Development of high-affinity nanobodies specific for NaV1.4 and NaV1.5 voltage-gated sodium channel isoforms

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.

The nine human isoforms of voltage-gated sodium channels (Na V 1.1-1.9) rapidly respond to changes in cellular membrane potential by allowing the passage of Na + ions into the cell. They play an important role in the generation of the action potential in excitable tissues, such as skeletal muscle, heart, and nerves (1). Mutations in the cytoplasmic C-terminal (CT) region of these proteins have been implicated in human genetic diseases, such as generalized epilepsy with febrile seizures, hypokalemic periodic paralysis, myotonia, long-QT syndrome, and Brugada syndrome (2)(3)(4). Given the physiological importance of the Na V isoforms in normal physiology and disease, development of reagents for their study, such as antibodies (Abs), has been an important part of Na V research. Specifically targeting each individual isoform, however, has been challenging, because of their high sequence identity. To achieve tissue specificity and avoid off-target side effects of anti-Pan Na V Abs, there is an increasing need for biologicals with high solubility, stability, and specificity (5,6).
Nanobodies (Nbs) are single variable heavy-chain (VHH) immunoglobulin domains derived from heavy-chain-only Abs produced in camelids, such as camels, llamas (Lama glama), and alpacas. Nbs are small prolate-shaped molecules (15 kDa) that fully retain the epitope-recognizing function in a single-chain Ab. They may be selected to contain an extended and flexible complementarity-determining region 3 (CDR3) loop partly contributing to their high epitope affinity and their ability to better access smaller and cryptic epitopes (7). Moreover, VHH domains are amenable to cloning and protein modifications and can be produced in bacterial expression systems in scalable amounts. Nbs also display superior solubility, stability, in vivo half-lives, and pharmacodynamics compared with conventional Abs (8). For example, Nbs to P2x channel proteins have been shown to display greater therapeutic potential than conventional Abs for modulating channel function and reducing the in vivo inflammation caused by P2X7 (9)(10)(11)(12). Nbs have also been used as crystallization chaperones (7,13,14), visualization agents (15)(16)(17), in vivo radiotracers (18), pulldown baits (19,20), intracellular pathway modulators (21), virus neutralization agents (22,23), and therapeutics agents (24)(25)(26)(27). In this study, we have raised, selected, and characterized Nb clones that recognize the CT region of two Na V isoforms: Na V 1.4 (skeletal muscle) and Na V 1.5 (cardiac muscle). We selected the CT domain to serve as an antigen for Nb production since the sequences are more divergent than in other regions of the channels such as the pore-forming regions. Furthermore, the CT domain of the channel is the binding site for channelinteracting proteins, which regulate their activity. This would potentially allow the selection of Nbs that are specific for a given isoform or to a particular state of the channel. Moreover, the available cryo-EM structures have not fully resolved details of these functionally relevant CTNa V domains. Hence, Nbs targeted to the CT could potentially stabilize these regions, thereby aiding their structure elucidation. In this study, we detail the production, biophysical characterization, and unique structural features of two Nbs that recognize Na V 1. 4 and Na V 1.5 channels and show results highlighting their potential to serve as molecular tools to study Na V proteins in vitro as well as in cells and tissues.

Results
Generation and identification of Na V 1. 4

-specific Nbs
To facilitate the selection of Nbs with isoform specificity, we analyzed the homology amongst Na V isoforms to choose a divergent region. Sequence homology analysis amongst the Na V isoforms revealed that the linker between domains 1 and 2 (1-2 linker, amino acids 481-581 in Na V 1.4) is the most divergent with pairwise identity between 40 and 55%, whereas the CTNa V (1599-1836 amino acids in Na V 1.4) domain displays pairwise identity of 60 to 70% (Table S1). The fact that CTNa Vcalmodulin (CaM) is known to fold as a globular domain that can be purified and crystallized (28,29) cemented the CTNa V domain in complex with CaM (CTNa V -CaM) as the antigen of choice. To generate, select, and produce the Nbs (Fig. 1), a llama was immunized with purified CTNa V 1.4T in complex with CaM (CTNa V 1.4T-CaM, amino acids 1599-1764 CTNa V 1.4T, Table S2 and Fig. 1, step 1 and 2), and its humoral immune response was evaluated by ELISA (Figs. S1A and 1, steps 3 and 4) (29). mRNA was extracted from isolated peripheral blood lymphocytes (PBLs; Fig. 1, step 4) and PCR amplified (Fig. 1, step 5). The VHH complementary DNA (cDNA) was cloned into a phagemid vector pHEN4 to obtain the VHH phage library (Fig. 1, step 6).
To identify Nbs that target CTNa V 1.4-CaMs, we screened an Nb phage display library with an estimated complexity of >5.6 × 10 7 clones (Fig. 1, step 7). Following two rounds of positive selection against CTNa V 1.4T-CaM, a total of 87 randomly chosen clones were expressed and tested by ELISA (Fig. 1, step 8). Amongst them, 14 clones were found specific to CTNa V 1.4-CaM (Fig. S1B). We further classified the 14 clones into four different families based on the length and variability of their CDR2 and CDR3 regions (Fig. S1C). One representative clone from each family was chosen and screened by periplasmic ELISA against CTNa V 1.4T-CaM, Ca 2+ CaM, and apoCaM proteins ( Fig. 2A). Of these, Nb17, Nb30, and Nb82 were found to be specific to CTNa V 1.4T-CaM ("+" signs) but not to Ca 2+ CaM or apo-CaM. However, Nb26 recognized not only CTNa V 1.4T-CaM but also Ca 2+ CaM and apo-CaM. Based on this information, Nb17, Nb30, and Nb82 were chosen for expression and purification in Escherichia coli for further biophysical and biochemical characterization. The three selected Nb clones were subcloned into a pHEN6-His vector as a CT 6×-His tagged fusion protein and successfully expressed in the periplasm of E. coli Rosetta-gami 2(DE3) cells. The Nbs were extracted from the periplasm using a combination of thermal and osmotic shock methods (30). Of these, Nb30 was not pursued further because of its low expression levels. Nb17 and Nb82 were purified via nickel-nitrilotriacetic acid (Ni-NTA) chromatography, followed by size-exclusion chromatography (SEC; Figs.1, step 9 and 2, B-D). Both Nbs behave as monomers in solution and were detected as a single homogenous peak on SEC with retention volumes of 14.4 ml (Nb17) and 15.6 ml (Nb82) on a Superdex 75 10/300 GL column (Fig. 2D). These retention volumes are in agreement with those of Nbs reported in the literature (31,32).

