Development of high-affinity nanobodies specific for Na1.4 and Na1.5 voltage-gated sodium channel isoforms.

Voltage-gated sodium channels, Nas, are responsible for the rapid rise of action potentials in excitable tissues. Na 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 nanobodies (Nbs), Nb17 and Nb82, that recognize the Na1.4 (skeletal muscle) and Na1.5 (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 Na1.4. The Nbs were expressed in Escherichia coli, purified, and biophysically characterized. Development of high-affinity Nbs specifically targeting a given human Na isoform has been challenging because they usually show undesired crossreactivity for different Na isoforms. Our results show, however, that Nb17 and Nb82 recognize the CTNa1.4 or CTNa1.5 over other CTNav isoforms. Kinetic experiments by biolayer interferometry determined that Nb17 and Nb82 bind to the CTNa1.4 and CTNa1.5 with high affinity (K ∼ 40-60 nM). In addition, as proof of concept, we show that Nb82 could detect Na1.4 and Na1.5 channels in mammalian cells and tissues by Western blot. Furthermore, human embryonic kidney cells expressing holo Na1.5 channels demonstrated a robust FRET-binding efficiency for Nb17 and Nb82. Our work lays the foundation for developing Nbs as anti-Na reagents to capture Nas from cell lysates and as molecular visualization agents for Nas.