nb linker pctx1  (Alomone Labs)

Journal: eLife

Article Title: Structure and analysis of nanobody binding to the human ASIC1a ion channel

doi: 10.7554/eLife.67115

Figure Lengend Snippet: Two side, top and bottom views of superimposed structures of hASIC1a-NbC1 complex (red) with ( A ) MitTx-bound to chicken ASIC1 (4ntw) in open conformation (orange). In side views, the threefold axis of the channel is indicated by a dashed vertical line; in top and bottom views it is indicated by dotted triangles. ( B ) PcTx1-bound chicken ASIC1 (3s3x) (gray). ( C ) Mambalgin-1-bound human ASIC1 (7ctf) (blue). Only one subunit is shown for simplicity. Surface clashes are indicated by dashed rectangles. Nb.C1, MitTx- α, MitTx- β, PcTx1, Mambalgin-1 are shown as red, orange, light-orange, light-purple, marine respectively.

Article Snippet: PcTx1 was used at 10 nM and MitTx at 50 nM, both were purchased from Alomone Labs. Oocytes were pre-incubated with purified Nb or Nb-linker-PcTx1 (10 nM) for 20 min at RT before recording.

Techniques:

Journal: eLife

Article Title: Structure and analysis of nanobody binding to the human ASIC1a ion channel

doi: 10.7554/eLife.67115

Figure Lengend Snippet: ( A ) Representative currents of an oocyte expressing hASIC1a activated with pH 6.0 followed by a second activation with 50 nM MitTx at pH 7.4. ( B ) Same experiment after pre-incubation of the oocyte with 50 nM Nb.C1 for 15 min. ( C ) Summary of the peak currents from pH 6.0 and MitTx activations. In this and all traces, the conditioning pH is 7.4. The bars represent the mean±SD of currents, n=8 Nb control and n=6 Nb.C1. Asterisks indicate statistical significance in t-test, p<0.001. ( D ) Cartoon of the proposed mechanism of how Nb.C1 associated with hASIC1a may interfere with MitTx binding. ( E ) Whole-cell patch clamp of SH-SY5Y cells activated with pH 6.0 generates typical hASIC1a currents. Proton-induced currents are inhibited by PcTx and amiloride. ( F ) Immunofluorescence confocal image of SH-SY5Y cells incubated with Nb.C1-PcTx1-HA fusion and anti-HA antibody (green) shows cells decorated on the periphery. Nuclei were stained with DAPI (blue). Scale bar, 5 µm. ( G ) Cartoon representation showing the Nb.C1-PcTx1 polypeptide binding to two distinct sites on the surface of hASIC1a, accounting for a possible mechanism of toxin potentiation. ( H ) Confocal images of live HEK-293 cells transfected with hASCIC1a-Flag on coverslips incubated with Nb.C1-HA for 30 min and followed for 0, 1, 2, 3, and 4 hr at 18°C in DMEM containing HEPES. Three of the five time points are shown. At each 1 hr interval, all cells were washed except for the one dish of cells removed for fixation. All cells were processed for immunofluorescence with HA and Flag monoclonals to visualize Nb.C1-HA and hASIC1a-Flag, respectively. Nb.C1-HA labels only the cell surface whereas hASIC1a distributes in the plasma membrane and intracellular endoplasmic reticulum and perinuclear membrane. Scale bar, 5 µm. ( I ) Quantification of fluorescence intensity of Nb.C1 (red channel) normalized to time 0 hr ( t 0 ). For each time point 300 cells were analyzed. Columns are the mean ± SEM. ( J ) Coomassie blue SDS-PAGE of purified fusion proteins (Nb.C1-FlexLinker-PcTx and Nb.C1-RigidLinker-PcTx) and Nb.C1 alone. On the right a cartoon representation of the fusion proteins. ( K ) Representative examples of oocytes expressing hASIC1a exposed to 10 nM of PcTx1 or 10 mM of Nb.C1-Rigid-PcTx1 fusion for 60 s prior to serial activations with a change of pH from 7.35 to 6.0. Cells remained in the perfusion chamber throughout the experiment. ( L ) Time course of recovery of acid-induced currents in control (no pretreatment), and pretreatment with PcTx1, Nb.C1-Flex-PcTx, or Nb.C1-Rigid-PcTx1. Preconditioning pH 7.35, activation pH 6.0. Data were fit with a single exponential a ( 1 − e − t / τ ) where τ is 220 s for PcTx, 350 and 880 s for Nb.C1-Flex-PcTx and Nb.C1-Rigid-PcTx; a = 0.90 for PcTx, and 0.16 and 0.14 for the fusions, respectively. Data points represent the mean ± SD of 7–12 cells. Values of currents from each cell are shown in .

Article Snippet: PcTx1 was used at 10 nM and MitTx at 50 nM, both were purchased from Alomone Labs. Oocytes were pre-incubated with purified Nb or Nb-linker-PcTx1 (10 nM) for 20 min at RT before recording.

Techniques: Expressing, Activation Assay, Incubation, Binding Assay, Patch Clamp, Immunofluorescence, Staining, Transfection, Fluorescence, SDS Page, Purification

Journal: eLife

Article Title: Structure and analysis of nanobody binding to the human ASIC1a ion channel

doi: 10.7554/eLife.67115

Figure Lengend Snippet:

Article Snippet: PcTx1 was used at 10 nM and MitTx at 50 nM, both were purchased from Alomone Labs. Oocytes were pre-incubated with purified Nb or Nb-linker-PcTx1 (10 nM) for 20 min at RT before recording.

Techniques: Expressing, Recombinant, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Isolation, Mutagenesis, Magnetic Beads, Affinity Purification, Strep-tag, Software