Nb17 and Nb82 are thermally stable
The temperature stability of Nb17 and Nb82 was measured by differential scanning calorimetry (DSC). Nb17 is characterized by a T m of 76 C (Fig. 3A) and Nb82 by a T m of 66 C (Fig. 3B), suggesting both are relatively stable protein suitable for functional and structural characterization. Notably, Nb17 undergoes reversible temperature denaturation, whereas Nb82 undergoes irreversible denaturation. The different denaturation mechanisms probably originate from sequence differences. These different mechanisms are also reflected in the shape of the DSC curves (33). Also, Nb17 has a van't Hoff enthalpy of 118 kcal/mol that is close to what is expected for a protein of this size undergoing two-state unfolding. For Nb82, the irreversible transition is kinetically controlled, with a noticeable change in shape of the curve, and characterized by an activation energy of 86 kcal/mol.

Nb82 has an extended CDR3 loop
The structure of Nb82 was determined by X-ray crystallography to 2.0 Å resolution and refined to an R work /R free of 0.19/0.24 with excellent geometry (Protein Data Bank [PDB] ID: 7R63; Table 1 and Fig. 3, C-F). The asymmetric unit includes four copies of Nb82 with almost identical conformations (Cα RMSD <0.30 Å, pairwise among all four chains, including 117 Cα carbons). Nb82 bears the classical immunoglobulin fold (Fig. 3, C and D) with two β-sheets of four antiparallel β-strands (β1-β3-β8-β7 and β6-β5-β4-β9) and a smaller β-sheet made up of two parallel β-strands, β2 and β10, that elongate the β6-β5-β4-β9 sheet. The structure displays good electron density for all portions of the four molecules (Table 1 and Fig. 3, C and D). The fold is decorated by three π-helices, two of which are in the epitope-recognizing CDR1 and CDR3, formed between the β3-β4 and β9-β10 loops, respectively. The third π-helix is on the loop connecting strands β3 and β4. CDR π-helices are a characteristic feature observed in other Nbs (14). CDR3, the usual major contributor for antigen recognition and specificity, folds as a random coil with a π-helix of seven residues that folds back onto β6-β5-β4-β9 sheet. Notably, the CDR1 and CDR2 present a positively charged patch (Fig. 3, E and F) suggesting that Nb82 favors an interaction with a large negatively charged surface on the Na V . Comparison of the sequence of Nb82 with Nb17 and Nb30 (Fig. 3G) and its structure (PDB ID: 7R63) with other Nbs available (PDB IDs: 5LMJ, 6H6Y, and 5LZ0 (14)) ( Fig. S2) highlights the differences in the fold of their CDR3 (Fig. S2, A and B).
Nb17 and Nb82 specifically recognize CTNa V 1.4 and CTNa V 1.5 isoforms To evaluate the specificity of the two Nbs, we performed ELISA experiments (Fig. 4A) with the purified Nbs and purified CTNa V isoforms in complex with CaM (CTNa V 1.4-CaM, CTNa V 1.5-CaM, CTNa V 1.7-CaM) and with CTNa V 1.9, Step 8: Screening phage library by ELISA for high-binding Nb clones.
Step 11: Characterization of Nb binding to Na V 1.4 and Na V 1.5 isoforms. This rendition was contributed by Sora Ji (Sorajistudio.com). CaM, calmodulin.
which does not form a complex with CaM. We used a truncated (T) or full-length (FL) construct of the CTNa V isoforms (Fig. 4B). In ELISA experiments, Nb82 and Nb17 recognize both the T and FL versions of both CTNa V 1.4-CaM (skeletal) and CTNa V 1.5-CaM (cardiac). However, they do not recognize neuronal isoforms, CTNa V 1.7-CaM or CTNa V 1.9 (Fig. 4A). Importantly, they do not crossreact with free CaM (Fig. 4A).
Consistent with these findings, Western blot analysis demonstrated that His-tagged Nb82 as a primary Ab detected purified CTNa V 1.4T, CTNa V 1.4FL, CTNa V 1.5T, and CTNa V 1.5FL, resulting in signals corresponding to their respective molecular weights (Fig. 5A). On the other hand, no signal was detected for purified CTNa V 1.7, CTNa V 1.9, CaM alone, and glutathione-S-transferase (GST) (Fig. 5A), suggesting that Nb82 is specific to Na V 1.4 and Na V 1.5 channels.
To fully establish the selectivity of Nb17 and Nb82, we utilized a flow cytometry-based FRET two-hybrid assay to systematically probe the interaction of the two Nbs with CTNa V 1.1-CTNa V 1.9. We engineered Nb17 and Nb82 with a Cerulean fluorescent protein as a FRET donor and the CT domains of Na V 1.x with the Venus fluorescent protein, as FRET acceptor. Stochastic expression of the FRET pairs resulted in variable FRET efficiencies (E A ) in individual cells. A saturating binding relation was then constructed by correlating E A with the free donor concentration (D free ). Nb82 and Nb17 exhibited robust FRET with CTNa V 1.4 and CTNa V 1.5, whereas other isoforms showed qualitatively weaker FRET (Fig. 5, B and D). To quantify further, we calculated a relative association constant, K a,EFF (in arbitrary units) as 1/K d,EFF (the relative dissociation constant) from the fits in panels B and D. Indeed, both Nb17 and Nb82 demonstrated 7-fold to 100-fold higher K a,EFF for Na V 1.4 and Na V 1.5 as compared with other Na V isoforms (Fig. 5, C and E). Taken together, these results highlight the exquisite selectivity of Nb17 and Nb82 for the muscle Na V channels.
