pH of TGN and recycling endosomes of H+/K+-ATPase-transfected HEK-293 cells:: implications for pH regulation in the secretory pathway

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 (pH(TGN)) and recycling endosomes (pH(RE)) 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 pH(TGN) was similar in both H+/K+-alpha,beta cells (pH(TGN) 6.36) and vector-transfected ("mock") cells (pH(TGN) 6.34); pH(RE) was also similar in H+/K+-alpha,beta (pH(RE) 6.40) and mock cells (pH(RE) 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 pH(TGN) of 7.0-7.5, close to pH(cytosol). 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 (8,000) active in the TGN to balance a large, passive H+ leak (with P-H similar to 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 similar to4,000 active in the TGN) and other transporters have little effect on luminal pH as they traffic to the plasma membrane.