Exploring Volatile General Anesthetic Binding to a Closed Membrane-Bound Bacterial Voltage-Gated Sodium Channel via Computation

Abstract

Despite the clinical ubiquity of anesthesia, the molecular basis of anesthetic action is poorly understood. Amongst the many molecular targets proposed to contribute to anesthetic effects, the voltage gated sodium channels (VGSCs) should also be considered relevant, as they have been shown to be sensitive to all general anesthetics tested thus far. However, binding sites for VGSCs have not been identified. Moreover, the mechanism of inhibition is still largely unknown. The recently reported atomic structures of several members of the bacterial VGSC family offer the opportunity to shed light on the mechanism of action of anesthetics on these important ion channels. To this end, we have performed a molecular dynamics “flooding” simulation on a membrane-bound structural model of the archetypal bacterial VGSC, NaChBac in a closed pore conformation. This computation allowed us to identify binding sites and access pathways for the commonly used volatile general anesthetic, isoflurane. Three sites have been characterized with binding affinities in a physiologically relevant range. Interestingly, one of the most favorable sites is in the pore of the channel, suggesting that the binding sites of local and general anesthetics may overlap. Surprisingly, even though the activation gate of the channel is closed, and therefore the pore and the aqueous compartment at the intracellular side are disconnected, we observe binding of isoflurane in the central cavity. Several sampled association and dissociation events in the central cavity provide consistent support to the hypothesis that the “fenestrations” present in the membrane-embedded region of the channel act as the long-hypothesized hydrophobic drug access pathway.

Fluorine-19 NMR and computational quantification of isoflurane binding to the voltage-gated sodium channel NaChBac

“…F227 lines the pore near the cytoplasmic end and contributes to the channel activation gate (18,19). In previous MD simulations, the F224 and F227 rings were found to hinder the diffusion of water molecules and Na + ions (20) and at least one isoflurane molecule occupied this pore region (12). Thus, we anticipated observation of significant STD effects in the NMR experiments at this site.…”

“…Extracellular surface sites. Isoflurane was predicted to bind to sites near the extracellular surface of the channel at the interface between the P-loops (12). Both L179 and S208 are located in the extracellular interfacial region but have different microenvironments: S208 resides at the hinge of the P2 and S6 helices, whereas L179 is on the tip of the P1 helix with its side chain fully exposed to solvent (Fig.…”

“…Three regions, including the central cavity in the pore, the S4-S5 linker, and the extracellular surface of the channel near the selectivity filter, were identified. The simulations suggest that isoflurane binding affinities at these sites are physiologically relevant (12). All three regions are predicted to be important for channel gating and conduction.…”

“…It shares with Na V a similar pharmacological profile in response to volatile anesthetics, such as isoflurane, which inhibits inward currents of NaChBac in a concentrationdependent manner (10). The ease with which NaChBac can be produced in large quantity and high purity makes it a suitable model for high-resolution structural and functional studies of anesthetic binding in voltage-gated ion channels (11,12).…”

“…In silico screening of isoflurane binding sites and access pathways in NaChBac was performed previously on a closed-pore NaChBac structure model through molecular dynamics "flooding" simulations (12). Three regions, including the central cavity in the pore, the S4-S5 linker, and the extracellular surface of the channel near the selectivity filter, were identified.…”