Thus affirmed, we further characterized the CTNa V -CaM and Nb82 interactions by analyzing the CTNa V X-CaM + Nb mixtures (where X equals 1.4 or 1.5) by SEC (Fig. 6). In the profiles, we observed that the CTNa V 1.4T-CaM + Nb82 (Fig. 6, A and B) and CTNa V 1.4FL-CaM + Nb82 complexes ( Fig. 6, C and D) elute about 2 ml earlier than the equivalent complexes in the absence of Nb82. SDS-PAGE gels of the elution fractions show that the new peaks contain all three proteins; CTNa V 1.4, CaM, and the Nb82 (Fig. 6, B and D). Also, the complexes elute at retention volumes that correspond to the molecular weight of the CTNa V 1.4-CaM + Nb complex 1:1 stoichiometry. CTNa V 1.5 showed a similar behavior. The complexes CTNa V 1.5T-CaM + Nb82 and CTNa V 1.5FL-CaM + Nb82 elute 1.0 and 1.5 ml before the CTNa V 1.5T-CaM complex, respectively (Fig. 6, E and G). The SDS-PAGE gels of the peak fractions confirm the presence of all three proteins (CTNa V 1.5, CaM, and Nb) (Fig. 6, F and H).
The mixture of CTNa V 1.5-CaM + Nb17 on SEC does not show a fully resolved new peak, typical of the formation of a complex. However, in the CTNa V 1.5T-CaM + Nb17 (Fig. S3, A and B) and CTNa V 1.5FL-CaM + Nb17 (Fig. S3, C and D), peaks elute 0.5 ml before the CTNa V -CaMs without the Nb followed by an asymmetric Nb17 peak. Also, in the SDS-PAGE gels of the elution fractions, the peaks corresponding to the CT Na V 1.5-CaM + Nb17 complex shows the presence of all three proteins; CTNa V 1.5, CaM, and Nb17. These results imply that both Nb17 and Nb82 interact with the CTNa V -CaMs but affect their hydrodynamic radii differently.
Nbs bind with nanomolar affinities to Na V 1.4 and Na V 1.5 We further probed the molecular interactions of the CTNa V 1.4-CaM + Nb and CTNa V 1.5-CaM + Nb ([CTNa V X]-CaM + Nb; X = 1.4 or 1.5) complexes by determining the kinetic parameters of binding using biolayer interferometry (BLI) (Figs. 7 and 8). The change in resonance with time was recorded at different concentrations of purified CTNa V 1.XT-CaM proteins or isolated CaM. A 1:1 binding of the Nbs to CTNa V XT-CaM proteins was observed with signals reaching steady state in 300 s. Analysis of the BLI dose responses of association and dissociation curves indicates that Nb82 binds to CTNa V 1.4T-CaM and CTNa V 1.5T-CaM with  Table 2). However, no binding to Nb82 was observed when isolated CaM was used as an analyte (Fig. 7C) evidenced by a nonassociation/no-binding curve. In addition, as estimated from ELISA results, no binding to Nb82 was observed when CTNa V 1.7T-CaM or CTNa V 1.9T were used as analytes (Fig. 7, D and E). Similarly, with Nb17 ( Fig. 8, A and  B), BLI experiments show that it binds to CTNa V 1.4T-CaM and CTNa V 1.5T-CaM with dissociation constants (K D s) of 41.0 ± 0.1 nM and 60.5 ± 0.06 nM, respectively (Table 2 and Fig. 8C) but not to isolated CaM. Moreover, as expected, no binding to Nb17 was observed when CTNa V 1.7T-CaM or CTNa V 1.9T was used as analytes (Fig. 8, D and E). In these experiments, the lack of binding of Nb17 and Nb82 to isolated CaM and to CTNa V 1.7-CaM and CTNa V 1.9 indicates that the epitope for Nb82 binding is on the Na V 1.4(5) channels or on the Na V 1.4(5)-CaM interface in an area different enough from that of Na V 17 or Na V 1.9.

channel in live cells and tissue homogenates
To determine whether Nb17 and Nb82 interact with holo-Na V 1.5 channels in live cells, we utilized flow cytometry-based FRET two-hybrid assay (34). To do so, we utilized Ceruleantagged Nb17 and Nb82 as FRET donor, and we attached a Venus tag to the carboxy terminus of the FL Na V 1.5 (FRET acceptor) (Fig. 9A) and obtained fluorescence measurements using a flow cytometer. Stochastic expression of the FRET pairs resulted in variable FRET efficiencies (E A ) in individual cells. A saturating binding relation was then constructed by correlating E A with the free donor concentration (D free ). We observed robust FRET for both Nb17 (Fig. 9B) and Nb82 ( Fig. 9C) with Na V 1.5 confirming baseline association of the heterologously expressed Nb in live cells. By contrast, coexpression of Cerulean alone with Na V 1.5 did not show appreciable FRET (Fig. 9D). These findings demonstrate the robust interaction of Nb17 and Nb82 with Na V 1.5 in live cells, thus G, sequence alignment of Nb82, Nb30, and Nb17. The three CDR regions are color coded as CDR1 (blue), CDR2 (green), and CDR3 (magenta). The secondary structure elements of Nb82 are placed on top of the alignment. CDR, complementarity-determining region; DSC, differential scanning calorimetry.
furnishing a new avenue to probe and manipulate Na V channels in physiology.
Nb82 as a Na V 1.4 and Na V 1.5 molecular detection reagent To take advantage of the high affinity of the Nbs for CTNa V 1.4 and CTNa V 1.5, we used Nb82 as a molecular reagent to detect Na V s in cell and tissue lysates by Western blot. His-tagged Nb82 detects Na V 1.4 in mouse skeletal muscle homogenates in addition to Na V 1.5 in mouse heart homogenates as shown by a band at 220 kDa. Notably, Nb82 does not detect Na V s in brain tissue (Fig. 9E). The Western blot signal detected by the His-tagged Nb82 (Fig. 9E) is comparable to that detected by a commercially available anti-Pan Na V Ab (Fig. 9F) as observed by signals corresponding to the same molecular weight for skeletal muscle and heart tissue samples in both blots. Moreover, the anti-Pan Na v Ab detects a band at 250 kDa in mouse brain tissue (Fig. 9F) that is not observed with the Nb82 detection. Also, Nb82 detects Nav1.5 from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) differentiated from normal hiPSCs as observed by a band at 220 kDa (Fig. 9H). As a control, when the blots were reprobed with simply anti-His horseradish peroxidase (HRP) Ab (Fig. 9,G and I), no bands were observed at 220 to 250 kDa showing the specificity of detection by Nb82 as expected.

