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

Abstract

Voltage-gated sodium channels, Na_Vs, are responsible for the rapid rise of action potentials in excitable tissues. Na_V 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-Na_V nanobodies (Nbs), Nb17 and Nb82, that recognize the Na_V1.4 (skeletal muscle) and Na_V1.5 (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 Na_V1.4. The Nbs were expressed in Escherichia coli, purified, and biophysically characterized. Development of high-affinity Nbs specifically targeting a given human Na_V isoform has been challenging because they usually show undesired crossreactivity for different Na_V isoforms. Our results show, however, that Nb17 and Nb82 recognize the CTNa_V1.4 or CTNa_V1.5 over other CTNav isoforms. Kinetic experiments by biolayer interferometry determined that Nb17 and Nb82 bind to the CTNa_V1.4 and CTNa_V1.5 with high affinity (K_D ∼ 40-60 nM). In addition, as proof of concept, we show that Nb82 could detect Na_V1.4 and Na_V1.5 channels in mammalian cells and tissues by Western blot. Furthermore, human embryonic kidney cells expressing holo Na_V1.5 channels demonstrated a robust FRET-binding efficiency for Nb17 and Nb82. Our work lays the foundation for developing Nbs as anti-Na_V reagents to capture Na_Vs from cell lysates and as molecular visualization agents for Na_Vs.

Keywords: FRET; Lama glama; Na(V)1.4; Na(V)1.5; X-ray diffraction; biolayer interferometry; hIPSC-CM; nanobody; voltage-gated sodium channel.

Figure 1

Scheme describing the steps used for the selection of high-affinity nanobodies (Nbs) specific for voltage-gated sodium channels. Step 1: Production of CTNa_V1.4T–CaM to use as antigen. Step 2: Llama immunization with CTNa_V1.4T–CaM. Step 3: Collection of immune sera from llama on day 35 and day 40 postimmunization. Step 4: Isolation of lymphocytes to extract total RNA. Step 5: RT–PCR to obtain complementary DNA of VHH domains. Step 6: Cloning of VHH complementary DNA (750 bp) into Escherichia coli shuttle vector for phage display. Step 7: Phage display for selection of Nbs against CTNa_V1.4T–CaM in M13 phage. Step 8: Screening phage library by ELISA for high-binding Nb clones. Step 9: Cloning in pHEN6 vector and purification of selected Nb clones (Nb17, Nb30, and Nb82) in E. coli. Step 10: Crystallization of Nb82 using commercially available sparsematrix screens. Step 11: Characterization of Nb binding to Na_V1.4 and Na_V1.5 isoforms. This rendition was contributed by Sora Ji (Sorajistudio.com). CaM, calmodulin.

Figure 2

Identification of nanobodies (Nbs) specific for Na_V1.4.A, ELISA of the periplasmic extract using positively selected clones. Absorbances higher than 2 (orange dashed line) were considered positive. “+” signs over Nb17, Nb30, and Nb82 indicate that they are specific to CTNa_V1.4T–CaM but not to Ca²⁺ CaM or apo-CaM. B, SDS-PAGE gel of IMAC purification of Nb17. C, same as (B) for Nb82. D, size-exclusion chromatogram of Nb17 (17.4 kDa, purple) and Nb82 (16.8 kDa, yellow). Lysozyme (14 kDa, gray) used as gel filtration molecular weight standard is also included. CaM, calmodulin; E, elution fraction; FT, flow-through; IMAC, immobilized metal affinity chromatography; S, supernatant; W, wash.

Figure 3

Nanobody (Nb) thermal stability and crystal structure of Nb82.A, DSC curve showing the temperature denaturation of Nb17 undergoing reversible denaturation with T_m centered at 75.8 °C. B, same as A. Nb82 undergoes irreversible denaturation with T_m centered at 66.0 °C. C, cartoon representation of Nb82 (yellow) displaying the CDR1 (blue), CDR2 (green), and CDR3 (magenta). D, same as (A) with 180° rotation along the vertical axis. E, bird's eye view of Nb82 in (A) with surface coloring according to the CDRs. F, same as (C) with Nb82 surface colored according to the electrostatic charges. 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.

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_V1.4T–CaM, CTNa_V1.4FL–CaM, CTNa_V1.5T–CaM, and CTNa_V1.5FL–CaM. The gray box clusters the other Na_V isoforms tested, CTNa_V1.7T–CaM, CTNa_V1.7FL–CaM, CTNa_V1.9T, CTNa_V1.9FL, and CaM. Data are representative of three independent experiments. B, sequence alignment of CTNa_V1.4FL, CTNa_V1.5FL, CTNa_V1.7FL, and CTNa_V1.9FL proteins. The black trace indicates the limits of the T CTNa_VT–CaM constructs. CaM, calmodulin; FL, full length; Nb, nanobody; T, truncated.

