Production and Applications of Anti-Voltage-Gated Sodium Channel Nav1.4 Nanobodies

VANINA ALZOGARAY; L. MARIO AMZEL; JESSE YODER; FERNANDO GOLDBAUM; LAKSHMI SRINIVASAN; SEBASTIÁN KLINKE; SANDRA B. GABELLI

Baltimore

Congreso; 63 Annual Meetings of the Biophysical Society; 2019

Voltage-gated sodium channels (Navs) rapidly respond to changes in cellular membrane potential by allowing Na+ ions to move into cells. They play an important role in the generation of action potential in excitable tissues such as skeletal muscle, heart and the nerve. Mutations in these proteins have been implicated in human genetic diseases such as hypokalemic periodic paralysis, myotonia and Brugada syndrome. Nanobodies (Nbs), single chain antibody domains containing a single variable heavy chain (VHH) domain (~15kDa), are produced by antigen stimulation in camelids like llama or alpaca. Nanobodies exhibit high epitope affinity at least in part because they contain longer CDR3 loop than the regular VH. We have raised and selected Nanobody clones to the complex of the C-terminal of skeletal muscle Nav with Calmodulin (Nav1.4-CT-1764-CaM). Selected nanobody clones with high binding were cloned for periplasmic expression in E. coli. We expressed and purified two nanobodies and assessed their binding to Nav1.4 by ELISA and determined that they do not bind CaM. Interestingly, the nanobodies also recognize Nav1.5, albeit to a lesser extent. The ease of production of both nanobodies and the Navs combined with the stabilization of the Navs by the nanobodies in flexible regions of the Navs makes the Nav-Nab complexes ideal candidates for protein crystallography. The potential of such Nav-Nb structures can also be explored to identify or trap specific Nav1.4 or Nav1.5 conformations not accessible previously. Also, the high specificity and low immunogenicity of Nbs promises to serve as a beneficial pharmacodynamic property for potential drug delivery systems.