DESIGN OF NANOBODIES SPECIFIC TO MUSCLE ISOFORMS OF VOLTAGE-GATED SODIUM CHANNELS

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

San Diego

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

Biophysical Society 2020.

Nav1.4, in skeletal muscle, and Nav1.5, in cardiac muscle, are voltage-gated sodiumchannels (Navs) that play an important role in the generation of action potential inexcitable tissues. Navs rapidly respond to changes in cellular membrane potentialand control the passage of sodium ions into cells. Dysfunction of Navs proteinscaused by genetic mutations have been implicated in several human diseases such ashypokalemic periodic paralysis, myotonia and Brugada syndrome. Antigenstimulation of dromedary animals (llama and alpaca) and also sharks produces single,variable heavy chain only, (VHH) called nanobodies (Nbs), of small (15 kDa), VHdomains that exhibit high epitope-affinity. We have raised in llama and selected bypanning, high-affinity binding Nb clones to the complex of the C-terminal of skeletalmuscle Nav1.4 and Calmodulin (Nav1.4-CT-1764-CaM). Of these, two nanobodieswere expressed in the E. coli periplasm, purified to homogeneity and determined thestructure by x-ray crystallography to 2.8 Å resolution. ELISA and binding kineticexperiments using BLI (Bio-Layer Interferometry) show that the Nbs are selective forboth Nav muscle isoforms Nav1.4 and Nav1.5 with nM affinities. However, the Nbsdo not bind to CaM and neuronal Nav isoforms Nav1.7 and Nav1.9. Further, weanalyzed by Differential scanning fluorimetry that the anti-Nav Nbs are highlythermostable (Tm > 99°C) and stabilized Nav1.4-CT and Nav1.5-CT proteins asevidenced by a robust thermal shift (ΔTm = 10-15°C) of the Nav-CT-Nb complexes.