The human ATP1AL1-encoded protein (an ␣ subunit of the human non-gastric H,K-ATPase) has previously been shown to assemble with the gastric H,K-ATPase ␤ subunit (gH,K␤) to form a functionally active ionic pump in HEK 293 cells. This pump has been found to be sensitive to both SCH 28080 and ouabain. However, the 86 Rb ؉influx mediated by the ATP1AL1-gH,K␤ heterodimer in HEK 293 cells is at least 1 order of magnitude larger than the maximum ouabain-sensitive proton efflux detected in the same

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... s. In this study we find that the intracellular Na ؉ content in cells expressing ATP1AL1 and gH,K␤ is two times lower than that in control HEK 293 cells in response to incubation for 3 h in the presence of 1 M ouabain. Moreover, analysis of net Na ؉ efflux in HEK 293 expressing the ATP1AL1-gH,K␤ heterodimer reveals the presence of Na ؉ extrusion activity that is not sensitive to 1 M ouabain but can be inhibited by 1 mM of this drug. In contrast, ouabain-inhibitable Na ؉ efflux in control HEK 293 cells is similarly sensitive to either 1 M or 1 mM ouabain. Finally, 86 Rb ؉ influx through the ATP1AL1-gH,K␤ complex is comparable to the 1 mM ouabain-sensitive Na ؉ efflux in the same cells. The data presented here suggest that the enzyme formed by ATP1AL1 and the gastric H,K-ATPase ␤ subunit in HEK 293 cells mediates primarily Na ؉ ,K ؉ rather than H ؉ ,K ؉ exchange.