The phospholipid flippase activity of gastric vesicles.

We found that isolated gastric vesicles contain a novel Mg2+-ATP-dependent phospholipid translocation (flippase) activity. Fluorescence analogue of phosphatidylcholine, 2-(12-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3- phosphocholine, was ATP-dependently translocated from the outer (cytosolic) to inner (luminal) leaflet of the lipid membrane bilayer of hog gastric vesicles. The translocation was saturable and depended on time and the ATP concentration (Km = 3.1 microM). The basal Mg2+-ATPase activity of gastric vesicles in the absence of K+ showed high (Km = 1.6 microM) and low (Km = 80 microM) affinities for ATP, indicating that the present flippase activity is driven mostly by the high affinity Mg2+-ATPase activity. It required Mg2+ but not K+. Verapamil, which is an inhibitor of mouse mdr2 phosphatidylcholine flippase, did not inhibit the present flippase activity. Isolated sarcoplasmic reticulum vesicles that contain Ca2+-ATPase did not show any flippase activity. Fluorescence analogues of phosphatidylserine and phosphatidylethanolamine were similarly translocated by the gastric flippase. These phospholipid flippase activities were inhibited by 2-methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine-3-acetonitrile (SCH 28080) (IC50 = 0.14-0.25 microM), a specific K+-ATPase inhibitor of gastric H+,K+-ATPase rich in gastric vesicles. IC50 value for the SCH 28080-inhibitable Mg2+-ATPase activity was about 0.13 microM, indicating that the phospholipid translocation was driven mostly by the SCH 28080-sensitive Mg2+-ATPase activity. Possible physiological roles of flippases were discussed in relation with the gastric acid secretory and cytoprotective mechanisms.