Artículo
Development of high-affinity nanobodies specific for NaV1.4 and NaV1.5 voltage-gated sodium channel isoforms
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
Fecha de publicación:
04/2022
Editorial:
American Society for Biochemistry and Molecular Biology
Revista:
Journal of Biological Chemistry (online)
ISSN:
0021-9258
Idioma:
Inglés
Tipo de recurso:
Artículo publicado
Clasificación temática:
Resumen
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.
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Articulos(IIBBA)
Articulos de INST.DE INVEST.BIOQUIMICAS DE BS.AS(I)
Articulos de INST.DE INVEST.BIOQUIMICAS DE BS.AS(I)
Articulos(SEDE CENTRAL)
Articulos de SEDE CENTRAL
Articulos de SEDE CENTRAL
Citación
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
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