Discussion
This study describes the generation and characterization of two Nbs, Nb17 and Nb82, that recognize and bind to the voltage-gated sodium channel Na V 1.4 and Na V 1.5 isoforms. The Nbs do not recognize isolated CaM, Na V 1.7, or Na V 1.9 channels showing their specificity for the Na V muscle isoform, Na V 1.4 (5). Both Nbs have structural stabilities similar to those observed for other globular proteins. According to the survey of Robertson and Murphy (35), the average T m for globular proteins is approximately 68 C compared with the T m s of 76 and 66 C for Nb17 and Nb82, respectively. Also, according to the survey of Robertson and Murphy, the mean enthalpy for a protein of 15 kDa at 70 C is 115 kcal/mol, which is close to the van't Hoff enthalpy of Nb17 suggesting structuring interactions similar to those observed for other proteins. The denaturation transition of Nb82 is irreversible at pH 8.0, which is not unseen for other proteins (36) that nevertheless undergo reversible denaturation at lower pH values.
The crystal structure of Nb82 reveals a long (15 amino acids) CDR3 for a llama-derived Nb that could possibly underlie the nanomolar binding affinities observed for the Nbs to Na V 1.4 and Na V 1.5.
Nb82 promises to be a high-affinity Na V 1.4(5) protein detection reagent. Our SEC experiments demonstrate formation of stable CTNa V 1.4(5)-CaM + Nb complexes. Importantly, binding kinetic experiments by BLI show nanomolar binding affinities for the Nbs to CTNa V 1.4-CaM and CTNa V 1.5-CaM proteins and no binding to CaM or CTNa V 1.7-CaM and CTNa V 1.9 proteins. Furthermore, flowcytometric FRET measurements in human embryonic kidney 293 (HEK293) cells expressing FL Na V 1.5 channel display high efficiency of binding of Nb17 and Nb82 to Na V 1.5 channels on the cell surface but no binding to CaM-expressing cells. In HEK293 cells expressing CTNa V 1.4 or CTNa V 1.5 isoforms, flow-cytometric FRET measurements show binding to Nb17 or Nb82. In contrast, cells expressing any of the other CTNa V 1.x isoforms displayed negligible binding. We further demonstrate that Nb82 can be used as a molecular reagent to detect Na V 1.4(5) proteins in various types of cell and tissue lysates since it recognizes endogenous Na V 1.4 channel from mammalian skeletal muscle in addition to Na V 1.5 from mammalian heart and hiPSC-CMs.
Nbs have been selected for other voltage-gated channelrelated subunits. For instance, the auxillary subunit of Ca V channel, Ca v β, has been targeted to design a potent genetically encoded high-voltage-activated calcium channel inhibitor (37). It is worth highlighting that the anti-Na V 1.4 and anti-Na V 1.5 Nbs produced as part of this study carry a simple CT 6×Histidine tag, ensuring ease of production and scale up of anti-Na V Nbs to serve as Na V detection reagents, crystallization chaperones, and as in vitro and in vivo research tools and molecular probes.

CT Na V 1.4-CaM protein expression for llama immunization
The GST-tagged CT region of Na V 1.4 (amino acids 1599-1764), in complex with CaM (CTNa V 1.4T-CaM), was expressed and purified from BL21-CodonPlus RIL E. coli cells using a GST-sepharose. Reduced L-glutathione eluted fractions were proteolyzed with PreScission protease followed by anion exchange chromatography and a final gel filtration chromatography as described by Yoder et al. (29). Purified CTNa V 1.4T-CaM at 56 mg/ml was used for generation of single-chain Abs by llama immunization.

Llama immunization and construction of Nb library
The immunization protocol and the construction of the library were done as previously described (30,38). In brief, llama was immunized three times intramuscularly every 15 days with 100 μg of purified CTNa V 1.4T-CaM emulsified with complete Freund's adjuvant. The humoral immune response in the sera was monitored by ELISA performed in plates coated with CTNa V 1.4T-CaM. Forty-five days after the first immunization (D45), the animal was bled. PBLs were isolated from 300 ml of blood by Ficoll-Paque gradient centrifugation. Total RNA was purified from these cells and subjected to cDNA synthesis. The Nb coding regions were amplified by PCR using specific primers: forward (fwd_001): 5 0 -GTCCTGGCTGCTCTT CTACAAGG-3 0 ; reverse (rvd_002): 5 0 -GGTACGTGCTGTT-GAACTGTTCC-3 0 . The amplicons were purified from agarose gels, digested with PstI and NotI, and cloned into the pHEN4 phagemid vector downstream of the PelB-leader peptide and upstream of the hemagglutinin (HA) tag. E. coli TG1 cells were transformed with this vector for obtaining a phage display library. About 15 clones randomly chosen from the library were used for plasmid DNA preparation and run on agarose gel electrophoresis for qualitative analysis, where ˃70% of clones were found to be positive for Nbs.

