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. 2017 Sep 5;114(39):10491–10496. doi: 10.1073/pnas.1707873114

Fig. 6.

Fig. 6.

BTX-resistant and BTX-sensitive phenotypes in single and combination mutants. (A) Superimposed traces of N1584T-rNav1.4 Na+ currents were recorded during repetitive pulses (+50 mV for 20 ms at 2 Hz). The arrows indicate the current traces after 1 pulse and 1,500 pulses, respectively. Little or no noninactivating persistent currents were evident after 1,500 pulses (1.8 ± 0.6%; n = 8). A small residual slowing effect on the decaying phase of the transient Na+ currents was detected in all recorded cells (dashed arrow). This slow decaying component could be best fitted by an exponential function with a τ-value of 0.55 ± 0.03 ms (n = 8; at 1,500 pulses), whereas the fast component had a τ-value of 0.25 ± 0.02 ms (n = 8). (B) Superimposed traces of A445D/V1583I/N1584T-rNav1.4 Na+ currents were recorded during repetitive pulses (+50 mV for 20 ms at 2 Hz). A complete BTX-resistant phenotype of this combination mutant was evident, which resembled that of the 5AA mutant shown in Fig. 3A. The arrows indicate the current traces after 1 pulse and 2,000 pulses, respectively. The maintained current was about 1% of the peak amplitude after 1,500 pulses (0.8 ± 0.4%; n = 8). (C) Superimposed traces of S429A/I433V/A445D-rNav1.4 Na+ currents were recorded during repetitive pulses (+50 mV for 20 ms at 2 Hz). Most transient Na+ currents were converted to noninactivating after 1,500 pulses (85% of the peak amplitude; 86.0 ± 2.8%; n = 5). This BTX-sensitive phenotype of S429A/I433V/A445D-rNav1.4 Na+ currents resembled that of wild-type rNav1.4 counterparts shown in Fig. 2A. Current traces correspond to 1, 100, 300, 600, 1,000, and 1,500 pulses applied. (D) After 1,500 pulses, ramp Na+ currents through the S429A/I433V/A445D-rNav1.4 mutant channels were recorded using a 1-s ramp stimulus from −140 mV to +50 mV and plotted against voltage. Inward BTX-modified Na+ currents were activated near −105 mV (−104.6 ± 2.0 mV; n = 5) and reached the maximal value of 9 nA (13.9 ± 2.2 nA; n = 5). The pipette solution contained 5 μM BTX. p, pulse(s).