Multiple Mechanisms of Propofol Inhibition of the Voltage-Gated Sodium Channel Nachbac: A 19F NMR Investigation

Voltage-gated sodium channels are important molecular targets for anesthetics. The intravenous general anesthetic propofol was found to inhibit NaChBac, a prokaryotic sodium channel from Bacillus halodurans, but little is known about where propofol binds and how propofol binding inhibits the function of this channel. Here, we used 19F-NMR saturation transfer difference (STD) spectroscopy to quantify propofol interactions with NaChBac and to understand the inhibitory mechanisms by propofol. 19F probes were introduced, one at a time, by tagging 1,1,1-trifluoro-3-bromo acetone to site-directed single cysteine mutations that had been computationally predicted to be involved in propofol binding. Quantitative 19F-NMR STD between the 19F probes and 4-fluoropropofol, a fluorinated propofol analog show that propofol binds with different affinities to the sites in different regions of NaChBac. The order of fluoropropofol binding strength, reflected in the cross relaxation rate constant (σ), from the strongest to the weakest, is as follows: the channel activation gate at the cytoplasmic end of the pore, the voltage sensing domain near the gating charge-carrying residues, the ion selectivity filter at the end of the P1 loop, the S4-S5 linker, and the central cavity of the pore. No STD was detectable in the extracellular interface of channel. The finding of multiple binding sites is consistent with the previous observations with the volatile anesthetics isoflurane and sevoflurane. Our results suggest that propofol inhibits NaChBac through multiple sites with distinct mechanisms. As a channel blocker, propofol interferes with the activation gate and selectivity filter and obstructs conductance by binding to the inner pore. As an inhibitory modulator, propofol binding interrupts charge movement in the voltage-sensing domain and restricts the pivot motion of the S4-S5 linker. The study provides a molecular basis for the understanding of propofol inhibitory action on voltage-gated sodium channels. This research was supported by grants from the NIH.