Riluzole's Mechanism of Action on Voltage-Gated Sodium Channels Elucidated
核心洞察
Researchers have determined the structural basis for riluzole's interaction with voltage-gated sodium channels (搜索) (VGSCs) using bacterial models and molecular dynamics.
Riluzole binds to a hydrophobic pocket within the fenestration of VGSCs, stabilizing the inactivated state of the channel without directly blocking the pore.
The study identifies a key residue in the local anesthetic binding site crucial for riluzole's action, confirming its importance in stabilizing inactivation.
Researchers have elucidated the structural mechanism by which riluzole, a neuroprotective drug, interacts with voltage-gated sodium channels (搜索) (VGSCs). The study, published in Nature Communications, combines X-ray crystallography, nuclear magnetic resonance (NMR), electrophysiology, and molecular dynamics simulations to reveal how riluzole stabilizes the inactivated state of VGSCs, offering insights into its therapeutic effects in conditions like amyotrophic lateral sclerosis (搜索) (ALS (搜索)) and myotonia (搜索).
Structural Insights into Riluzole Binding
The team utilized NavMs (搜索), a prokaryotic VGSC from Magnetococcus marinus (搜索), as a structural model due to its similarity to eukaryotic VGSCs. Through Saturation Transfer Difference NMR (STD-NMR), they confirmed direct interaction between riluzole and NavMs. Subsequent X-ray crystallography revealed that riluzole binds to a hydrophobic pocket within the fenestration of NavMs, making contacts with the S6 helix and selectivity filter (SF) residues from two consecutive domains.
Functional Validation and Mechanism of Action
Electrophysiological experiments using HEK293T cells expressing NavMs (搜索) showed that riluzole dose-dependently shifted steady-state inactivation (SSI) in the hyperpolarized direction, with an IC50 of 2 μM, and slowed recovery from inactivation (RFI). These effects mirror those observed in eukaryotic VGSCs, suggesting that NavMs is a valid functional model. The binding site does not occlude the Na+ conduction pathway, explaining why riluzole stabilizes inactivation without causing channel block at therapeutic concentrations.
Role of the Local Anesthetic Binding Site
The study highlights the importance of a conserved phenylalanine residue in the local anesthetic (LA) binding site of eukaryotic VGSCs for riluzole's action. Mutating the corresponding threonine residue (T207) in NavMs (搜索) to alanine (T207A) abrogated the effect of riluzole on SSI. Molecular dynamics simulations further showed that a T207A mutation reduced riluzole's binding affinity, while a T207F mutation, mimicking the human phenylalanine, enhanced riluzole binding through π–π stacking interactions.
Impact on Human Nav1.4 and Disease Variants
Molecular dynamics simulations with human Nav1.4 (hNav1.4) revealed that riluzole binds in the DIII-DIV fenestration, interacting with key residues. The researchers also investigated the effect of riluzole on the hNav1.4 variant P1158S, which causes myotonia (搜索) and periodic paralysis and exhibits a pathologically increased late sodium current (INaL). The addition of 1 μM riluzole to cells expressing hNav1.4 P1158S stabilized inactivation and restored the elevated INaL to wild-type levels.
Implications for Therapeutic Intervention
These findings provide a detailed structural and functional understanding of how riluzole modulates VGSC activity. By stabilizing the inactivated state of sodium channels, riluzole can reduce cellular excitability, which is particularly relevant in conditions characterized by hyperexcitability, such as ALS (搜索) and myotonia (搜索). The study supports the use of riluzole in managing these disorders and provides a framework for developing novel therapeutics targeting VGSCs.