Development of high-affinity nanobodies specific for NaV1.4 and NaV1.5 voltage-gated sodium channel isoforms

Autores
Srinivasan, Lakshmi; Alzogaray, Vanina Andrea; Selvakumar, Dakshnamurthy; Nathan, Sara; Yoder, Jesse B.; Wright, Katharine M.; Klinke, Sebastian; Nwafor, Justin N.; Labanda, María Soledad; Goldbaum, Fernando Alberto; Schön, Arne; Freire, Ernesto; Tomaselli, Gordon F.; Amzel, León Mario; Ben-Johny, Manu; Gabelli, Sandra
Año de publicación
2022
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Voltage-gated sodium channels, NaVs, are responsible for the rapid rise of action potentials in excitable tissues. NaV channel mutations have been implicated in several human genetic diseases, such as hypokalemic periodic paralysis, myotonia, and long-QT and Brugada syndromes. Here, we generated high-affinity anti-NaV nanobodies (Nbs), Nb17 and Nb82, that recognize the NaV1.4 (skeletal muscle) and NaV1.5 (cardiac muscle) channel isoforms. These Nbs were raised in llama (Lama glama) and selected from a phage display library for high affinity to the C-terminal (CT) region of NaV1.4. The Nbs were expressed in Escherichia coli, purified, and bio-physically characterized. Development of high-affinity Nbs specifically targeting a given human NaV isoform has been challenging because they usually show undesired cross-reactivity for different NaV isoforms. Our results show, however, that Nb17 and Nb82 recognize the CTNaV1.4 or CTNaV1.5 over other CTNav isoforms. Kinetic experiments by biolayer interferometry determined that Nb17 and Nb82 bind to the CTNaV1.4 and CTNaV1.5 with high affinity (KD ~ 40-60 nM). In addition, as proof of concept, we show that Nb82 could detect NaV1.4 and NaV1.5 channels in mammalian cells and tissues by Western blot. Furthermore, human embryonic kidney cells expressing holo NaV1.5 channels demonstrated a robust FRET-binding efficiency for Nb17 and Nb82. Our work lays the foundation for developing Nbs as anti-NaV reagents to capture NaVs from cell lysates and as molecular visualization agents for NaVs.
Fil: Srinivasan, Lakshmi. University Johns Hopkins; Estados Unidos
Fil: Alzogaray, Vanina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina
Fil: Selvakumar, Dakshnamurthy. Fortébio; Estados Unidos
Fil: Nathan, Sara. University Johns Hopkins; Estados Unidos
Fil: Yoder, Jesse B.. University Johns Hopkins; Estados Unidos
Fil: Wright, Katharine M.. University Johns Hopkins; Estados Unidos
Fil: Klinke, Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina
Fil: Nwafor, Justin N.. University Johns Hopkins; Estados Unidos
Fil: Labanda, María Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina
Fil: Goldbaum, Fernando Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina
Fil: Schön, Arne. University Johns Hopkins; Estados Unidos
Fil: Freire, Ernesto. University Johns Hopkins; Estados Unidos
Fil: Tomaselli, Gordon F.. University Johns Hopkins; Estados Unidos
Fil: Amzel, León Mario. University Johns Hopkins; Estados Unidos
Fil: Ben-Johny, Manu. Columbia University; Estados Unidos
Fil: Gabelli, Sandra. University Johns Hopkins; Estados Unidos
Materia
VOLTAGE-GATED SODIUM CHANNELS
NAVS
HIGH-AFFINITY ANTI-NAV NANOBODIES
ANTI-NAV REAGENTS
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/200892
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/200892
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Development of high-affinity nanobodies specific for NaV1.4 and NaV1.5 voltage-gated sodium channel isoformsSrinivasan, LakshmiAlzogaray, Vanina AndreaSelvakumar, DakshnamurthyNathan, SaraYoder, Jesse B.Wright, Katharine M.Klinke, SebastianNwafor, Justin N.Labanda, María SoledadGoldbaum, Fernando AlbertoSchön, ArneFreire, ErnestoTomaselli, Gordon F.Amzel, León MarioBen-Johny, ManuGabelli, SandraVOLTAGE-GATED SODIUM CHANNELSNAVSHIGH-AFFINITY ANTI-NAV NANOBODIESANTI-NAV REAGENTShttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Voltage-gated sodium channels, NaVs, are responsible for the rapid rise of action potentials in excitable tissues. NaV channel mutations have been implicated in several human genetic diseases, such as hypokalemic periodic paralysis, myotonia, and long-QT and Brugada syndromes. Here, we generated high-affinity anti-NaV nanobodies (Nbs), Nb17 and Nb82, that recognize the NaV1.4 (skeletal muscle) and NaV1.5 (cardiac muscle) channel isoforms. These Nbs were raised in llama (Lama glama) and selected from a phage display library for high affinity to the C-terminal (CT) region of NaV1.4. The Nbs were expressed in Escherichia coli, purified, and bio-physically characterized. Development of high-affinity Nbs specifically targeting a given human NaV isoform has been challenging because they usually show undesired cross-reactivity for different NaV isoforms. Our results show, however, that Nb17 and Nb82 recognize the CTNaV1.4 or CTNaV1.5 over other CTNav isoforms. Kinetic experiments by biolayer interferometry determined that Nb17 and Nb82 bind to the CTNaV1.4 and CTNaV1.5 with high affinity (KD ~ 40-60 nM). In addition, as proof of concept, we show that Nb82 could detect NaV1.4 and NaV1.5 channels in mammalian cells and tissues by Western blot. Furthermore, human embryonic kidney cells expressing holo NaV1.5 channels demonstrated a robust FRET-binding efficiency for Nb17 and Nb82. Our work lays the foundation for developing Nbs as anti-NaV reagents to capture NaVs from cell lysates and as molecular visualization agents for NaVs.Fil: Srinivasan, Lakshmi. University Johns Hopkins; Estados UnidosFil: Alzogaray, Vanina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Selvakumar, Dakshnamurthy. Fortébio; Estados UnidosFil: Nathan, Sara. University Johns Hopkins; Estados UnidosFil: Yoder, Jesse B.. University Johns Hopkins; Estados UnidosFil: Wright, Katharine M.. University Johns Hopkins; Estados UnidosFil: Klinke, Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Nwafor, Justin N.. University Johns Hopkins; Estados UnidosFil: Labanda, María Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Goldbaum, Fernando Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Schön, Arne. University Johns Hopkins; Estados UnidosFil: Freire, Ernesto. University Johns Hopkins; Estados UnidosFil: Tomaselli, Gordon F.. University Johns Hopkins; Estados UnidosFil: Amzel, León Mario. University Johns Hopkins; Estados UnidosFil: Ben-Johny, Manu. Columbia University; Estados UnidosFil: Gabelli, Sandra. University Johns Hopkins; Estados UnidosAmerican Society for Biochemistry and Molecular Biology2022-04info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/200892Srinivasan, Lakshmi; Alzogaray, Vanina Andrea; Selvakumar, Dakshnamurthy; Nathan, Sara; Yoder, Jesse B.; et al.; Development of high-affinity nanobodies specific for NaV1.4 and NaV1.5 voltage-gated sodium channel isoforms; American Society for Biochemistry and Molecular Biology; Journal of Biological Chemistry (online); 298; 4; 4-2022; 1-160021-9258CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S0021925822002034info:eu-repo/semantics/altIdentifier/doi/10.1016/j.jbc.2022.101763info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-22T11:42:09Zoai:ri.conicet.gov.ar:11336/200892instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-10-22 11:42:10.099CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Development of high-affinity nanobodies specific for NaV1.4 and NaV1.5 voltage-gated sodium channel isoforms
title Development of high-affinity nanobodies specific for NaV1.4 and NaV1.5 voltage-gated sodium channel isoforms
spellingShingle Development of high-affinity nanobodies specific for NaV1.4 and NaV1.5 voltage-gated sodium channel isoforms
Srinivasan, Lakshmi
VOLTAGE-GATED SODIUM CHANNELS
NAVS
HIGH-AFFINITY ANTI-NAV NANOBODIES
ANTI-NAV REAGENTS
title_short Development of high-affinity nanobodies specific for NaV1.4 and NaV1.5 voltage-gated sodium channel isoforms
title_full Development of high-affinity nanobodies specific for NaV1.4 and NaV1.5 voltage-gated sodium channel isoforms
title_fullStr Development of high-affinity nanobodies specific for NaV1.4 and NaV1.5 voltage-gated sodium channel isoforms
title_full_unstemmed Development of high-affinity nanobodies specific for NaV1.4 and NaV1.5 voltage-gated sodium channel isoforms
title_sort Development of high-affinity nanobodies specific for NaV1.4 and NaV1.5 voltage-gated sodium channel isoforms
dc.creator.none.fl_str_mv Srinivasan, Lakshmi
Alzogaray, Vanina Andrea
Selvakumar, Dakshnamurthy
Nathan, Sara
Yoder, Jesse B.
