Stable Expression of Gastric Proton Pump Activity at the Cell Surface

Stable cell lines expressing the gastric proton pump a-and/or β-subunits were constructed. The cell line co-expressing the a- and β-subunits showed inward Rb+transport, which was activated by Rb* in a concentration-dependent manner. In the α+βP-express-ing cell line, rapid recovery of intracellular pH was also observed after acid load, indicating that this cell line transported protons outward. These ion transport activities were inhibited by a proton pump inhibitor, 2-methyl-8-(phenylmethoxy)imidazo[l,2-α]pyridine-3-acetonitrile (SCH 28080). In a membrane fraction of the α+β-expressing cell line, K+-stimulated ATPase (K+-ATPase) activity and the acylphosphorylation of the α-subunit were observed, both of which were also inhibited by SCH 28080. The specific activity and properties of the K+-ATPase were comparable to those found in the native gastric proton pump. In the stable cell lines, the α-subunit was retained in the intracellular compartment and was unstable in the absence of the (β-subunit, but it was stabilized and reached the cell surface in the presence of the (β-subunit. On the other hand, the β-subunit was stable and able to travel to the cell surface in the absence of the α-subunit. These cell lines are ideal for the structure-function study of ion transport by the gastric proton pump as well as for characterization of the cellular regulation of surface expression of the functional proton pump.