Conditions that Stabilize Membrane Domains Also Antagonize n-Alcohol Anesthesia

Abstract

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.

Figure 1

Determination of the average critical temperature or pressure of DiIC₁₂-labeled GPMVs through fluorescence imaging. (A) Fields containing multiple GPMVs were imaged over a range of temperatures and at fixed pressure, with representative subsets of images shown on the right. At high temperatures, most GPMVs appear uniform, whereas an increasing fraction of vesicles appear phase separated as the temperature is lowered, with phase-separated vesicles indicated by yellow arrows. From these images, we manually tabulate the fraction of GPMVs that contain two coexisting liquid phases as a function of temperature, constructing the plot on the left. These points are fit to the sigmoid function described in Materials and Methods to determine the extrapolated temperature at which 50% of vesicles contain coexisting liquid phases. (B) Fields containing multiple GPMVs were imaged over a range of pressures at fixed temperature, and representative subsets of images are shown on the right. At low pressure, most GPMVs appear uniform, whereas an increasing fraction of vesicles appear phase separated as pressure is increased. As with the fixed-pressure data in (A), these points are fit to the sigmoid function described in Materials and Methods to determine the extrapolated pressure at which 50% of vesicles contain coexisting liquid phases. To see this figure in color, go online.

Figure 2

Hexadecanol raises $T_{\text{c}}$ in GPMVs from rat (RBL) and Xenopus (XTC-2) cell lines and can counteract the T_c-lowering effects of ethanol. (A) Values indicate the average shift in $T_{\text{c}}$$\left(\Delta T_{\text{c}}\right)$ in a population of vesicles upon treatment with the compounds indicated. Solutions containing hexadecanol were prepared to be supersaturated, as described in Materials and Methods. Each point represents a single measurement, and error bounds represent the 68% confidence interval on the extrapolated $\Delta T_{\text{c}}$. (B) Plots showing the fraction of phase-separated vesicles versus temperature for the three points inside the gray box in (A). To see this figure in color, go online.

Figure 3

(A) (Upper) Tadpole LRR for a titration of ethanol alone and combinations of ethanol and hexadecanol (EtOH+Hex) or ethanol and tetradecanol (EtOH+Tet) measured after 1 h incubation in equilibrated solutions. At a given ethanol concentration, the fraction of tadpoles that respond to stimulus increases in the presence of hexadecanol (EtOH+Hex; red circles, upper horizontal axis). The EtOH+Tet combination contains either 5 or 10 μM tetradecanol (green triangles). (Lower) $\Delta T_{\text{c}}$ in RBL-derived GPMVs is shown for identical titrations. All solutions contain the ethanol concentration indicated by the lower horizontal axis. (B) Time course of LRR for one ethanol and EtOH+Hex combination. (C) (Left) Points in (A) replotted as LRR versus $\Delta T_{\text{c}}$, including additional experiments with other n-alcohol combinations, as indicated in the legend. (Center) LRR plotted versus aggregate hydrophobicity, tabulated by summing the concentration of each n-alcohol present normalized by its ${\text{AC}}_{50}$ (22), using 3 μM as a proxy ${\text{AC}}_{50}$ for hexadecanol and 5 μM as a proxy ${\text{AC}}_{50}$ for tetradecanol. (Right) LRR plotted versus the net anesthetic concentration, tabulated by summing the concentration of each n < 14 alcohol anesthetic present normalized by its ${\text{AC}}_{50}$ (22). (Top row) In each case, the dashed line represents a best fit to the data, with R² values giving the fraction of explained variance and f values comparing the quality of the fit to a null model where all points have the same probability. All fits are linear and constrained to go through the origin. (Bottom row) In each case, the black line is fit to all conditions that exclude hexadecanol, the red line is fit to all conditions that include hexadecanol, and the gray dashed line is fit to all points, as in the top row. These fits are substantially different from each other, except when plotted versus $\Delta T_{\text{c}}$, indicating that $\Delta T_{\text{c}}$ has the most quantitative predictive power across chemical species. To see this figure in color, go online.

Figure 4

RO15-4513 and dihydromyricetin block the acute toxicity and intoxicating effect of ethanol. Each raises $T_{\text{c}}$ and cancels the effects of ethanol when added to GPMVs at the same concentration at which they are effective in vivo.

Figure 5

(A) The fraction of vesicles that are macroscopically phase separated is plotted as a function of hydrostatic pressure at three different temperatures for both control vesicles and vesicles incubated with 12 mM butanol (BtOH). In each case, increasing the pressure leads to an increase in the fraction of vesicles that are macroscopically phase separated. (B) $T_{\text{c}}$ rises with increasing hydrostatic pressure in both control GPMVs and GPMVs incubated in BtOH. Here, 240 ± 30 bar of hydrostatic pressure is required to reverse the effects of 12 mM BtOH (shaded region). Solid symbols are obtained by extrapolating to find $T_{\text{c}}$ from the data acquired at constant pressure, whereas open symbols are obtained by extrapolating to find $P_{\text{c}}$ at constant temperature. At temperatures above $T_{\text{c}}$, most vesicles are composed of a macroscopically uniform single liquid, whereas below $T_{\text{c}}$, most are separated into two coexisting liquid phases. To see this figure in color, go online.

Figure 6

Anesthetics lower the transition temperature of a biologically tuned critical point, which could lead to misregulation of ion channels and other membrane-bound proteins. (A) Schematic phase diagrams for the plasma membrane of an untreated and an n-alcohol-treated cell. Guided by experiments (19), we hypothesize that the plasma membrane lies in the white region where lipids, proteins, and other membrane components are well mixed macroscopically into a single two-dimensional liquid phase. However, due to their close proximity to the critical point (star), thermal fluctuations lead to relatively large domains enriched in particular components. When cooled into the gray two-phase region, GPMVs separate into two coexisting liquid phases termed liquid-ordered and liquid-disordered. Several n-alcohol general anesthetics lower the critical temperature of the membrane (21), changing the distance above the critical point, $T-T_c$. Here, we also show that treatments that antagonize anesthetic action raise critical temperatures, reversing the effects of n-alcohols on $T_{\text{c}}$. (B) Under normal conditions, a hypothetical ion channel (large blue inclusion) has a tendency to inhabit relatively large domains enriched in particular lipids and proteins. When the membrane is taken away from the critical point, the structure of these domains is altered, possibly leading to changes in ion channel gating and function through a variety of mechanisms discussed in the text. To see this figure in color, go online.