Previous work on membrane effects of ethanol focused on fluidization of the bulk membrane lipid bilayer. That work was extended in the present study to an examination of ethanol's effect on lipid domains. Two independent methods were developed to examine the effects of ethanol on the inner and outer leaflets of synaptic plasma membranes (SPM). First, differential polarized phase and modulation fluorometry and selective quenching of diphenyl-1,3,5-hexatriene (DPH) were used to examine individual leaflets. Both limiting anisotropy and rotational relaxation time of DPH in SPM indicated that the outer leaflet was more fluid than the inner leaflet. Second, plasma membrane sidedness selective fluorescent DPH derivatives, cationic 1-[4-(trimethylammonio)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) and anionic 3-[p-6-phenyl)-1,3,5-hexatrienyl]phenylpropionic acid (PRO-DPH), confirmed this transmembrane fluidity difference. TMA-DPH and PRO-DPH preferentially localized in the inner and outer leaflets of SPM, respectively. Ethanol in vitro had a greater fluidizing effect in the outer leaflet as compared to the inner leaflet. Thus, ethanol exhibits a specific rather than nonspecific fluidizing action within transbilayer SPM domains. This preferential fluidization of the SPM outer leaflet may have a role in ethanol affecting transmembrane signaling in the nervous system.
Bibliographical noteFunding Information:
This work wa~ supported in part by grants for the USPHS, National Institutes of Health Oraat GM31651 (to F.S.) and National Institute on AI- cohol Abuse and Alcoholism Grants AA07054 and AA07292 (to W.G.W.), the Medical Research Service and the Geriatric Research, Education and Clixtical Center of the Veterans Adn-J~ztration. The authors wish to thank Ms. Shannon Lawrence for helpful technical assistance and Ms. Yvonne Young for excellent secretarial assistance.
- Fluorescence probe
- Membrane asymmetry
- Membrane domain
- Membrane fluidity
- Synaptic plasma membrane