Electron Spin Changes During General Anesthesia in Drosophila
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Electron spin changes during general anesthesia in Drosophila Luca Turina,1, Efthimios M. C. Skoulakisa, and Andrew P. Horsfieldb aDivision of Neuroscience, Biomedical Sciences Research Centre Alexander Fleming, 16672 Vari, Greece; and bDepartment of Materials, Imperial College London, London SW7 2AZ, United Kingdom Edited* by Mark A. Ratner, Northwestern University, Evanston, IL, and approved July 15, 2014 (received for review March 10, 2014) We show that the general anesthetics xenon, sulfur hexafluoride, Other theories were also proposed, involving the formation of nitrous oxide, and chloroform cause rapid increases of different gas hydrates (27), proton leaks (28), hydrogen bonds (29, 30), magnitude and time course in the electron spin content of Dro- and membrane dipoles (31). In the 1980s, however, after the sophila. With the exception of CHCl3, these changes are reversible. discovery of an effect of anesthetics on firefly luciferase (32), Anesthetic-resistant mutant strains of Drosophila exhibit a differ- Franks and Lieb (33) first showed enzyme inhibition by GAs (34) ent pattern of spin responses to anesthetic. In two such mutants, and then differences in potency between GAs enantiomers (35, the spin response to CHCl3 is absent. We propose that these spin 36). This finding pointed to a protein site of action, likely changes are caused by perturbation of the electronic structure of a weakly chiral hydrophobic pocket (37–39). Indeed, GAs are proteins by general anesthetics. Using density functional theory, now believed to act on proteins (40–42) and have now been seen we show that general anesthetics perturb and extend the highest in just such sites in protein structures, where they exert small but occupied molecular orbital of a nine-residue α-helix. The calculated definite effects on protein (43) and ion channel (44) conforma- perturbations are qualitatively in accord with the Meyer–Overton tion. The Meyer–Overton rule then becomes all the more sur- relationship and some of its exceptions. We conclude that there prising, because protein binding sites are usually highly selective may be a connection between spin, electron currents in cells, and for ligand shape and size. the functioning of the nervous system. However, if, indeed, both the Meyer–Overton rule and the Franks–Lieb protein hypothesis are taken to be correct, a single eneral anesthesia (GA) is both indispensable and fascinat- mechanism should be shared by all GAs at the protein binding Ging. Millions of surgical procedures are performed each site(s). Then, the small GAs, especially xenon, drastically con- year, most of which would be unthinkable if GAs did not exist. strain the range of possibility. Xenon is uniquely slippery and However, although the first clinical anesthesia with diethyl ether falls outside the normal confines of molecular recognition. It has was reported over 160 y ago (1), the mechanism by which the no shape, because it is a perfect sphere of electron density. It has same GAs act on animals as far apart in evolution as paramecia no chemistry either at any rate under conditions found in the and man (2)—and even plants (3–5)—is still unclear. In 2005, brain. However, and this is the hitherto overlooked starting point GA was included in a Science list of major unsolved problems in of the ideas developed in this paper, xenon has physics: like many the august company of cancer, quantum gravity, and high- other elements and molecules, it is capable of facilitating elec- temperature superconductivity (6). Today, GA remains an in- tron transfer between conductors: recall the iconic photograph of tellectual challenge and arguably, one of the few experimental the IBM logo written in Xe atoms in a scanning tunneling mi- inroads to consciousness (7–9). croscope (STM) (45). Each Xe atom is a bump, because it The mystery of GA resides in a uniquely baffling structure– facilitates tunneling from substrate to tip, and the tip must rise activity relationship: the range of compounds capable of acting above it to keep the current constant (46). Indeed, among the as GAs makes no pharmacological sense. Adrien Albert (10) many molecules that have been imaged in the STM are several called it “biological activity unrelated to structure” (10). In GAs or close congeners other than xenon: nitrous oxide (47), number of atoms, the simplest of the GAs is xenon (11, 12), a monoatomic noble gas, and the most complex is alfaxalone Significance (3-hydroxypregnane-11,20-dione), a 56-atom steroid (13), span- ning a 35-fold range in molecular volume. In between is a host of One hundred sixty years after its discovery, the molecular molecules of widely different structures: nitrous oxide, haloge- mechanism of general anesthesia remains a notable mystery. A nated compounds [sulfur hexafluoride (SF6), chloroform, halo- very wide range of agents ranging from the element xenon to thane, etc.], strained