Diverse molecules induce general anesthesia with potency strongly correlated with both 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 elevated 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 ΔTc predicts anesthetic potency for n-alcohols better than hydrophobicity in a range of contexts, supporting a mechanistic role for membrane heterogeneity in general anesthesia.

Spot variation fluorescence correlation spectroscopy (svFCS) was developed to study the movement and organization of single molecules in plasma membranes. This experimental technique varies the size of an illumination area while measuring correlations in time using standard fluorescence correlation methods. Frequently, this data is interpreted using the assumption that correlation measurements reflect the dynamics of single molecule motions, and not motions of the average composition. Here, we explore how svFCS measurements report on the dynamics of components diffusing within simulations of a 2D Ising model with a conserved order parameter. Simulated correlation functions report on both the fast dynamics of single component mobility and the slower dynamics of the average composition. Over a range of simulation conditions, a conventional svFCS analysis suggests the presence of anomalous diffusion even though single molecule motions are nearly Brownian in these simulations. This misinterpretation is most significant when the surface density of the fluorescent label is elevated, therefore we suggest future measurements be made over a range of tracer densities. Some simulation conditions reproduce qualitative features of published svFCS experimental data. Overall, this work emphasizes the need to probe membranes using multiple complimentary experimental methodologies in order to draw conclusions regarding the nature of spatial and dynamical heterogeneity in these systems.

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 (T c ) 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 T c . 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 T c 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 T c in vesicles with a magnitude that counters the effect of butanol at relevant concentrations and pressures. Taken together, these results demonstrate that ∆T c predicts anesthetic potency for n-alcohols better than hydrophobicity in a range of contexts, supporting a mechanistic role for membrane heterogeneity in general anesthesia.