Stabilizing membrane domains antagonizes 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 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 test the correlation between the anesthetic potency of n-alcohols and effects on Tc. First we show that hexadecanol acts oppositely to 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 in vesicles and counter ethanol’s effects in vesicles, mimicking the findings of previous electrophysiological 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 an anesthetic at relevant concentrations and pressures. Taken togetner, these results demonstrate that ∆Tc predicts anesthetic potency for n-alcohols better 000 00(00) 2–15 ar X iv :1 60 4. 00 41 2v 1 [ ph ys ic s. bi oph ] 1 A pr 2 01 6 000 Volume: 00 Month Year 2–15 than hydrophobicity in a range of contexts, supporting a mechanistic role for membrane heterogeneity in general anesthesia. Insert Received for publication Date and in final form Date. ∗To whom correspondence should be addressed; E-mail: sveatch@umich.edu. 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 anesthetics (7), where extensive structural work (8–10) suggests that anesthetic effects are mediated by specific residues in hydrophobic, membrane spanning regions. Our understanding of the structure and function of the animal plasma membrane has grown dramatically since most membrane theories of anesthesia were put forward. It is now appreciated that animal plasma membranes have a thermodynamic tendency to separate into coexisting liquid domains, sometimes referred to as ‘lipid rafts’ or ‘lipid shells’ (11, 12) and that this heterogeneity localizes and regulates ion channels, sometimes in a subtype specific manner (13). Much of this regulation likely arises from the membrane’s unusual thermodynamic properties. Cholesterol containing membranes of purified lipids can support two distinct liquid phases (14), and giant plasma membrane vesicles (GPMVs) isolated from mammalian cell lines display analogous phase coexistence at low temperature (15). Remarkably, GPMVs are near the critical point of this transition (16), a non-generic region of phase space distinguished by large correlation times and domain sizes that requires fine tuning both composition and temperature in synthetic systems (17). We recently found (18) that incubating several liquid general anesthetics with isolated GPMVs lowered their critical temperatures (Tc) in a way that scales well with their anesthetic dose as previously measured in tadpole loss of righting reflex (LRR) assays (19). While our assay measures a change in Tc somewhat below growth temperature, we predict that at higher temperatures these treatments would destabilize sub-micron liquid domains both in vesicles and the intact plasma membranes from which they were derived. While suggestive, we wanted to rule out the © 2013 The Authors 0006-3495/08/09/2624/12 $2.00