Figure 5

Selectivity of Nb17 and Nb82 in detecting Na_V1.4 and Na_V1.5 channels.A, Western blot of purified CTNa_V–CaM proteins, CaM alone, and Nb17-His, Nb82-His, CaM, and GST alone showing positive signals for CTNa_V1.4T/FL (1.4T/1.4FL), CTNa_V1.5T/FL (1.5T/1.5FL), and Nb17, Nb82-His and Nb82-StrepII (Nb82S). No signal is observed in the lanes that contained CTNa_V1.7T/FL and CTNa_V1.9T/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_V1.xT (1.xT) and CTNa_V1.xFL (1.xFL). B, Nbs tethered to Cerulean serve as a FRET donor, whereas Venus attached to amino-terminal region of CTNa_V1.x serves as a FRET acceptor. Robust FRET is observed between Nb17 and CTNa_V1.4/5. Other CTNa_Vs 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_V1.4/1.5 over other Na_V isoforms. D, analysis of Nb82 shows strong FRET with CTNa_V1.4/5. Other Venus-CTNa_V1.x isoforms exhibit weaker binding. E, the K_a,EFF values confirm strong preference of Nb82 for CTNa_V1.4/5 over other CTNa_V1.x isoforms. CaM, calmodulin; FL, full length; GST, glutathione-S-transferase; HRP, horseradish peroxidase; Nb, nanobody; T, truncated.

Figure 6

Nb82 forms a complex with CTNa_V1.4–CaM and CTNa_V1.5–CaM detected by size-exclusion chromatography (SEC).A, SEC profile for CTNa_V1.4T–CaM + Nb82 (solid blue line) showing the appearance a new peak to the left of the CTNa_V1.4T–CaM peak (dashed line) indicating complex formation. B, SDS-PAGE gel showing elution fractions from (A). The CTNa_V1.4T–CaM + Nb82 complex elutes at 8.7 ml. C and D, same as A and B, using construct CTNa_V1.4FL–CaM. The CTNa_V1.4FL–CaM + Nb82 elutes at 8.9 ml. E, SEC profile for CTNa_V1.5T–CaM + Nb82 (solid green line) showing the appearance of the peak of the complex to the left at 9.1 ml compared with CTNa_V1.5T–CaM (dashed line) at 10.6 ml. F, SDS-PAGE gel showing elution fractions from (E). G and H, same as (E) and (F) using construct CTNa_V1.5FL–CaM. The CTNa_V1.5FL–CaM + Nb82 complex elutes at 8.4 ml. Gel filtration molecular weight standards. BSA (66 kDa, dashed gray line) and lysozyme (14 kDa, solid gray line) are included in A, C, E, and G. BSA, bovine serum albumin; CaM, calmodulin; FL, full length; Nb, nanobody; T, truncated.

Figure 7

Nb82 binds to CTNa_V1.4T–CaM and CTNa_V1.5T–CaM with nanomolar affinity.A, BLI sensorgram of Nb82 titrated with CTNa_V1.4T–CaM at concentrations 6.25, 12.5, 25, 50, 100, and 200 nM plotted as nanometer shift with time. B, same sensorgram as A but for Nb82 titrated with CTNa_V1.5T–CaM. C–E, sensorgrams showing no binding of Nb82 to CaM alone, CTNa_V1.7T–CaM, or CTNa_V1.9T, respectively. BLI, biolayer interferometry; CaM, calmodulin; Nb, nanobody; T, truncated.

Figure 8

Nb17 binds to CTNa_V1.4T–CaM and CTNa_V1.5T–CaM with nanomolar affinity.A, BLI sensorgram of Nb17 titrated with CTNa_V1.4T–CaM at concentrations 6.25, 12.5, 25, 50, 100, and 200 nM plotted as nanometer shift with time. B, same sensorgram as (A) but for Nb17 titrated with CTNa_V1.5T–CaM. C–E, sensorgrams showing no binding of Nb17 to CaM alone, CTNa_V1.7T–CaM, or CTNa_V1.9T, respectively. BLI, biolayer interferometry; CaM, calmodulin; Nb, nanobody; T, truncated.

Figure 9

Nanobodies (Nbs) as tools to detect Na_Vchannels from live cells and tissue homogenates.A, schematic of FRET two-hybrid assay to probe live-cell binding of Nbs to holo-Na_V1.5 channels. Nbs tethered to Cerulean serve as a FRET donor, whereas Venus attached to Na_V1.5 serves as a FRET acceptor. B, robust FRET is observed between Nb17 and Na_V1.5. 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_V1.5. D, no appreciable FRET is observed between Na_V1.5-Venus and Cerulean alone. E, Western blot showing Nb82-His used as the primary antibody recognizing Na_V1.4(5) channels from tissues; mouse skeletal muscle, mouse heart, and brain. Blot developed using an anti-His-HRP antibody. F, same as (E) developed using the Pan-Na_V antibody (Sigma). G, same as (E) and (F) developed using only anti-His HRP antibody as a control. H, Western blot showing Nb82-His used as the primary antibody recognizing Nav1.5 from hiPSC–CMs. I, same as (H) developed using anti-His HRP antibody as a control. CM, cardiomyocyte; hiPSC, human-induced pluripotent stem cell; HRP, horseradish peroxidase.