Combined Stopped-Flow and Electrophysiological Experiments Suggest Direct Sodium Channel Inhibition by Model Fluorobenzene Anesthetics

General anesthetics are often proposed to affect membrane properties through interactions with the lipid bilayer. Though recent studies have shown that inhaled anesthetics can interact more specifically with certain membrane proteins such as ion channels. For example, volatile anesthetics inhibit voltage-gated sodium channels to reduce neurotransmitter release. Fluorobenzenes (FBs), once considered for clinical use, were abandoned due to their flammability and toxicity but are still valuable model anesthetics for investigating the molecular mechanisms of anesthetics. We examined the properties of four FB compounds, 1,2-DiFB, 1,4-DiFB, 1,3,5-TriFB and HexaFB on lipid bilayer and sodium channel function at equipotent clinically relevant concentrations. Effects on lipid bilayer properties were tested using a gramicidin channel based stopped-flow fluorescence assay for lipid bilayer perturbation; effects on sodium channel function were tested using whole-cell voltage-clamp electrophysiology on neuronal cells (ND7/23). The stopped-flow results showed that all four FBs minimally affected lipid bilayer properties, whereas the sodium channels were strongly inhibited by all four anesthetics. Inhibition of peak sodium current was voltage-dependent as a pre-pulse to a voltage at which half the channels were in the fast inactivated state (V1/2) revealed strong inhibition compared to a pre-pulse to a voltage at which the majority of the channels were in the resting state (V0). The FBs produce a left-shift in the voltage of half-maximal inactivation (V1/2, also known as h∞ or availability), with 1,2-DiFB showing the greatest and HexaFB the least shift; these changes are comparable to those observed with modern inhaled anesthetics such as isoflurane. Together these results suggest that these compounds alter sodium channel function through direct interactions with the channels, though we cannot exclude that membrane effects may become involved at high, supra-pharmacological concentrations.