Wright, Katharine M.
Klinke, Sebastian
Nwafor, Justin N.
Labanda, María Soledad
Goldbaum, Fernando Alberto
Schön, Arne
Freire, Ernesto
Tomaselli, Gordon F.
Amzel, León Mario
Ben-Johny, Manu
Gabelli, Sandra
author Srinivasan, Lakshmi
author_facet Srinivasan, Lakshmi
Alzogaray, Vanina Andrea
Selvakumar, Dakshnamurthy
Nathan, Sara
Yoder, Jesse B.
Wright, Katharine M.
Klinke, Sebastian
Nwafor, Justin N.
Labanda, María Soledad
Goldbaum, Fernando Alberto
Schön, Arne
Freire, Ernesto
Tomaselli, Gordon F.
Amzel, León Mario
Ben-Johny, Manu
Gabelli, Sandra
author_role author
author2 Alzogaray, Vanina Andrea
Selvakumar, Dakshnamurthy
Nathan, Sara
Yoder, Jesse B.
Wright, Katharine M.
Klinke, Sebastian
Nwafor, Justin N.
Labanda, María Soledad
Goldbaum, Fernando Alberto
Schön, Arne
Freire, Ernesto
Tomaselli, Gordon F.
Amzel, León Mario
Ben-Johny, Manu
Gabelli, Sandra
author2_role author
author
author
author
author
author
author
author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv VOLTAGE-GATED SODIUM CHANNELS
NAVS
HIGH-AFFINITY ANTI-NAV NANOBODIES
ANTI-NAV REAGENTS
topic VOLTAGE-GATED SODIUM CHANNELS
NAVS
HIGH-AFFINITY ANTI-NAV NANOBODIES
ANTI-NAV REAGENTS
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Voltage-gated sodium channels, NaVs, are responsible for the rapid rise of action potentials in excitable tissues. NaV channel mutations have been implicated in several human genetic diseases, such as hypokalemic periodic paralysis, myotonia, and long-QT and Brugada syndromes. Here, we generated high-affinity anti-NaV nanobodies (Nbs), Nb17 and Nb82, that recognize the NaV1.4 (skeletal muscle) and NaV1.5 (cardiac muscle) channel isoforms. These Nbs were raised in llama (Lama glama) and selected from a phage display library for high affinity to the C-terminal (CT) region of NaV1.4. The Nbs were expressed in Escherichia coli, purified, and bio-physically characterized. Development of high-affinity Nbs specifically targeting a given human NaV isoform has been challenging because they usually show undesired cross-reactivity for different NaV isoforms. Our results show, however, that Nb17 and Nb82 recognize the CTNaV1.4 or CTNaV1.5 over other CTNav isoforms. Kinetic experiments by biolayer interferometry determined that Nb17 and Nb82 bind to the CTNaV1.4 and CTNaV1.5 with high affinity (KD ~ 40-60 nM). In addition, as proof of concept, we show that Nb82 could detect NaV1.4 and NaV1.5 channels in mammalian cells and tissues by Western blot. Furthermore, human embryonic kidney cells expressing holo NaV1.5 channels demonstrated a robust FRET-binding efficiency for Nb17 and Nb82. Our work lays the foundation for developing Nbs as anti-NaV reagents to capture NaVs from cell lysates and as molecular visualization agents for NaVs.
