The effects of lipid composition on the binding of lasalocid A to small unilamellar vesicles

Abstract

The binding of the carboxylic ionophore lasalocid A (X537A) to small unilamellar phospholipid vesicles of varying composition was examined in an effort to determine what structural features of the phospholipid membrane influence the ionophore-membrane interaction. Apparent dissociation constants ( K app) were calculated for both the acidic and anionic forms of the ionophore using the change in fluorescence intensity observed for lasalocid A upon addition of phospholipid vesicles. The K app for binding to fluid phase dimyristoylphosphatidylcholine (DMPC) vesicles is 46 μM for the anion and 14 μM for the acid. While the phase transition of DMPC had no effect on the K app of the anion, an increase was observed in the K app of the acid below the phase transition temperature. The K app of the anion was not affected by the incorporation of 10% dimyristoylphosphatidylethanolamine (DMPE), but increased slightly upon incorporation of cholesterol. The p K a values of the ionophore were the same in DMPC and DMPC/DMPE membranes. Incorporation of the negative lipids phosphatidylglycerol, phosphatidic acid, or phosphatidylethanolamine (at pH 9.4 where PE carries a negative charge) decreases binding of the anion in accord with the increase in surface potential estimated from Gouy-Chapman theory. The CD spectrum of membrane-bound lasalocid A anion indicated the ionophore to be in an extended acyclic conformation on the membrane surface with the C-1 carboxylate rotated out of the plane of the salicylate ring. The out-of-plane rotation of the carboxylate may be the result of facial binding by the amphiphilic ionophore on the membrane surface or of weak ion pairing to the polar lipid head groups. These results suggest that the primary determinants of binding of the anionic ionophore on the membrane surface are packing density of the polar head groups and membrane surface potential. There is no evidence of strong hydrogen bond formation between the lipid polar head groups and the ionophore as has previously been suggested.