Figure 6
Nematocysts extract (NE) from B. annulata potently inhibited INav elicited by opening of hNav1.6 and hNav1.5. (a) HEK293 cells expressing hNav1.6 channels were recorded and INav was activated applying voltage steps from − 100 to − 20 mV, using a similar protocol to that used in Fig. 3c. In the experiment illustrated, 1 mg ml−1 NE was applied as indicated by the bar in green after a control recording of INav (traces in gray); during NE superfusion INav decreased progressively (traces in green) and recovered during wash. (b) Traces show the effect of 50 nM TTX (purple traces) using the same protocol in cells from the same cultures. The graph in (c) shows the time-course of the inhibitory effect produced by each treatment (mean ± S.D. from 6 to 10 cells). (d,e) HEK293 cells expressing hNav1.5 channels were recorded in similar manner and tested for either NE (traces in green) or TTX (traces in purple); as expected TTX did not affect INav, but NE strongly inhibited the response. The inhibition time-course produced by NE is illustrated in (f) together with the lack of effect by TTX; individual data points are the mean ± S.D. (5 cells in each case). (g) A dose–response relationship was built for the NE effect on the INav in cells expressing hNav1.6 channels (top graph); distinct NE concentrations from 1 to 0.05 mg ml−1 were applied to 6 different cells. Individual data points are the mean ± S.D. of the effect. Then, the normalized peak inhibition for each concentration was plotted (insert graph) and data points were fitted to a dose–response curve (green dashed line, EC50 = 155 ± 34 μg ml−1). (h) The graph shows the time-course of INav inhibition produced by 1 mg ml−1 NE (green circles), and the effect produced by NE from the same batch that was boiled by 15 min (light blue circles) or NE samples that were incubated at 37 °C overnight (pink circles); individual data points are the mean ± S.D. from 5 cells in each case.
