Design of nanobodies specific to muscle isoforms of voltage-gated sodium channels (Póster)

LAKSHMI SRINIVASAN; VANINA A. ALZOGARAY; JESSE YODER; SARA NATHAN; SEBASTIÁN KLINKE; FERNANDO A. GOLDBAUM; GORDON F. TOMASELLI; L. MARIO AMZEL; SANDRA GABELLI

San Diego

Congreso; 64th Annual Meeting of the Biophysical Society; 2020

Biophysical Society (EEUU)

Nav1.4, in skeletal muscle, and Nav1.5, in cardiac muscle, are voltage-gated sodium channels (Navs) that play an important role in the generation of action potential in excitable tissues. Navs rapidly respond to changes in cellular membrane potential and control the passage of sodium ions into cells. Dysfunction of Navs proteins caused by genetic mutations have been implicated in several human diseases such as hypokalemic periodic paralysis, myotonia and Brugada syndrome. Antigen stimulation of dromedary animals (llama and alpaca) and also sharks produces single variable heavy chain only (VHH) called nanobodies (Nbs) of small (15 kDa) VH domains that exhibit high epitope-affinity. We have raised in llama and selected by panning, high-affinity binding Nb clones to the complex of the C-terminal of skeletal muscle Nav1.4 and Calmodulin (Nav1.4-CT-1764-CaM). Of these, two nanobodies were expressed in the E. coli periplasm, purified to homogeneity and determined the structure by x-ray crystallography to 2.8 A resolution. ELISA and binding kinetic experiments using BLI (Bio-Layer Interferometry) show that the Nbs are selective for both Nav muscle isoforms Nav1.4 and Nav1.5 with nM affinities. However, the Nbs do not bind to CaM and neuronal Nav isoforms Nav1.7 and Nav1.9. Further, we analyzed by Differential scanning fluorimetry that the anti-Nav Nbs are highly thermostable (Tm > 99 °C) and stabilized Nav1.4-CT and Nav1.5-CT proteins as evidenced by a robust thermal shift (deltaTm = 10-15 °C) of the Nav-CT-Nb complexes.