Propofol inhibits the voltage-gated sodium channel NaChBac at multiple sites
Yali WangElaine YangMarta M. WellsVasyl BondarenkoKellie A. WollVincenzo CarnevaleDaniele GranataMichael L. KleinRoderic G. EckenhoffWilliam P. DaileyManuel CovarrubiasPei TangYan Xu
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Voltage-gated sodium (NaV) channels are important targets of general anesthetics, including the intravenous anesthetic propofol. Electrophysiology studies on the prokaryotic NaV channel NaChBac have demonstrated that propofol promotes channel activation and accelerates activation-coupled inactivation, but the molecular mechanisms of these effects are unclear. Here, guided by computational docking and molecular dynamics simulations, we predict several propofol-binding sites in NaChBac. We then strategically place small fluorinated probes at these putative binding sites and experimentally quantify the interaction strengths with a fluorinated propofol analogue, 4-fluoropropofol. In vitro and in vivo measurements show that 4-fluoropropofol and propofol have similar effects on NaChBac function and nearly identical anesthetizing effects on tadpole mobility. Using quantitative analysis by 19F-NMR saturation transfer difference spectroscopy, we reveal strong intermolecular cross-relaxation rate constants between 4-fluoropropofol and four different regions of NaChBac, including the activation gate and selectivity filter in the pore, the voltage sensing domain, and the S4-S5 linker. Unlike volatile anesthetics, 4-fluoropropofol does not bind to the extracellular interface of the pore domain. Collectively, our results show that propofol inhibits NaChBac at multiple sites, likely with distinct modes of action. This study provides a molecular basis for understanding the net inhibitory action of propofol on NaV channels.Keywords:
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Figure 6 summarizes the present state of our knowledge on the sodium channel in myelinated nerve fibers. Two sites have been discussed in detail: a metal cation binding site accessible by tetrodotoxin and saxitoxin from the outside surface only; and a second site accessible from the inside surface with which local anesthetics combine. Hydrogen ions gain access to this region of the sodium channel (and hence determine the relative local concentration of protonated drug) more readily from the extracellular fluid than from the axoplasm (Schwarz et al 1977). In addition, a variety of other sites have been mentioned, binding of drugs to which alters selectively the kinetics of opening and closing of the h and m gates. In myelinated nerve fibers these channels are packed tightly on the nodal membrane. The highest estimate for the sodium channel density in the mammalian node is 10,000 micron2. A re-evaluation of the effective nodal area, however, might reduce this value to 3000-5000/micron 2. This would still leave the nodal membrane rather crowded with sodium channels. Furthermore, the channel density would still be greater than the density of particles, sometimes believed to be sodium channels seen in freeze fracture studies (Rosenbluth 1976). One possibility for resolving this problem is that the units detected by X-ray inactivation (Levinson & Ellory 1973), and those seen in freeze-fracture studies (Rosenbluth 1976) represent not single sodium channels but groups of three. Catterall & Morrow (1978) in a comparison of the binding of saxitoxin and Leiurus sculpturatus scorpion toxin venom have concluded that there are three saxitoxin binding sites for each scorpion toxin binding site. On this basis, three saxitoxin molecules might act to block independently each of the three openings of the channels; while the the conformational change produced by the scorpion venom molecule would affect the inactivation process of all three channels.
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Background Molecular theories of general anesthesia often are divided into two categories: (1) Anesthetics may bind specifically to proteins, such as ionic channels, and alter their function directly, and (2) anesthetics may alter the functions of integral membrane proteins indirectly through modification of the physical properties of the membrane. Recent studies have provided evidence that anesthetics can bind to proteins and modify their function directly, bringing into question the role of the membrane in anesthetic interactions. To reexamine the role of membrane lipids in anesthetic interactions, an experimental approach was used in which the membrane lipid composition could be systematically altered and the impact on anesthetic interactions with potential targets examined. Methods Sodium channels from human brain cortex were incorporated into planar lipid bilayers with increasing cholesterol content. The anesthetic suppression of these channels by pentobarbital was quantitatively examined by single channel measurements under voltage-clamp conditions. Results Changes in cholesterol content had no effect on measured channel properties in the absence of anesthetic. In the presence of pentobarbital, however, cholesterol inhibited anesthetic suppression of channel ionic currents, with 1.9% (weight/weight, corresponding to 3.5 mol%) cholesterol decreasing anesthetic suppression of sodium channels by half. Conclusions These results support a critical role for the lipid membrane in some anesthetic actions and further indicate that differences in lipid composition must be considered in the interpretation of results when comparing the anesthetic potencies of potential targets in model systems.
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Sodium channels are the major proteins that underlie excitability in nerve, heart, and skeletal muscle. Chemical reaction rate theory was used to analyze the blockage of single wild-type and mutant sodium channels by cadmium ions. The affinity of cadmium for the native tetrodotoxin (TTX)-resistant cardiac channel was much higher than its affinity for the TTX-sensitive skeletal muscle isoform of the channel (μl). Mutation of Tyr 401 to Cys, the corresponding residue in the cardiac sequence, rendered μl highly susceptible to cadmium blockage but resistant to TTX. The binding site was localized approximately 20% of the distance down the electrical field, thus defining the position of a critical residue within the sodium channel pore.
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Abstract Propofol, one of the most commonly used intravenous general anesthetics, modulates neuronal function by interacting with ion channels. The mechanisms that link propofol binding to the modulation of distinct ion channel states, however, are not understood. To tackle this problem, we investigated prokaryotic ancestors of eukaryotic voltage-gated Na + channels (Navs) using unbiased photoaffinity labeling with a photoacitivatable propofol analog (AziP m ), electrophysiological methods and mutagenesis. The results directly demonstrate conserved propofol binding sites involving the S4 voltage sensors and the S4-S5 linkers in NaChBac and NavMs, and also suggest state-dependent changes at these sites. Then, using molecular dynamics simulations to elucidate the structural basis of propofol modulation, we show that the S4 voltage sensors and the S4-S5 linkers shape two distinct propofol binding sites in a conformation-dependent manner. These interactions help explain how propofol binding promotes activation-coupled inactivation to inhibit Nav channel function.
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Acid-sensing ion channels (ASICs) are neuronal sodium-selective channels activated by reductions in extracellular pH. Structures of the three presumptive functional states, high-pH resting, low-pH desensitized, and toxin-stabilized open, have all been solved for chicken ASIC1. These structures, along with prior functional data, suggest that the isomerization or flipping of the β11–12 linker in the extracellular, ligand-binding domain is an integral component of the desensitization process. To test this, we combined fast perfusion electrophysiology, molecular dynamics simulations and state-dependent non-canonical amino acid cross-linking. We find that both desensitization and recovery can be accelerated by orders of magnitude by mutating resides in this linker or the surrounding region. Furthermore, desensitization can be suppressed by trapping the linker in the resting state, indicating that isomerization of the β11–12 linker is not merely a consequence of, but a necessity for the desensitization process in ASICs.
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