Abstract
SummaryAcidification 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.
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