Abstract
Acidification in intracellular organelles is tightly linked to the influx of Cl- counteracting proton translocation by the electrogenic V-ATPase. We quantified the dynamics of Cl- transfer accompanying cargo incorporation into single phagosomes in alveolar macrophages (AMs). Phagosomal Cl- concentration and acidification magnitude were followed in real time with maximal acidification achieved at levels of approximately 200 mM. Live cell confocal microscopy verified that phagosomal Cl- influx utilized predominantly the Cl- channel CFTR. Relative levels of elemental chlorine (Cl) in hard X-ray fluorescence microprobe (XFM) analysis within single phagosomes validated the increase in Cl- content. XFM revealed the complex interplay between elemental K content inside the phagosome and changes in Cl- during phagosomal particle uptake. Cl- -dependent changes in phagosomal membrane potential were obtained using second harmonic generation (SHG) microscopy. These studies provide a mechanistic insight for screening studies in drug development targeting pulmonary inflammatory disease.