentamicin (5 µM), at a 10-fold lower concentration (0.5 µM), two of the derivatives protect OHCs from cell death caused by 48 h-exposure to 5 µM gentamicin.
published_date 2023-10-10T15:25:31Z
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