Phage display selection of Na V 1.4-CaM-specific Nbs
The panning was performed in MaxiSorp plates. Briefly, wells were sensitized with either 1 μg of CTNa V 1.4T-CaM + 1 mM DTT or uncoated to serve as negative controls. After blocking with 3% skimmed milk in PBS, 1 × 10 12 phages in PBS were added to each test and control coated wells and incubated for 2 h at room temperature (RT). Wells were then extensively washed with 25 mM Tris, 150 mM NaCl, 1 mM DTT, 0.05% Tween-20, pH 8.0, and bound phages were eluted with 0.25 mg/ml trypsin. Eluted phages were titrated and subjected to rounds of panning following the same procedure. Output phage titers were estimated by infection of TG1 cells and plating them on LB with 100 μg/ml ampicillin and 2% glucose. A total of 87 randomly chosen clones were grown in deep-well plates containing 1 ml of 2× TY (Tryptone 16 g, yeast extract 10 g, NaCl 5 g per L) medium added with 100 μg/ ml ampicillin and 0.1% glucose for 3 h at 37 C and 200 rpm until cell growth reached the exponential phase. The expression of Nbs was induced by adding 1 mM IPTG per well with shaking at 200 rpm for 4 h at 28 C. The Nbs were obtained from the periplasm and tested by ELISA on plates sensitized with 1 μg of CTNa V 1.4T-CaM. After washing with 25 mM Tris, 150 mM NaCl, 1 mM DTT, 0.05% Tween-20, pH 8.0, Nbs were detected by incubation with anti-HA Ab and developed using antirat IgG peroxidase conjugate. About 14 positive clones were selected for sequencing using universal M13 A B Figure 4. Nb17 and Nb82 recognize the Na V -muscle isoforms. A, ELISA bar graphs using purified Nb17 (magenta), the absence of Nb17 (green), Nb82 (orange-yellow), and the absence of Nb82 (gray). The red box clusters Na V proteins that represent muscle isoforms CTNa V 1.4T-CaM, CTNa V 1.4FL-CaM, CTNa V 1.5T-CaM, and CTNa V 1.5FL-CaM. The gray box clusters the other Na V isoforms tested, CTNa V 1.7T-CaM, CTNa V 1.7FL-CaM, CTNa V 1.9T, CTNa V 1.9FL, and CaM. Data are representative of three independent experiments. B, sequence alignment of CTNa V 1.4FL, CTNa V 1.5FL, CTNa V 1.7FL, and CTNa V 1.9FL proteins. The black trace indicates the limits of the T CTNa V T-CaM constructs. CaM, calmodulin; FL, full length; Nb, nanobody; T, truncated. reverse as primer and then classified in families based on the length and variability of their CDR2 and CDR3. Multiple sequence alignments were done with Clustal Omega (39). One representative clone for each family was selected for periplasmic Nb expression, purification, and further characterization. To determine whether the clones from the four families of Nbs recognize CTNa V 1.4-CaM or apo CaM, periplasmicextract ELISA was performed. MaxiSorp plates were sensitized with 0.1 μg of either CTNa V 1.4T-CaM, Ca 2+ CaM, or apoCaM for 2 h at RT and then blocked with 5% bovine serum albumin (BSA) for 2 h at RT. After removal of blocking solution, 50 μl of a 1:100 dilution of TG1 periplasmic extracts expressing each Nb (Nb17, Nb26, Nb30, and Nb82) were incubated overnight at 4 C (30). Nbs were detected using anti-HA-tag high-affinity secondary Ab by ELISA developed using an antirat IgG peroxidase-conjugated Ab. Nb17, Nb30, and Nb82 were then subcloned in pHEN6 vector. The coding regions were amplified by PCR using the following specific primers: forward (A6E): 5 0 -GATGTGCAGCTGCAG-GAGTCTGGRGGAGG-3';reverse (p38): 5 0 -GGACTAGTG CGGCCGCTGGAGACGGTGACCTGGGT-3'. The amplicons were purified from agarose gels, digested with restriction enzymes PsTI and BstEII, and cloned into the pHEN6-TEV-His vector for periplasmic expression of recombinant Nbs in E. coli Rosetta-gami 2 (DE3) cells. Nb82-His cloned into pHEN6-TEV-His was subcloned to generate Nb82-His-StrepII.

