Inhibition of Nav1.4 pore by CBD, F1586A reduces inhibition. (a) Side view of CBD docked into the pore of the human Nav1.4 structure. The structure is colored by domain. DIV is colored in deep blue. (b) Zoomed-in side view in which F1586 is colored yellow. (c and d) Representative families of macroscopic current traces from WT-Nav1.4 and F1586A. (e) Voltage dependence of activation as normalized conductance plotted against membrane potential (Nav1.4: V_1/2 = −19.9 ± 2.7 mV, z = 2.8 ± 0.3, n = 5; F1586A: V_1/2 = −22.4 ± 2.2 mV, z = 3.0 ± 0.3, n = 7; P > 0.05 for both V_1/2 and z). (f) Voltage dependence of SSFI as normalized current plotted against membrane potential. Channels were held at −130 mV for 200 ms (Nav1.4: V_1/2 = −64.1 ± 2.4 mV, z = −2.7 ± 0.3, n = 8; F1586A: V_1/2 = −63.3 ± 3.0 mV, z = −3.5 ± 0.3, n = 8; P > 0.05 for both V_1/2 and z). (g and h) Lidocaine/CBD inhibition of Nav1.4 and F1586A from −110 mV (rest) at 1 Hz with a 20-ms depolarizing pulse (lidocaine-Nav1.4: mean block = 60.6 ± 2.3%, n = 3; lidocaine-F1586A: mean block = 24.6 ± 9.3%, n = 3, #, P = 0.020; CBD-Nav1.4: mean block = 47.3 ± 3.7%, n = 5; CBD-F1586A: mean block = 25.3 ± 4.8%, n = 3, *, P = 0.037). Sample traces before and after compound perfusion are shown. All values in e–h are reported as mean ± SEM.