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

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

JOURNAL OF BIOLOGICAL CHEMISTRY

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Lugar: Bethesda, Maryland; Año: 2022

0021-9258

Voltage-gated sodium channels,NaVs, areresponsible for therapidrise of action potentials inexcitable tissues.NaVchannelmutations have beenimplicated in several human genetic diseases such ashypokalemic periodic paralysis, myotonia,andLongQT and Brugada syndromes.Here wegenerated highaffinity, anti-NaVnanobodies (Nbs), Nb17 and Nb82that recognize theNaV1.4 (skeletal muscle) andNaV1.5 (cardiac muscle) channel isoforms.TheseNbs were raised in llamaandselected from a phagedisplay library for high affinity to the C-terminalregion (CT) ofNaV1.4. The Nbs wereexpressed inE. coli, purified and biophysically characterized.Development of high-affinity nanobodiesspecificallytargetinga given humanNaVisoform hasbeen challenging because they usually showundesired cross-reactivity for differentNaVisoforms.Our results show, however, that Nb17 and Nb82recognize the CTNaV1.4orCTNaV1.5overotherCTNav isoforms.Kinetic experiments by Bio-Layer-Interferometry determined that Nb17 and Nb82 bindto the CTNaV1.4 and CTNaV1.5 with high affinity(KD~ 40-60nM).In addition, as proof of concept,we show that Nb82 could detectNaV1.4 andNaV1.5channels in mammalian cells and tissues by Westernblot.Furthermore, HEK cells expressingholoNaV1.5 channelsdemonstrated arobustFRETbinding efficiencyfor Nb17 and Nb82.Our worklays the foundation for developing Nbs as anti-NaVreagents to captureNaVs from cell lysatesandasmolecular visualization agents for NaVs.