Bioc himica et Biophysica Acta, 1067 (1991) 131-138 © 1991 Elsevier SciencePublishersB.V. 0005-2736/91/$03.50 ADONIS 0005273691003025 131 BBAMEM75316 pH-dependent lipid packing, membrane permeability and fusion in phosphatidylcholine vesicles Stefano Massari, Eleonora Folena, Valeria Ambrosin, Giampietro Schiavo and Raffaele Colonna C.N.R. Unit for the Study of PI,ysiology of Mitochondria. Laboratory of Biophysics and Molecular Biology, Institute of General Pathology, Unirersity of Padova. Padova (Italy) (Received 18 March 19911 Keywords: Phospholipid vesicle: Fusion:Lipidpacking:Membrane permeability: pH dependence: Lipidarrangement We have studied the rate of membrane fusion, the lipid dynamics and order and the membrane permeability of phosphatidylcholine vesicles as a function of pH. Acidification induced very different effects depending on the state of the bilayer. In liquid-crystalline hilayers, acidification decreased the rate of membrane fusion, the acyl chain motion and disorder and the rate of K + release, whereas in solid bilayers acidification increased the rate of membrane fusion, the lipid aryl chain disorder and the rate of K + release. These pH-dependent modifications are interpreted in terms of conformational and/or packing changes of the phosphatidylcholine head group in the membrane. In solid Uilayers, these changes are not easily accommodated by the rigid structure, and the resulting stress leads to an unstable bilayer. Introduction In recent years the effect of pH changes on several membrane- related phenomena, such as binding of external molecules to the membrane, solute diffusion, membrane fusion, and distribution of lipid components in the bilayer has been the subject of considerable interest. Current interpretations of these pH-in- fluenced processes have considered almost exclusively the role of acidification on the polar head of negative phospholipids and phosphatidylethanolamine, or on the molecules interacting with the bilayer. Insufficient at- tention, however, has been paid to the effect of pH on the zwitterionic lipid phosphatidylcholine (PC), which was practically considered as an inert molecule. Abbreviations: PC, phosphatidylcholine;DMPC, dimyristoylphos- phatidylcholinc:DPPC, dipalmitoylphosphatidylcholine; DSPC, dis- tearoylphosphatidylcholine; DOPC, dioleoylphosphatldylcholine: SUV, small unilamellarvesicles:DPH, 1,6-diphenyl-1,3,5-hexatriene; TMA-DPH, 1 -(4-trimethylammoniumphenyl)-6-phenyl- 1,3,5-hexa- triene: N-NBD-PE, N-(7-nitrobenz-2-oxa-l,3-diazol-4yl)dipalmitoyl- phosphatidylethanolamine; N-Rh-PE, N-(lissamine Rhodamine B sulfonyl)dipalmitoylphosphatidylethanolamine;Tris, tris(hydrox'y- methyl)aminomethane; Hepes, N.2.hydro~ethylpiperazinc.N'-2- ethanesulfonic acid. Correspondence: S. Massari,Instituteof General Pathology, Univer- sity of Padova,Via Trieste 75, 35121 Padova,Italy. We present experimental evidence that acidification modifies the fusion capacity of PC bilayers, the lipid mobility and order, and the permeability properties of the membrane, presumably as a consequence of modi- fications of lipid packing. Molecular packing of the constituents of biological membranes is critical for several events such as receptor function [1], enzymatic activity [2], solute diffusion [3], segregation of mem- brane components [4], incorporation of proteins into membranes [5] or action of anesthetics [6]. Our results indicate that care should be taken in interpreting pH- dependent phenomena in PC-containing membranes. Materials and Methods Phospholipids, 1,6-diphenyl-l,3,5-hexatriene (DPH) and perylene were supplied by Sigma. in order to improve the purity, the lipids were precipitated from acetone as described by Massari and Colonna [7]. N-(7- Nitrobenz-2-oxa- 1,3-diazol-4-yl)dipalmitoylphosphatid- ylethanolamine (N-NBD-PE) and N-(lissamine Rho- damine B suffonyDdipalmitoylphosphatidylethanol- amine (N-Rh-PE) were synthetized in the laboratory of Dr. Montccucco (this Institute). 1-(4-Trimethylam- moniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) was supplied by Molecular Probes, Inc. Small unilamellar vesicles (SUV) were obtained by sonication in a medium containing 0,1 M KCI and I0