Fil: Srinivasan, Lakshmi. University Johns Hopkins; Estados Unidos
Fil: Alzogaray, Vanina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina
Fil: Selvakumar, Dakshnamurthy. Fortébio; Estados Unidos
Fil: Nathan, Sara. University Johns Hopkins; Estados Unidos
Fil: Yoder, Jesse B.. University Johns Hopkins; Estados Unidos
Fil: Wright, Katharine M.. University Johns Hopkins; Estados Unidos
Fil: Klinke, Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina
Fil: Nwafor, Justin N.. University Johns Hopkins; Estados Unidos
Fil: Labanda, María Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina
Fil: Goldbaum, Fernando Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina
Fil: Schön, Arne. University Johns Hopkins; Estados Unidos
Fil: Freire, Ernesto. University Johns Hopkins; Estados Unidos
Fil: Tomaselli, Gordon F.. University Johns Hopkins; Estados Unidos
Fil: Amzel, León Mario. University Johns Hopkins; Estados Unidos
Fil: Ben-Johny, Manu. Columbia University; Estados Unidos
Fil: Gabelli, Sandra. University Johns Hopkins; Estados Unidos
description Voltage-gated sodium channels, NaVs, are responsible for the rapid rise of action potentials in excitable tissues. NaV channel mutations have been implicated in several human genetic diseases, such as hypokalemic periodic paralysis, myotonia, and long-QT and Brugada syndromes. Here, we generated high-affinity anti-NaV nanobodies (Nbs), Nb17 and Nb82, that recognize the NaV1.4 (skeletal muscle) and NaV1.5 (cardiac muscle) channel isoforms. These Nbs were raised in llama (Lama glama) and selected from a phage display library for high affinity to the C-terminal (CT) region of NaV1.4. The Nbs were expressed in Escherichia coli, purified, and bio-physically characterized. Development of high-affinity Nbs specifically targeting a given human NaV isoform has been challenging because they usually show undesired cross-reactivity for different NaV isoforms. Our results show, however, that Nb17 and Nb82 recognize the CTNaV1.4 or CTNaV1.5 over other CTNav isoforms. Kinetic experiments by biolayer interferometry determined that Nb17 and Nb82 bind to the CTNaV1.4 and CTNaV1.5 with high affinity (KD ~ 40-60 nM). In addition, as proof of concept, we show that Nb82 could detect NaV1.4 and NaV1.5 channels in mammalian cells and tissues by Western blot. Furthermore, human embryonic kidney cells expressing holo NaV1.5 channels demonstrated a robust FRET-binding efficiency for Nb17 and Nb82. Our work lays the foundation for developing Nbs as anti-NaV reagents to capture NaVs from cell lysates and as molecular visualization agents for NaVs.
publishDate 2022
dc.date.none.fl_str_mv 2022-04
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/200892
Srinivasan, Lakshmi; Alzogaray, Vanina Andrea; Selvakumar, Dakshnamurthy; Nathan, Sara; Yoder, Jesse B.; et al.; Development of high-affinity nanobodies specific for NaV1.4 and NaV1.5 voltage-gated sodium channel isoforms; American Society for Biochemistry and Molecular Biology; Journal of Biological Chemistry (online); 298; 4; 4-2022; 1-16
0021-9258
CONICET Digital
CONICET
url http://hdl.handle.net/11336/200892
identifier_str_mv Srinivasan, Lakshmi; Alzogaray, Vanina Andrea; Selvakumar, Dakshnamurthy; Nathan, Sara; Yoder, Jesse B.; et al.; Development of high-affinity nanobodies specific for NaV1.4 and NaV1.5 voltage-gated sodium channel isoforms; American Society for Biochemistry and Molecular Biology; Journal of Biological Chemistry (online); 298; 4; 4-2022; 1-16
0021-9258
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S0021925822002034
info:eu-repo/semantics/altIdentifier/doi/10.1016/j.jbc.2022.101763
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
application/pdf
dc.publisher.none.fl_str_mv American Society for Biochemistry and Molecular Biology
publisher.none.fl_str_mv American Society for Biochemistry and Molecular Biology
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
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instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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