Purification of Nbs Nb17 and Nb82
The expression and purification of the Nbs was performed as described previously with minor modifications (30). Transformed E. coli Rosetta-gami 2 (DE3) cells were grown in LB medium supplemented with 100 μg/ml of carbenicillin, 50 μg/ml of kanamycin, and 20 μg/ml of chloramphenicol in addition to 1 mM MgCl 2 and 0.1% glucose. Proteins were expressed by inducing the cultures with 1 mM IPTG at an absorbance of 0.8 to 0.9 at 600 nm at 18 C at 200 rpm overnight. Cells were harvested by centrifugation and frozen at −80 C for at least 2 h before proceeding to thawing and No signal is observed in the lanes that contained CTNa V 1.7T/FL and CTNa V 1.9T/FL suggesting that Nb82 does not recognize these two isoforms. Western blot was developed using Nb82-His as primary antibody and anti-HisHRP antibody as secondary. All Western blot data show one representative experiment of three. CTNa V -CaM proteins are labeled CTNa V 1.xT (1.xT) and CTNa V 1.xFL (1.xFL). B, Nbs tethered to Cerulean serve as a FRET donor, whereas Venus attached to amino-terminal region of CTNa V 1.x serves as a FRET acceptor. Robust FRET is observed between Nb17 and CTNa V 1.4/5. Other CTNa V s demonstrate reduced binding. FRET efficiency (E A ) is plotted against the free donor concentration (D free ). Each cell represents data from a single cell. C, the relative association constant, K a,EFF (in arbitrary units) computed as 1/Kdeff from the fits in (A), demonstrates the preference of Nb17 for CTNa V 1. 4   Nanobodies against Na v 1.4 and Na v 1.5 (TN buffer). Beads were washed in a gravity flow column using 40 ml of TN buffer (pH 8.0) with 10 mM imidazole, and Nbs were eluted using 40 ml of TN buffer (pH 8.0) with 300 mM imidazole. Purification fractions were loaded on an any kilodalton gel and bands analyzed by SDS-PAGE, and the Nbcontaining elution fractions were pooled, dialyzed at 4 C into low-salt TN buffer (20 mM Tris-HCl, 50 mM NaCl, 2 mM DTT, pH 7.5), and concentrated to 1 mg/ml before loading on a Superdex 75 column using TN buffer. The peak fractions from gel filtration containing the Nbs were then concentrated using a 3.5 kDa cutoff filtering units to a final concentration of 10 to 11 mg/ml as 24 μl aliquots and flash frozen and stored at −80 C.
Purification of CT regions of Na V 1.4, Na V 1.5, Na V 1.7, and Na V 1.9 in complex with CaM (CTNa V 1.X-CaM) CT regions of Na V 1.4(UniProtKb:P35499), Na V 1.5 (Uni-ProtKb:Q14524), Na V 1.7 (UniProtKb:Q15858), and Na V 1.9 (UniProtKb:Q9UI33) in complex with CaM (UniProtKb:P0DP23) (CTNa V 1.X-CaM) were used. The constructs of the CT regions of the voltage-gated sodium channel isoforms were named consistently as "truncated (T)" for constructs ending at equivalent residue of Na V 1.4 1764 and "FL" for constructs ending at equivalent residue of Na V 1.4 1836 . The constructs are CTNa V 1.4T with amino acids 1599 to 1764, CTNa V 1.4FL with amino acids 1599 to 1836, CTNa V 1.5T with amino acids 1775 to 1940, CTNa V 1.5FL with amino acids 1775 to 2016, CTNa V 1.7T with amino acids 1761 to 1928, CTNa V 1.7FL with amino acids 1761 to 1988, CTNa V 1.9T with mino acids 1605 to 1768, and CTNa V 1.9FL with amino acids 1605 to 1791 (Table S1 and Fig. 3B). Each construct was coexpressed with mammalian CaM and purified from BL21-CodonPlus RIL cells using a GST-sepharose column followed by anion exchange chromatography and a final gel filtration chromatography step as described by Yoder et al. (29) with minor modifications. In brief, cells were grown overnight at 37 C in 100 ml of LB medium supplemented with 50 μg/ml kanamycin, 20 μg/ml chloramphenicol, and 100 μg/ml carbenicillin. About 10 ml of the overnight culture was used to inoculate 1 l of LB media containing the same antibiotics. The cells were grown at 37 C to an absorbance of 0.8 to 0.9 at 600 nm, and protein expression was induced with 1 mM IPTG.  The cells were grown overnight at 18 C (approximately 18 h), centrifuged, and the cell pellet was frozen at −80 C. After thawing, pellets were resuspended with PBS at 5× volume/ weight ml/g of cells. DNAse was added, the cells were lysed using a microfluidizer, and the lysate was clarified at 27,500g. The supernatant was loaded on to a 3 ml GST resin using gravity flow. The column was washed with 30 ml wash buffer (PBS added with 100 mM NaCl), and free CaM was purified from this fraction for other experiments. The CTNa V T-CaM and CTNa V FL-CaM complexes were eluted in aliquots of 5 ml with an elution buffer containing 10 mM reduced L-glutathione in 50 mM Tris-HCl (pH 8.0). Eluted fractions containing protein were pooled, and 5 μg of PreScission protease was added per milligram of CTNa V -CaM for cleaving the GST tag. Dialysis was performed against 2 l of buffer containing 20 mM Tris, 50 mM NaCl, 1 mM DTT, and pH 7.4. The buffer was changed twice, and the final dialysis was allowed to proceed overnight at 4 C. The dialyzed and PreScission proteasecleaved protein was loaded on a 15 ml Source Q anion exchange column (GE). Elution was performed using buffer 20 mM Tris, 1 mM DTT, pH 7.4 and a gradient of 50 to 500 mM NaCl. Free and cleaved GST eluted at 8 mS/cm, and CTNa V -CaM complexes eluted at between 14 and 27 mS/cm conductance that varied depending on the Na V isoform.
Fractions were judged to be >95% pure by SDS-PAGE gel and then pooled and concentrated to 15 to 20 mg/ml and flash frozen and stored at −80 C. In the case of CTNa V 1.9T and CTNa V 1.9FL, CaM did not coelute with the CTNa V s unlike the other cases.
Detection of Nb specificity for Na V s by ELISA Purified Nbs were assessed for recognition of Na V proteins in high-binding 96-well plates. The analytes, purified CTNa V T-CaM and CTNa V FL-CaM protein isoforms (CTNa V 1.4T-CaM, CTNa V 1.4FL-CaM, CTNa V 1.5T-CaM, CTNa V 1.5FL-CaM, CTNa V 1.7T-CaM, and CTNa V 1.7FL-CaM), CTNa V 1.9T, CTNa V 1.9FL, and CaM, were diluted in PBS and coated (1 μg/well) to a 96-well plate using carbonatebicarbonate buffer, pH 9.5, at 4 C overnight. Next, the plate was washed five times using 100 μl/well wash buffer (PBS added with 0.1% Tween-20). Then, protein-coated wells were incubated with 100 μl/well of 0.01 μg of Nb17 or Nb82 diluted in 1× blocking buffer (PBS added with 2% BSA) for 2 h at RT on a shaking platform. The plates were then washed five times using 100 μl/well of wash buffer and incubated with mouse anti-His-peroxidase Ab at 100 mU/ml in blocking buffer for 1 h at RT. The plates were finally washed in wash buffer five times, and peroxidase activity was assayed using 100 μl/well anti His Figure 9. Nanobodies (Nbs) as tools to detect Na V channels from live cells and tissue homogenates. A, schematic of FRET two-hybrid assay to probe live-cell binding of Nbs to holo-Na V 1.5 channels. Nbs tethered to Cerulean serve as a FRET donor, whereas Venus attached to Na V 1.5 serves as a FRET acceptor. B, robust FRET is observed between Nb17 and Na V 1.5. FRET efficiency (E A ) is plotted against the free donor concentration (D free ). Each cell represents data from a single cell. C, analysis of Nb82 also shows strong FRET with Na V 1.5. D, no appreciable FRET is observed between Na V 1.5-Venus and Cerulean alone. E, Western blot showing Nb82-His used as the primary antibody recognizing Na V 1.4(5) channels from tissues; mouse skeletal muscle, mouse heart, and brain. Blot developed using an anti-His-HRP antibody. o-phenyl diamine in 150 mM citrate phosphate buffer and 30% H 2 O 2 . The plates were incubated in the dark for a few minutes until a yellow color developed in at least one of the wells. The reaction was stopped by the addition of 100 μl/well of 2 N H 2 SO 4 , and the absorbance was read at 450 nm (20 flashes) using a microplate reader.

