Development of High affinity Nanobodies Specific for NaV1.4 and NaV1.5 Voltage-Gated Sodium Channel Isoforms

LAKSHMI SRINIVASAN; VANINA ALZOGARAY; DAKSHNAMURTHY SELVAKUMAR; SARA NATHAN; JESSE YODER; KATHARINE M. WRIGHT; SEBASTIÁN KLINKE; JUSTIN N. NWAFOR; MARÍA S. LABANDA; FERNANDO A. GOLDBAUM; ARNE SCHÖN; ERNESTO FREIRE; GORDON F. TOMASELLI; L. MARIO AMZEL; MANU BEN-JOHNY; SANDRA GABELLI

JOURNAL OF BIOLOGICAL CHEMISTRY

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Lugar: Bethesda, Maryland; Año: 2022 vol. 298

0021-9258

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 biophysically characterized. Development of high-affinity Nbs specifically targeting a given human NaV isoform has been challenging because they usually show undesired crossreactivity 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 about 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.