pH gradients across phospholipid membranes caused by fast flip-flop of un-ionized fatty acids.

Abstract

A central, unresolved question in cell physiology is how fatty acids move across cell membranes and whether protein(s) are required to facilitate transbilayer movement. We have developed a method for monitoring movement of fatty acids across protein-free model membranes (phospholipid bilayers). Pyranin, a water-soluble, pH-sensitive fluorescent molecule, was trapped inside well-sealed phosphatidylcholine vesicles (with or without cholesterol) in Hepes buffer (pH 7.4). Upon addition of a long-chain fatty acid (e.g., oleic acid) to the external buffer (also Hepes, pH 7.4), a decrease in fluorescence of pyranin was observed immediately (within 10 sec). This acidification of the internal volume was the result of the "flip" of un-ionized fatty acids to the inner leaflet, followed by a release ofprotons from -50% of these fatty acid molecules (apparent pKa in the bilayer = 7.6). The proton gradient thus generated dissipated slowly because of slow cyclic proton transfer by fatty acids. Addition of bovine serum albumin to vesicles with fatty acids instantly removed the pH gradient, indicating complete removal of fatty acids, which requires rapid "flop" of fatty acids from the inner to the outer monolayer layer. Using a four-state kinetic diagam of fatty acids in membranes, we conclude that un-ionized fatty acid flip-flops rapidly (t,/2 s 2 sec) whereas ionized fatty acid flip-flops slowly (t112 of minutes). Since fatty acids move across phosphatidylcboline bilayers spontaneously and rapidly, complex mechanisms (e.g., transport proteins) may not be required for translocation of fatty acids in biological membranes. The proton movement accompanying fatty acid ifip-flop is an important consideration for fatty acid metabolism in normal physiology and in disease states such as cardiac ischemia.Unesterified fatty acids are a key intermediate in lipid metabolism. In the well-oxygenated heart, fatty acids constitute the primary fuel, and the availability of fatty acids in plasma is a controlling factor in the rate of intracellular fatty acid utilization (1). Recently, fatty acids have also been found to have diverse biological activities, serving as second messengers (2), K+ channel activators (3), inhibitors of the binding of plasma low density lipoproteins to receptors (4), and uncouplers of oxidative phosphorylation (5, 6). Fatty acids are either delivered to a membrane [e.g., from serum albumin (7)] or generated within it [e.g., by phospholipase A2 (8)] in one leaflet only. Movement of fatty acids across the membrane may be essential for their utilization.At present there is controversy as to the mechanism and rate of transbilayer movement offatty acids in membranes (7,9). Investigations with cell systems have led to conflicting conclusions, suggesting either rapid passive diffusion ["flipflop" (10)] across the plasma membrane (11, 12) or proteinmediated transport (13-15), although the latter studies have not distinguished whether the protein(s) sequesters fatty acids from the plasma into the membrane or actually tra...

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