Characterizing fenestration size in sodium channel subtypes and their accessibility to inhibitors

Voltage-gated sodium channels (Nav) underlie the electrical activity of nerve and muscle cells. Humans have nine different subtypes of these channels, which are the target of small-molecule inhibitors commonly used to treat a range of conditions. Structural studies have identified four lateral fenestrations within the Nav pore module that have been shown to influence Nav pore blocker access during resting-state inhibition. However, the structural differences among the nine subtypes are still unclear. In particular, the dimensions of the four individual fenestrations across the Nav subtypes and their differential accessibility to pore blockers is yet to be characterized. To address this, we applied classical molecular dynamics simulations to study the recently published structures of Nav1.1, Nav1.2, Nav1.4, Nav1.5, and Nav1.7. Although there is significant variability in the bottleneck sizes of the Nav fenestrations, the subtypes follow a common pattern, with wider DI-II and DIII-IV fenestrations, a more restricted DII-III fenestration, and the most restricted DI-IV fenestration. We further identify the key bottleneck residues in each fenestration and show that the motions of aromatic residue sidechains govern the bottleneck radii. Well-tempered metadynamics simulations of Nav1.4 and Nav1.5 in the presence of the pore blocker lidocaine also support the DI-II fenestration being the most likely access route for drugs. Our computational results provide a foundation for future in vitro experiments examining the route of drug access to sodium channels. Understanding the fenestrations and their accessibility to drugs is critical for future analyses of diseases mutations across different sodium channel subtypes, with the potential to inform pharmacological development of resting-state inhibitors and subtype-selective drug design. SIGNIFICANCE Voltage-gated sodium channels initiate electrical signaling in nerve and muscle tissue. Nine different subtypes are expressed in humans. These are critical targets of drugs for treating pain, epilepsy, and cardiac arrhythmias, but it is unclear whether differential access of drugs to their binding sites could be harnessed to target medications to enter just one sodium channel subtype, reducing side effects. We compare multiple pathways for drug access (fenestrations) in individual subtypes and among subtypes over time to identify likely entry routes. Although fenestration dimensions are variable, one appears most favorable for drug passage in all subtypes, confounding the possibility of subtype-selective drug access.

Automated summary

Voltage-gated sodium channels play a crucial role in the electrical activity of nerve and muscle cells, with humans having nine different subtypes. Research indicates that there are four lateral fenestrations within these channels influencing the access of pore blockers. Although there are structural differences among the subtypes, a common pattern in the dimensions of the fenestrations has been observed, with one particular fenestration appearing most favorable for drug access in all subtypes. These findings are significant for the development of pharmacological treatments and the analysis of disease mutations in sodium channel subtypes.