(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 (t0). 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 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 Source data 3.