DSC experiments
DSC experiments were performed using a MicroCal capillary DSC instrument. Purified Nbs, Nb17 and Nb82, were dialyzed into buffer containing 10 mM PBS, pH 8.0, with 1 mM Tris(2carboxyethyl)phosphine, and further diluted with the dialysate to an experimental concentration of 0.15 mg/ml. The exact concentration of each protein solution was determined from the absorbance at 280 nm. Each solution was thoroughly degassed before loading of the calorimetric cell to have an effective volume of 138 μl. The reference cell was loaded with the dialysate. Thermal scans were conducted from 10 to 90 C at a rate of 1 C/min. The raw data were collected and processed using the software provided with the instrument. Data were analyzed after baselines were subtracted as described by Freire (40). The resulting transition excess heat capacity curves were fitted by nonlinear least squares in terms of reversible or irreversible denaturation models depending on whether the transitions were reversible or irreversible (33). For reversible transitions, the fitting procedure yields the calorimetric enthalpy, van't Hoff enthalpy, and transition temperature. For irreversible transitions, the fitting procedure yields the calorimetric enthalpy, activation energy, and transition temperature.

Crystallization of Nb82
Purified Nb82 was used at 10 mg/ml for all crystallization experiments. Sparse matrix commercial crystallization screens were used to find conditions in hanging-drop vapor diffusion by mixing equal volumes of Nb82 and reservoir solution. Nb82 crystallized in 2% (w/v) PEG monomethyl ether 550, 1.8 M ammonium sulfate, 0.1 M Bis-Tris, pH 6.5 was used for X-ray diffraction experiments. Crystals appeared as needles after 1 day of equilibration at 20 C and reached 100 μm in their longest dimension on day 30. These needles were used to macro seed into 2 μl hanging-drop vapor diffusion plates with drops containing equal volumes of 10 mg/ml Nb82 and reservoir conditions optimized around the original crystallization condition varying the concentrations of PEG and ammonium sulfate. New crystals appeared in 2% (w/v) PEG monomethyl ether 550, 1.5 to 1.8 M ammonium sulfate, 0.1 M Bis-Tris, pH 6.5, on day 5. The crystals grew into cubes with largest samples being 125 μm in their longest dimension and were harvested on day 30 postseeding from the mother liquor mixed with 1 M lithium sulfate as the cryoprotectant into Hampton loops and plunge-frozen into liquid nitrogen.
Data collection and structure refinement X-ray diffraction data of the Nb82 crystal were collected at 100 K at the NSLS II 17-ID-1 (AMX) on a DECTRIS Eiger 9M. Data were processed with fastdp (41) and XDS (42) and scaled (42) using XSCALE. Initial phases were obtained by molecular replacement using an Nb structure as search model (PDB ID: 5LMJ (14)) with the CCP4 program PHASER (43). Initial models were improved with multiple rounds of rebuilding using Coot (44) and refinement using REFMAC, version 5.8 (45). The quality of the model was assessed with Coot (44) validation tools and the wwPDB validation servers (46). Statistics are shown in Table 1. The final model contains four Nb82 molecules in the asymmetric unit.
Nb-mediated shift of Na V s by SEC Mobility in-gel filtration chromatography was performed to verify formation of CTNa V -CaM + Nb complexes using purified proteins (Figs. 5 and 6). Nbs and Na V s were mixed in a 1.2:1 M ratio, incubated for 2 h to overnight at 4 C, and run on a Superdex 75 column using 20 mM Tris, 50 mM NaCl, and pH 7.4. Chromatograms were exported as Excel files to GraphPad Prism (GraphPad Software, Inc) for analysis and plotting. About 0.5 mg of egg-white lysozyme (14 kDa) and 0.5 mg of BSA (66 kDa) were run to serve as molecular weight gel filtration standards using the same experimental conditions to evaluate the molecular weight of the peak elution fractions.
Nb17 and Nb82 binding kinetics using BLI Nb17 and Nb82 binding to CTNa V 1.4 and CTNa V 1.5 was measured by BLI. Data were acquired using the Data acquisition software, version 11.0, and analyzed using the Data analysis software, version 11.0 (ForteBio, Sartorius Corp). The assay was performed in kinetics mode using 200 μl protein/ well in a 96-well plate format. His-tagged proteins, Nb17 and Nb82, or just buffer was immobilized on Ni-NTA biosensors. Nb17 and Nb82 loading was done at 2.5 μg/ml concentration for 300 s to prevent overcrowding and self-association of the ligand. CTNa V 1.4T-CaM, CTNa V 1.5T-CaM, CTNa V 1.7T-CaM, and CTNa V 1.9T-CaM were tested as analytes. To measure Nb association with Na V proteins, the Nb-loaded Ni-NTA sensors were first transferred to wells containing blocking reagent (0.1% biocytin) in assay buffer for 150 s to prevent nonspecific binding and then to wells containing assay buffer until a stable baseline was reached (100 s). Following this, sensors were dipped into wells with 1:2 serially diluted Na V proteins (at 200, 100, 50, 25, 12.5, and 6.25 nM concentrations) for 300 s followed by a 300 s dissociation step in assay buffer. All experiments were carried out at 25 C and acquisition standard kinetics at 5 Hz with the assay plate shaking at 1000 rpm.

