Abstract
Voltage-gated sodium channels, NaVs, are responsible for the rapid rise of action potentials in excitable tissues. NaV 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-NaV nanobodies (Nbs), Nb17 and Nb82, that recognize the NaV1.4 (skeletal muscle) and NaV1.5 (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 NaV1.4. The Nbs were expressed in Escherichia coli, purified, and biophysically characterized. Development of high-affinity Nbs specifically targeting a given human NaV isoform has been challenging because they usually show undesired crossreactivity for different NaV isoforms. Our results show, however, that Nb17 and Nb82 recognize the CTNaV1.4 or CTNaV1.5 over other CTNav isoforms. Kinetic experiments by biolayer interferometry determined that Nb17 and Nb82 bind to the CTNaV1.4 and CTNaV1.5 with high affinity (KD ∼ 40–60 nM). In addition, as proof of concept, we show that Nb82 could detect NaV1.4 and NaV1.5 channels in mammalian cells and tissues by Western blot. Furthermore, human embryonic kidney cells expressing holo NaV1.5 channels demonstrated a robust FRET-binding efficiency for Nb17 and Nb82. Our work lays the foundation for developing Nbs as anti-NaV reagents to capture NaVs from cell lysates and as molecular visualization agents for NaVs.
Highlights
14 clones were found potentially allow the selection of nanobodies that are specific to CTNaV1.4-CaM (Fig. S1B)
The three selected Nb clones were sub-cloned into a pHEN6-His vector as a C-terminal 6x-His tagged fusion protein and successfully expressed in the periplasm of E. coli Rosetta-gami 2(DE3) cells
Nb17 and Nb82 were purified via Ni-NTA chromatography, followed by size exclusion chromatography (Fig.[1] Step 9; Fig. 2B, C and D). Both Nbs behave as monomers in solution and were detected as a single, homogenous peak on size exclusion chromatography with retention volumes of 14.4 ml (Nb17) and 15.6 ml (Nb82) on a Superdex
Summary
To facilitate the selection of nanobodies with isoform specificity, we analyzed the homology amongst NaV isoforms to choose a divergent region. In the SDS-PAGE gels of the elution fractions, the peaks corresponding to the CT NaV1.5-CaM+Nb17 complex shows the presence of all three proteins; CTNaV1.5, CaM and Nb17 These results imply that both Nb17 and Nb82 interact with the CTNaV-CaMs but affect their hydrodynamic radii differently. Ab (Fig. 9F) as observed by signals corresponding to cells expressing full-length NaV1.5 channel display the same molecular weight for skeletal muscle and high efficiency of binding of Nb17 and Nb82 to heart tissue samples in both blots. As a control, when the blots were reprobed with molecular reagent to detect NaV1.4(5) proteins in anti-His HRP Ab (Fig. 9G and 9I), no bands various types of cell and tissue lysates since it were observed at 220-250 kDa showing the recognizes endogenous NaV1.4 channel from. Journal Pre-proof specificity of detection by Nb82 as expected
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