TY - JOUR
T1 - Development of high-affinity nanobodies specific for NaV1.4 and NaV1.5 voltage-gated sodium channel isoforms
AU - Srinivasan, Lakshmi
AU - Alzogaray, Vanina
AU - Selvakumar, Dakshnamurthy
AU - Nathan, Sara
AU - Yoder, Jesse B.
AU - Wright, Katharine M.
AU - Klinke, Sebastián
AU - Nwafor, Justin N.
AU - Labanda, María S.
AU - Goldbaum, Fernando A.
AU - Schön, Arne
AU - Freire, Ernesto
AU - Tomaselli, Gordon F.
AU - Amzel, L. Mario
AU - Ben-Johny, Manu
AU - Gabelli, Sandra B.
N1 - Funding Information:
Acknowledgments—We thank the technical assistance of Inmunova (Buenos Aires, Argentina) for supporting llama immunization. We thank Chulan Kwon for providing mouse tissue and David Suh for technical assistance. This work was funded by the National Institutes of Health (NIH)/National Heart, Lung, and Blood Institute (grant no.: HL128743). We thank Dr Robert Cole for help with mass spectrometry analysis carried out at the Johns Hopkins University School of Medicine Mass Spectrometry and Proteomics Facility, which is supported by the Sidney Kimmel Comprehensive Cancer Center (National Cancer Institute/NIH grant: 2P30 CA006973) and Hopkins Conte NIH/National Institute of Diabetes and Digestive and Kidney Diseases Basic and Translational Research Core Center (grant no.: P30 DK089502). Work at the AMX (17-ID-1) and FMX (17-ID-2) beamlines was supported by National Institute of General Medical Sciences, NIH (grant no.: P41GM111244) and by the US Department of Energy Office of Biological and Environmental Research grant (grant no.: KP1605010), and the National Synchrotron Light Source II at Brookhaven National Laboratory is supported by the Department of Energy Office of Basic Energy Sciences under contract number DE-SC0012704 (grant no.: KC0401040). Flow cytometry was performed in the CCTI Flow Cytometry Core, supported in part by the Office of the Director, NIH under awards S10RR027050. We acknowledge the use of the Eukaryotic Tissue Culture Facility at the Johns Hopkins University and the guidance of its manager, Dr Yana Li.
Funding Information:
Funding and additional information—J. N. N. was supported by the NIH grant (grant no.: R25 GM109441) and the Vivien Thomas Scholars Initiative at Johns Hopkins University. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.
Publisher Copyright:
© 2022 THE AUTHORS
PY - 2022/4/1
Y1 - 2022/4/1
N2 - 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.
AB - 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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U2 - 10.1016/j.jbc.2022.101763
DO - 10.1016/j.jbc.2022.101763
M3 - Article
C2 - 35202650
AN - SCOPUS:85127234733
SN - 0021-9258
VL - 298
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 4
M1 - 101763
ER -
