Terry Machen, Mary Jae
Leigh, Carmen Taylor, Tohru Kimura, Shinji Asano and Hsiao-Ping Moore
(2003). pH of TGN and recycling endosomes of H/K-ATPase-transfected
HEK 293 cells: implications for pH regulation in the secretory pathway.
Am. J. Physiol. 285, C205-C214.
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.