Development of high-affinity nanobodies specific for Na(V)1.4 and Na(V)1.5 voltage-gated sodium channel isoforms

Voltage-gated sodium channels, Na(V)s, are responsible for the rapid rise of action potentials in excitable tissues. Na(V) 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-Na(V) nanobodies (Nbs), Nb17 and Nb82, that recognize the Na(V)1.4 (skeletal muscle) and Na(V)1.5 (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 Na(V)1.4. The Nbs were expressed in Escherichia coli, purified, and biophysically characterized. Development of high-affinity Nbs specifically targeting a given human Na(V) isoform has been challenging because they usually show undesired crossreactivity for different Na(V) isoforms. Our results show, however, that Nb17 and Nb82 recognize the CTNa(V)1.4 or CTNa(V)1.5 over other CTNav isoforms. Kinetic experiments by biolayer interferometry determined that Nb17 and Nb82 bind to the CTNa(V)1.4 and CTNa(V)1.5 with high affinity (K(D) ∼ 40–60 nM). In addition, as proof of concept, we show that Nb82 could detect Na(V)1.4 and Na(V)1.5 channels in mammalian cells and tissues by Western blot. Furthermore, human embryonic kidney cells expressing holo Na(V)1.5 channels demonstrated a robust FRET-binding efficiency for Nb17 and Nb82. Our work lays the foundation for developing Nbs as anti-Na(V) reagents to capture Na(V)s from cell lysates and as molecular visualization agents for Na(V)s.