Tp1a activity on NaV channels evaluated by fluorescent imaging and electrophysiology represented as IC50 values
For fluorescent assays, hNaV recombinant cell lines were incubated with rGly-Tp1a, sTp1a-NH2, or sTp1a-OH for 30 minutes followed by activation with veratridine/OD1. Fluorescent intensity was normalized against positive and negative controls and maximum response from 5-minute reading after NaV activation used for plotting the concentration-response curves. For electrophysiology assays, NaV recombinant cell lines held at –80 mV were incubated with rGly-Tp1a, sTp1a-OH, or sTp1a- NH2 for 5 minutes followed by a prepulse of –120 mV for 200 milliseconds and activation with a single pulse of 0 mV for 20 milliseconds. I/Imax values were used for plotting the concentration-response curves. Values are from at least three independent experiments (mean ± S.D.).
| Toxin | hNaV1.1 | hNaV1.2 | hNaV1.3 | hNaV1.4 | hNaV1.5 | hNaV1.6 | hNaV1.7 | hNaV1.8 |
|---|---|---|---|---|---|---|---|---|
| Fluorescent imaging assay (μM) | ||||||||
| rGly-Tp1a | 2.8 ± 1.4 | 2.4 ± 0.4 | 13.7 ± 1.0 | >5 | >5 | 4.5 ± 1.3 | 1.12 ± 0.3 | >5 |
| sTp1a-OH | 1.5 ± 0.2 | 1.4 ± 0.7 | 12.1 ± 5.6 | >5 | >5 | 3.7 ± 1.1 | 1.5 ± 0.4 | >5 |
| sTp1a-NH2 | 0.5 ± 0.1 | 0.3 ± 0.1 | 0.9 ± 0.2 | >5 | >5 | 0.29 ± 0.05 | 0.22 ± 0.05 | >5 |
| Electrophysiology (nM) | ||||||||
| rGly-Tp1a | 60 ± 26 | ND | 21.9 ± 4.0 | ND | >500 | ND | 9.5 ± 3.4 | ND |
| sTp1a-OH | 101 ± 30 | ND | 41.3 ± 4.7 | ND | >500 | ND | 11.5 ± 3.9 | ND |
| sTp1a-NH2 | 11.3 ± 1.2 | ND | 11.5 ± 3.0 | ND | >50 | ND | 2.5 ± 0.8 | ND |
ND, not determined.