Stabilizing membrane domains antagonizes n-alcohol anesthesia

Diverse molecules induce general anesthesia with potency strongly correlated both with their hydrophobicity and their effects on certain ion channels. We recently observed that several n-alcohol anesthetics inhibit heterogeneity in plasma membrane derived vesicles by lowering the critical temperature (Tc) for phase separation. Here we exploit conditions that stabilize membrane heterogeneity to further test the correlation between the anesthetic potency of n-alcohols and effects on Tc. First we show that hexadecanol acts oppositely to n-alcohol anesthetics on membrane mixing and antagonizes ethanol induced anesthesia in a tadpole behavioral assay. Second, we show that two previously described ‘intoxication reversers’ raise Tc and counter ethanol’s effects in vesicles, mimicking the findings of previous electrophysiological and behavioral measurements. Third, we find that hydrostatic pressure, long known to reverse anesthesia, also raises Tc in vesicles with a magnitude that counters the effect of butanol at relevant concentrations and pressures. Taken together, these results demonstrate that ∆Tc predicts anesthetic potency for n-alcohols better than hydrophobicity in a range of contexts, supporting a mechanistic role for membrane heterogeneity in general anesthesia. INTRODUCTION The potencies of many general anesthetics are roughly proportional to their oil:water partition coefficient over more than five orders of magnitude in overall concentration (1). This Meyer-Overton correlation suggests membrane involvement, and anesthetics have been shown to decrease lipid chain ordering, lower the main chain melting temperature, and increase membrane spontaneous curvature, fluidity, and conductance (2–4). However, these effects are small (5) and often cannot account for those molecules which deviate from Meyer-Overton (6). Most recent attention focuses on the ion channels known to be most sensitive to these compounds (7), where extensive structural work (8–10) suggests that anesthetic effects are mediated by specific residues in hydrophobic, membrane spanning regions. High potency general anesthetics such as etomidate and barbiturates are more effective at potentiating channels and producing anesthesia than predicted from their hydrophobicity alone, and evidence is accumulating that these compounds bind directly and specifically to channels at the interface between subunits (11). Many researchers also favor a direct binding mechanism of anesthetic action for lower potency anesthetics, including the n-alcohol anesthetics investigated here (8). However, channels are proposed to contain more numerous binding sites for these compounds (11–13), each with low affinity, leaving open the possibility that these anesthetics interact with channels more as a solvent than as a ligand. 000 00(00) 1–20 not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/057257 doi: bioRxiv preprint first posted online Jun. 7, 2016;