Effect of the volatile anesthetic agent isoflurane on lateral diffusion of cell membrane proteins

Abstract

The volatile anesthetic isoflurane (ISO) has previously been shown to increase the fluidity of artificial lipid membranes, but very few studies have used biological cell membranes. Therefore, to investigate whether ISO affects the mobility of membrane proteins, fluorescence-labeled transferrin receptor (TfR) and glycosylphosphatidylinositol (GPI)-anchored protein were expressed in human embryonic kidney 293T cells and neural cells and lateral diffusion was examined using fluorescence recovery after photobleaching. Lateral diffusion of the TfR increased with ISO treatment. On the other hand, there was no effect on GPI-anchored protein. We also used GC/MS to confirm that there was no change in the concentration of ISO due to vaporization during measurement. These results suggest that ISO affects the mobility of transmembrane protein molecules in living cells.

Keywords: live cell imaging; membrane fluidity; volatile anesthetic agent.

Figure 1

Anesthetic agents and cell morphology before and after anesthesia. (A) Chemical structure of ISO and MDZ. (B) Cell morphology before and after anesthesia (1 mm ISO, or 60 μm MDZ, 30 min) in FRAP analysis. The cDNA plasmids of the HaloTag‐TfR were transiently transfected into each cell. After 1 day, 1 μm cell membrane impermeability ligand Alexa Fluor®488–HaloTag ligand (Promega) was added, and after 60 min, expression of the fluorescence probe was observed by confocal laser scanning microscopy (LSM710; Zeiss). No change in morphology was observed using FRAP even after anesthetic action in HEK293T cells and neural cells, in any imaging period. Pre, prebleaching; bleach, just after bleaching; recovery, 60 s after bleaching; red circle, bleaching area (ROI); blue circle, background; green square, measurement area. Scale bar: 2 μm.

Figure 2

FRAP analysis of HEK293T cells and neural cells. Influence of anesthetic on the recovery of fluorescence intensity in HEK293T cells (A) and neural cells (C). The t _1/2 of fluorescence recovery rate of Alexa488‐TfR in HEK293T cells (B) and neural cells (D) treated with 1 mm ISO for 30 min or 60 μm MDZ for 30 min. Blue circle: control; orange circle: 1 mm ISO; gray circle: 60 μm MDZ; dot line: t _1/2, *P < 0.05 compared with control and MDZ groups.