The CFTR gene encodes a transmembrane conductance regulator, which is dysfunctional in patients with cystic fibrosis (CF). The mechanism by which defective CFTR (CF transmembrane conductance regulator) leads to undersialylation of plasma membrane glycoconjugates, which in turn promote lung pathology and colonization with Pseudomonas aeruginosa causing lethal bacterial infections in CF, is not known. Here we show by ratiometric imaging with lumenally exposed pH-sensitive green fluorescent protein that dysfunctional CFTR leads to hyperacidification of the trans-Golgi network (TGN) in CF lung epithelial cells. The hyperacidification of TGN, glycosylation defect of plasma membrane glycoconjugates, and increased P. aeruginosa adherence were corrected by incubating CF respiratory epithelial cells with weak bases. Studies with pharmacological agents indicated a role for sodium conductance, modulated by CFTR regulatory function, in determining the pH of TGN. These studies demonstrate the molecular basis for defective glycosylation of lung epithelial cells and bacterial pathogenesis in CF, and suggest a cure by normalizing the pH of intracellular compartments.

Both lysosomes and endosomes are acidified by an electrogenic proton pump, although studies in intact cells indicate that the steady-state internal pH (pHi) of lysosomes is more acid than that of endosomes. We undertook the present study to examine in detail the acidification mechanism of purified rat liver secondary lysosomes and to compare it with that of a population of early endosomes. Both endosomes and lysosomes exhibited ATP-dependent acidification, but proton influx rates were 2.4- to 2.7-fold greater for endosomes than for lysosomes because of differences in both buffering capacity and acidification rates, suggesting that endosomes exhibited greater numbers or rates of proton pumps. Lysosomes, however, exhibited a more acidic steady-state pHi due in part to a slower proton leak rate. Changes in medium Cl- increased acidification rates of endosomes more than lysosomes, and the lysosome ATP-dependent interior-positive membrane potential was only partially eliminated by high-Cl- medium. Permeability studies suggested that lysosomes were less permeable to Na+, Li+, and Cl- and more permeable to K+ and PO4(2-) than endosomes. Na-K-adenosine-triphosphatase did not appear to regulate acidification of either vesicle type. Endosome and lysosome acidification displayed similar inhibition profiles to N-ethylmaleimide, dicyclohexyl-carbodiimide, and vanadate, although lysosomes were somewhat more sensitive [concentration producing 50% maximal inhibition (IC50) 1 nM] to bafilomycin A1 than endosomes (IC50 7.6 nM). Oligomycin (1.5-3 microM) stimulated lysosome acidification due to shunting of membrane potential. Overall, acidification of endosomes and lysosomes was qualitatively similar but quantitatively somewhat different, possibly related to differences in the density or rate of proton pumps as well as vesicle permeability to protons, anions, and other cations.

Endocytosed ligands move through a series of progressively more acidic vesicles. These differences in pH (pHi) could reflect differences in ion transport mechanisms. Vesicles representing three stages of endocytosis, compartment for uncoupling of receptor and ligand (CURL), multivesicular bodies (MVB), and receptor recycling compartment (RRC), were studied, and all exhibited ATP-dependent electrogenic acidification that was a saturable function of medium chloride. Initial rates of acidification differed (RRC > CURL > MVB), and proton influx was similar for CURL and RRC but slower for MVB. Steady-state ATP-dependent pHi in the three vesicles was more similar. Vesicle membrane potential was substantial (+41 to +69 mV) in low-chloride medium and greatest for RRC but was low (-6 to +6 mV) in 140 mM KCl. These vesicles also exhibited -22 to -37 mV Donnan potentials. Steady-state pump-generated proton electrochemical gradients (delta mu H+) ranged from 114 to 175 mV and were greater for CURL and RRC than for MVB; however, delta mu H+ changed little over a 140-fold difference in chloride concentration. Proton leak rates were faster in CURL and RRC than in MVB, but proton efflux was similar. Finally, proton fluxes and permeabilities, calculated with regard to surface area, differed in the opposite direction (MVB > CURL > RRC). Thus, for the endocytic vesicles studied, intrinsic differences in proton flux and in vesicle geometry could be demonstrated that contributed to differences in pre-steady-state vesicle pHi.

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