An Exploration of Charge Compensating Ion Channels across the Phagocytic Vacuole of Neutrophils

Abstract

Neutrophils phagocytosing bacteria and fungi exhibit a burst of non-mitochondrial respiration that is required to kill and digest the engulfed microbes. This respiration is accomplished by the movement of electrons across the wall of the phagocytic vacuole by the neutrophil NADPH oxidase, NOX2. In this study, we have attempted to identify the non-proton ion channels or transporters involved in charge compensation by examining the effect of inhibitors on vacuolar pH and cross-sectional area, and on oxygen consumption. The chloride channel inhibitors 4-[(2-Butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid (DCPIB) and flufenamic acid (FFA) were the most effective inhibitors of alkalinisation in human neutrophil vacuoles, suggesting an efflux of chloride from the vacuole. The proton channel inhibitor, zinc (Zn2+), combined with DCPIB caused more vacuolar swelling than either compound alone, suggesting the conductance of osmotically active cations into the vacuole. Support for cation influx was provided by the broad-spectrum cation transport inhibitors anandamide and quinidine which inhibited vacuolar alkalinisation and swelling when applied with zinc. Oxygen consumption was generally unaffected by these anion or cation inhibitors alone, but when combined with Zn2+ it was dramatically reduced, suggesting that multiple channels in combination can compensate the charge. In an attempt to identify specific channels, we tested neutrophils from knock-out mouse models including CLIC1, ClC3, ClC4, ClC7, KCC3, KCNQ1, KCNE3, KCNJ15, TRPC1/3/5/6, TRPA1/TRPV1, TRPM2, and TRPV2, and double knockouts of CLIC1, ClC3, KCC3, TRPM2, and KCNQ1 with HVCN1, and humans with channelopathies involving BEST1, ClC7, CFTR, and MCOLN1. No gross abnormalities in vacuolar pH or area were found in any of these cells suggesting that we had not tested the correct channel, or that there is redundancy in the system. The respiratory burst was suppressed in the KCC3-/- and enhanced in the CLIC1-/- cells, but was normal in all others, including ClC3-/-. These results suggest charge compensation by a chloride conductance out of the vacuole and by cation/s into it. The identity of these channels remains to be established.

K+-Cl− cotransporter 1 (KCC1): a housekeeping membrane protein that plays key supplemental roles in hematopoietic and cancer cells

“…As mentioned, KCC1 is ubiquitously expressed and could thus play roles in many other cell types within the hematopoietic lineage. In this regard, KCC3 has been found to sustain hypochlorite synthesis by white blood cells through its Cl − cotransporter function in phagosomes [103, 104]. Along the same line, it is particularly intriguing that KCC1 is expressed in a variety of leukemic cells but that it is virtually absent from most types of lymphoma cells.…”

The LRRC8A Mediated “Swell Activated” Chloride Conductance Is Dispensable for Vacuolar Homeostasis in Neutrophils

“…Foote et al (2017) have demonstrated that a range of Cl - channel blocker can reduce this alkalinisation, but tamoxifen, which strongly blocks the swell activated channel, was clearly unable to do so ( Figure 9A ). It was also without effect on the volume of the phagocytic vacuole ( Figure 9B ).…”

“…Cl - appears to play an important role in this process because certain Cl - channel blockers inhibit this elevation in pH ( Foote et al, 2017 ). There are two competing theories as to the direction of the movement of Cl - .…”

Macrophage Polarization Alters Postphagocytosis Survivability of the Commensal Streptococcus gordonii

“…Because we preactivated the RAW264.7 macrophages prior to performing the killing assays, which increases phagosomal ROS production (48-50), we next tested the requirement for ROS production by macrophages for the survival of S. gordonii. When the macrophages were treated with diphenyleneiodonium (DPI), a NOX2 inhibitor that prevents phagosomal ROS production (51)(52)(53)(54)(55), intracellular survival of the pathogenic strain DL1 decreased to levels not significantly different from that of the nonpathogenic strain SK12 ( Fig. 2A).…”