Flow cytometric FRET two-hybrid assay
Flow cytometric FRET two-hybrid assay for detecting Nb interaction with Na V 1.5 was performed as in previous studies (34). Briefly, HEK293 cells were cultured in 12-well plates and transfected with PEI 25 kDa linear polymer (Polysciences; catalog no.: 2396602). For each experiment, we cotransfected: 0.5 μg of Nb17 or Nb82 fused to Cerulean; 2 μg of Venustagged FL Na V 1.5; and 0.5 μg of t-Antigen. To test the interaction of Na V 1.x isoforms to the Nbs, we cotransfected 0.5 μg of Nb17 or Nb82 fused to Cerulean, 2 μg of Venus-tagged CTNa V 1.x; and 0.5 μg of t-Antigen. The cDNA pairs were mixed together in 200 μl of serum-free Dulbecco's modified Eagle's medium (DMEM) media, and 5 μl of PEI was added into each sterile tube. Following 15 min of incubation, PEI-cDNA mixtures were added to the 12-well plates, and cells were cultured for 2 days prior to experimentation. Protein synthesis inhibitor, cycloheximide (100 μM), was added to cells 2 h prior to experimentation to enhance fluorophore maturation. For FRET measurements, we utilized an LSR II (BD Biosciences) flow cytometer equipped with 405, 488, and 633 nm lasers for excitation and 18 different emission channels as previously described (34). Forward and side scatter signals were detected and used to gate for single and healthy cells. Fluorescence emission from three different channels (BV421, FITC, and BV510) were used to estimate fluorescence emission in the donor, acceptor, and FRET channels. Data were analyzed using custom MATLAB software (Mathworks).
Western blots using Nb82 to detect purified CTNa V -CaM proteins Western blot analysis was performed to assess whether Nb82 can detect CTNa V 1.4 and CTNa V 1.5 proteins using Nb82 as the primary Ab. For this, 1 μg/lane of purified CTNa V 1.4T-CaM, CTNa V 1.4FL-CaM, CTNa V 1.5T-CaM, CTNa V 1.5FL-CaM, CTNa V 1.7T-CaM, CTNa V 1.7FL-CaM, CTNa V 1.9T, CTNa V 1.9FL, CaM, Nb82-His, Nb17-His proteins were run on an Any kD Mini-PROTEAN TGX gel at 180 V for 30 min. Proteins were transferred using the P3 program (20 V for 10 min) of the iBlot2 protein transfer system and blocked with blocking buffer (1× PBS with Tween-20 [PBST], pH 7.4 with 5% nonfat dry milk) for 27 C for 1 h and then washed five times in PBST (0.1% Tween-20). Proteins were incubated with Nb82-His (1 μg/ml) in blocking buffer shaking in the cold room overnight. Blots were further washed five times in PBST and probed using an anti-His-HRP-Ab at 1:500 dilutions in blocking buffer by incubating at 27 C for 1 h with shaking. Western blot was developed and visualized using the GeneSys image acquisition software to obtain exposures at 20 s, 2 min, and 5 min. The blots were exported as tiff images and viewed and edited using ImageStudioLite (Li-COR Biosciences).
Western blots using Nb82 to detect endogenous Na V 1.4 and Na V 1.5 Western blot analysis was performed to assess whether Nb82 could detect Na V 1.4 in skeletal muscle homogenates and Nav1.5 in heart of adult mouse and also Nav1.5 in cell lysate from hiPSC-CM. Heart muscle, brain, and quadriceps femoris muscle were collected from a 16-week-old female C57BL/6 mouse and snap frozen before analysis by Western blot. Tissue samples were crushed using a tissue-grinder pestle and resuspended in 1× PBS (pH 7.4) with 500 mM NaCl and protease inhibitors and sonicated in the cold room with a 10 s pulse-20 s pause program for a total of 10 min. Following sonication, 2% (v/v) Triton X-100 was added to the lysate and incubated on ice for 30 min. The lysate was then centrifuged in a table-top centrifuge at 10,000g at 4 C for 30 min. The supernatants were mixed with equal volumes of 2× SDS-loading buffer and loaded at 40 μl/lane and run on an NuPAGE 4 to 12% Bis-Tris gel using 1× MES-SDS running buffer (Novagen) at 230 V for 72 min. Proteins were transferred, and blot was developed as described previously using blocking buffer (1× PBST [pH 7.4] with BSA). The blots were probed with Nb82 followed by anti-His-HRP detection or by pan-Na V Ab and antimouse IgG-HRP or using anti-His-HRP Ab alone as control.

Llama immunization and Nb phage library
Freund's adjuvant (Sigma), ELISA of immune sera using MaxiSorp plates (Thermo Scientific), PBL cells isolated using Ficoll-Paque (GE Healthcare), total RNA isolation using RNeasy Midi Kit (Qiagen), restriction enzymes for cDNA amplification, PstI and NotI (New England Biolabs), E. coli Funding and additional information-J. N. N. was supported by the NIH grant (grant no.: R25 GM109441) and the Vivien Thomas Scholars Initiative at Johns Hopkins University. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.