The proton‐pumping H+,K+‐adenosinetriphosphatase (H,K‐ATPase), responsible for acid secretion by the gastric parietal cell, faces a harshly acidic environment, with some pepsin from neighboring chief cells, at its luminal surface. Its large catalytic α‐subunit is mostly oriented cytoplasmically. The smaller β‐subunit (HKβ), is mainly extracellular, with one transmembrane domain and a small cytoplasmic domain. Seven N‐linked oligosaccharides in the extracellular domain of HKβ are thought to contribute to protection of the H,K‐ATPase, since previous work has shown that their complete removal, by peptide N‐glycosidase F (PNGase F), greatly increased susceptibility of HKβ to proteolysis. The possibility of graded protection by different numbers of oligosaccharides was investigated here with the use of mutant HKβ cDNA, having various N‐glycosylation sites mutated (Asn to Gln), transfected into HEK‐293 cells. Membrane preparations, two days after transfection, were solubilized in 1% Triton X‐100 and subjected to trypsinolysis (pH 8, 37°C, trypsin:protein 1:10–1:25). Relative amounts of HKβ remaining after 20 min trypsin were determined, after sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and probing of Western blots with an antibody to the HKβ extracellular domain, by chemiluminescent development of blots and densitometry of resulting films. Maturely glycosylated HKβ was made significantly more susceptible to trypsin than wild type when at least five oligosaccharides were deleted, while the high‐mannose form (pre‐β), from the endoplasmic reticulum, became significantly more susceptible than wild‐type pre‐β with removal of only two or more oligosaccharides. For each mutant, and wild type, pre‐β was consistently more susceptible than the mature form. While the number, and kind, of oligosaccharides seem to affect protection for HKβ against trypsinolysis, other aspects of protein maturation, including proper folding of peptide domains and possible subtle alterations of conformation during Golgi processing, are also likely to contribute to this protection.

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ABSTRACT The influences of the gastric H,-α,βcells). The pH’s of trans-Golgi network (pHTGN) and

The influences of the gastric H+/K+ pump on organelle pH during trafficking to and from the plasma membrane were investigated using HEK-293 cells stably expressing the alpha- and beta-subunits of human H+/K+-ATPase (H+/K+-alpha,beta cells). The pH values of trans-Golgi network (pHTGN) and recycling endosomes (pHRE) were measured by transfecting H+/K+-alpha,beta cells with the pH-sensitive GFP pHluorin fused to targeting sequences of either TGN38 or synaptobrevin, respectively. Immunofluorescence showed that H+/K+-ATPase was present in the plasma membrane, TGN, and RE. The pHTGN was similar in both H+/K+-alpha,beta cells (pHTGN 6.36) and vector-transfected ("mock") cells (pHTGN 6.34); pHRE was also similar in H+/K+-alpha,beta (pHRE 6.40) and mock cells (pHRE 6.37). SCH28080 (inhibits H+/K+-ATPase) caused TGN to alkalinize by 0.12 pH units; subsequent addition of bafilomycin (inhibits H+ v-ATPase) caused TGN to alkalinize from pH 6.4 up to a new steady-state pHTGN of 7.0-7.5, close to pHcytosol. Similar results were observed in RE. Thus H+/K+-ATPases that trafficked to the plasma membrane were active but had small effects to acidify the TGN and RE compared with H+ v-ATPase. Mathematical modeling predicted a large number of H+ v-ATPases (8000) active in the TGN to balance a large, passive H+ leak (with PH approximately 10-3 cm/s) via unidentified pathways out of the TGN. We propose that in the presence of this effective, though inefficient, buffer system in the Golgi and TGN, H+/K+-ATPases (estimated to be approximately 4000 active in the TGN) and other transporters have little effect on luminal pH as they traffic to the plasma membrane.

Stable cell lines expressing the gastric proton pump alpha- and/or beta-subunits were constructed. The cell line co-expressing the alpha- and beta-subunits showed inward Rb(+) transport, which was activated by Rb(+) in a concentration-dependent manner. In the alpha+beta-expressing 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[1,2-a]pyridine-3-acetonitrile (SCH 28080). In a membrane fraction of the alpha+beta-expressing cell line, K(+)-stimulated ATPase (K(+)-ATPase) activity and the acylphosphorylation of the alpha-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 alpha-subunit was retained in the intracellular compartment and was unstable in the absence of the beta-subunit, but it was stabilized and reached the cell surface in the presence of the beta-subunit. On the other hand, the beta-subunit was stable and able to travel to the cell surface in the absence of the alpha-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.