PHOSPHOLIPID SUBCLASS-SPECIFIC ALTERATIONS IN THE PASSIVE ION PERMEABILITY OF MEMBRANE BILAYERS - SEPARATION OF ENTHALPIC AND ENTROPIC CONTRIBUTIONS TO TRANSBILAYER ION FLUX

Alterations in phospholipid class, subclass, and individual molecular species contribute to the diversity of biologic membranes, but their e ffects on membrane passive ion penneability have not been systematical ly studied. Herein, we developed a simple and efficient fluorescence t echnique based upon the loss of valinomycin-inducible membrane potenti al to characterize the passive flux of ions across phospholipid bilaye rs. Detailed kinetic characterization of ion flux across membrane bila yers composed of discrete chemical entities demonstrated that the clas s, subclass, and individual molecular species of each phospholipid hav e substantive effects on membrane passive ion permeability properties. Increasing the degree of unsaturation in either the sn-1 or sn-2 alip hatic chains in phosphatidylcholine markedly enhanced transmembrane io n flux, with over 10-fold differences in the first-order rate constant manifested in molecular species containing four double bonds in compa rison to those possessing three double bonds (e.g., k(app) = 0.0014 mi n(-1) for 1-octadec-9'-enoyl-2-octadec-9', 12'-dienoyl-sn-glycero-3-ph osphocholine (18:1-18:2 phosphatidylcholine) while k(app) = 0.021 min( -1) or octadec-9',12'-dienoyl-sn-glycero-3-phosphocholine (18:2-18:2 p hosphatidylcholine)). Moreover, although the apparent first-order rate constants for transmembrane ion flux in vesicles composed of phosphat idylcholine or plasmanylcholine containing palmitate at the sn-1 posit ion and arachidonate at the sn-2 position were similar (k(app) = 0.04 min(-1) at 22 degrees C for both), the k(app) for corresponding vesicl es composed of plasmenylcholine was 20-fold less (k(app) = 0.002 min(- 1) at 22 OC). Examination of the temperature dependence of passive mem brane ion penneability demonstrated that altered ion flux across membr anes composed of choline glycerophospholipids was primarily due to ent ropic effects without substantial changes in the activation energy for ion translocation. For example, E-a = 19.7 +/- 0.5 and 20.7 +/- 0.6 k cal mol(-1) for 1-hexadecanoyl-2-eicosa-5',8', 11',14'- tetraenoyl-sn- glycero-3-phosphocholine (16:0-20:4 phosphatidylcholine) and ,8',11',1 4'-tetraenoyl-sn-glycero-3-phosphocholine (16:0-20:4 plasmenylcholine) l respectively, while their difference in the entropies of activation (Delta S) was 4.3 +/- 0.5 cal.mol(-1).K-1. Collectively, these results identify substantial differences in the membrane passive ion permeabi lity properties of phospholipid classes, subclasses, and molecular spe cies present in biologic membranes of eukaryotic cells and identify en tropic alterations as an important contributor to these differences.