CBD interactions with and through Nav fenestrations. (a) Side view of CBD docked into the human Nav1.4 structure. The structure is colored by domain (matched color to domain is shown in f). CBD is represented in purple. (b) Side view of all four sides of human Nav1.4 (colored by domain). Nav1.4 fenestrations are highlighted in red, along with the position of respective residues that were mutated into tryptophans (W). (c) Computationally predicted mutagenesis of fenestrations results two full (pose 1) and two partial (pose 2) occlusions/alterations (paralleled domains). (d) Lidocaine (1.1 mM) inhibition of Nav1.4 and WWWW from −110 mV (rest) at 1 Hz 20 ms depolarizing pulse (Nav1.4: mean block = 60.6 ± 2.3%, n = 3; WWWW: mean block = 53.6 ± 11.7%, n = 3; P > 0.05), flecainide (350 µM) inhibition (Nav1.4: mean block = 64.6 ± 6.0%, n = 3; WWWW: mean block = 76.4 ± 11.3%, n = 3; P > 0.05), and CBD (10 µM) inhibition (Nav1.4: mean block = 47.3 ± 3.7%, n = 5; WWWW: mean block = 6.4 ± 1.3%, n = 5; *, P = 0.0001). Traces before and after compound perfusion are shown. Values are reported as mean ± SEM. (e) CBD pathway through the Nav1.4 fenestration from side view, as predicted by MD simulations. Red and blue correlate with CBD being inside and outside the fenestration, respectively (see Videos 2 and 3). (f) CBD pathway from top view of the channel. (g) Progressive snapshots of the movement of CBD over time from inside to outside the channel.