Protonation state of inhibitors determines interaction sites within voltage-gated sodium channels.

Voltage-gated sodium channels are essential for carrying electrical signals throughout the body, and mutations in these proteins are responsible for a variety of disorders, including epilepsy and pain syndromes. As such, they are the target of a number of drugs used for reducing pain or combatting arrhythmias and seizures. However, these drugs affect all sodium channel subtypes found in the body. Designing compounds to target select sodium channel subtypes will provide a new therapeutic pathway and would maximize treatment efficacy while minimizing side effects. Here, we examine the binding preferences of nine compounds known to be sodium channel pore blockers in molecular dynamics simulations. We use the approach of replica exchange solute tempering (REST) to gain a more complete understanding of the inhibitors' behavior inside the pore of NavMs, a bacterial sodium channel, and NavPas, a eukaryotic sodium channel. Using these simulations, we are able to show that both charged and neutral compounds partition into the bilayer, but neutral forms more readily cross it. We show that there are two possible binding sites for the compounds: (i) a site on helix 6, which has been previously determined by many experimental and computational studies, and (ii) an additional site, occupied by protonated compounds in which the positively charged part of the drug is attracted into the selectivity filter. Distinguishing distinct binding poses for neutral and charged compounds is essential for understanding the nature of pore block and will aid the design of subtype-selective sodium channel inhibitors.