Effects of Various Small-Molecule Anesthetics on Vesicle Fusion: A Study Using Two-Photon Fluorescence Cross-Correlation Spectroscopy

Abstract

Currently, the molecular mechanism for membrane fusion remains unconfirmed. The most compelling suggested mechanism is the stalk hypothesis, which states that membrane fusion proceeds via stalk formation/hemifusion, among other steps. Because the stalk would have a very high radius of curvature, small lipophilic molecules could enhance fusion by lowering the energy barrier to stalk formation. We previously showed that the general anesthetic halothane is capable of inducing membrane fusion in 1,2-dileoyl-sn-3-glycero-3-phospocholine (DOPC) vesicles. In the present study, we examined other small molecules, general anesthetics (chloroform, isoflurane, enflurane, and sevoflurane), to determine whether they exhibit fusion properties with model lipid membranes similar to those of halothane. We employed both two-photon excitation fluorescence cross-correlation spectroscopy (TPE-FCCS) and steady-state fluorescence dequenching (FD) assays. Using volatile general anesthetics as novel fusion agents, we also aimed to gain a better understanding of the membrane fusion mechanism at a molecular level. We found that lipid mixing or lipid rearrangement, which is required for the formation of the fusion-state intermediates and the fusion pore, rather than the association of lipid vesicles, is rate-limiting. In addition, halothane and chloroform were found to induce lipid mixing (rearrangement) to a greater extent than isoflurane, enflurane, and sevoflurane. Finally, it is proposed that the efficiency of these general anesthetics as fusion agents is related to their partition coefficients, water solubilities, polarities, and molecular volumes, all of which affect the ability of each anesthetic to perturb the contacting bilayer membranes of fusing vesicles.