alkanes (cyclopropane), phenols (propofol) steroids can act as general anesthetics on all animals from (14), ethers (diethyl ether and sevoflurane), amides (urethane), protozoa to man, suggesting that a basic cellular mechanism is sulfones (tetronal), pyrimidines (barbiturates), etc. If one adds involved. In this paper, we show that volatile general anes- gases, like dioxygen and nitrogen, that cause narcosis under thetics cause large changes in electron spin in Drosophila fruit pressure and volatile solvents used as inhalational recreational flies and that the spin responses are different in anesthesia- drugs, the list is longer still. What property can all these mole- resistant mutants. We propose that anesthetics perturb electron cules possibly have in common that causes GA? currents in cells and describe electronic structure calculations A partial answer has been known for nearly a century. GAs are on anesthetic–protein interactions that are consistent with this lipid-soluble, and their potency, regardless of structure, is ap- mechanism and account for hitherto unexplained features of proximately proportional to lipid solubility [with some exceptions general anesthetic pharmacology. (15)], a relationship known as the Meyer–Overton rule (16, 17) that is reviewed in ref. 1. This relationship implies, surprisingly in Author contributions: L.T. and E.M.C.S. designed the fly experiments; L.T. and A.P.H. light of their diverse structures, that, after they have arrived at designed the density functional theory calculations; L.T. performed experiments and cal- their destination, all GAs are equally effective. Accordingly, culations; and L.T., E.M.C.S., and A.P.H. analyzed data and wrote the paper. because GAs dissolve in the oily core of the lipid bilayer, they The authors declare no conflict of interest. were long thought to perturb the featureless dielectric in which *This Direct Submission article had a prearranged editor. ion channels, receptors, and pumps are embedded (18–24), al- Freely available online through the PNAS open access option. though an action on proteins could never be ruled out (25, 26). 1To whom correspondence should be addressed. Email: [email protected]. E3524–E3533 | PNAS | Published online August 11, 2014 www.pnas.org/cgi/doi/10.1073/pnas.1404387111 Downloaded by guest on October 2, 2021 PNAS PLUS piece of filter paper with 20 μL solution of 1 μM1-λ1-oxidanyl-2,2,6,6- tetramethylpiperidin-4-ol in water. The amplitude of the calibration signal was ∼3,000 arbitrary units at the gain settings used in the continuous measurements reported in this paper. The data were processed with Lab- View (www.ni.com) and plotted with Igor (www.wavemetrics.com). Density Functional Theory Calculations. The Amsterdam Density Functional (ADF) (www.scm.com) density functional theory (DFT) package was used in the calculations. All structures were optimized using the Perdew–Burke– Ernzerhof functional and core double zeta, valence triple zeta, polarized basis set (TZP) (helix alone) or a dispersion-corrected PBE-D3 functional and TZP basis set (helix and anesthetic). Orbitals were then recalculated at the final geometry using B3LYP-TZP. Some semiempirical preoptimizations as Fig. 1. (Left) Experimental setup for continuous spin measurements on live well as orbital volume calculations were performed in MacSpartan 14 (www. flies; ∼28–32 Drosophila are housed in a polytetrafluoroethylene tube inserted wavefun.com). Calculations were done on a 12-core Apple MacPro. into a quartz tube and positioned in the RF resonator cavity of an ESR spec- trometer. A hollow polyethylene plug at the bottom allows the temperature- controlled cooling gas coming up from below (blue arrow) to enter the fly Results tube if the other end is left open. Anesthetics are introduced in the chamber ESR Measurements in Drosophila. Organic free radicals are so re- by connecting a syringe to the top plug and cannula. The total chamber vol- active that their concentration in living cells is generally found to ume is about 1.5 mL. (Right) Continuous measurements of spin at a fixed value be subnanomolar and therefore, unmeasurable by ESR, which of magnetic field corresponding to the peak of the intrinsic spin resonance has a detection limit that is approximately three orders of mag- (Inset). In each 2-min trace, the sample chamber is filled with SF6 for 36 s from nitude higher (55). In the absence of spin traps, which integrate the 24-s time mark. At other times, temperature-controlled nitrogen at 6 °C the free radical signal over time, the cellular component con- flows through the chamber. Trace 1, empty chamber (signal unaffected by SF ); traces 2–4, recording from W1118 WT flies. The raw data (trace 2) are tributing most of the free electron spin signal (i.e., with a g factor 6 18 baseline-corrected (baseline corr.) by fitting a straight line to the period before near 2.0) will be melanins, which typically contain 10 spins/g SF6 exposure and subtracting that line from the entire trace, yielding trace 3.