ClC‐3 and LRRC8A interact physically and co‐localize with endosomal superoxide production.

Both the ClC‐3 Cl − /H + antiporter and LRRC8A anion channels are required for TNFα‐induced, Nox1‐dependent superoxide (O 2 −• ) production and subsequent signaling in vascular smooth muscle cells (VSMC). Both proteins have been associated with the swelling‐activated anion conductance (ICl swell ) which can modulate Nox1 activity, and thereby TNFα signaling. It is now clear that LRRC8 family (LRRC8A‐E) proteins constitute ICl swell channels. However, it is not known why ClC‐3 expression also affects the magnitude of this anion current. We hypothesized that a physical relationship and functional interdependence between ClC‐3 and LRRC8A explains this finding. Confocal microscopy and immunoprecipitation were used to localize expressed ClC‐3 and LRRC8A with O 2 −• (ROSstar 550) in HEK293 cells expressing fluorescent fusion proteins. The endogenous proteins were localized by immunostaining of cultured human aortic VSMC. As previously demonstrated, ClC‐3‐GFP localizes to the membrane of very large intracellular vesicles (1–10 μm diameter). These compartments are now revealed to be multivesicular bodies, and the smaller vesicles within (0.1–0.6 μm) are a prominent site of ROSstar staining. When expressed alone, GFP‐LRRC8A localizes to small cytoplasmic vesicles which also are strongly ROSstar positive, suggesting that this is an important site of O 2 −• production. Co‐expression of ClC‐3‐GFP and mCherry‐LRRC8A reveals that small vesicles, which are rich in LRRC8A, are contained within ClC‐3‐expressing multivesicular bodies. In VSMC, antibodies to ClC‐3, LRRC8A and Nox1 stain both localized regions of the plasma membrane and scattered intracellular vesicles in the 0.1–0.60 μm range. Overlapping staining for all three proteins is seen in a subset of these vesicles, and in scattered multivesicular clusters. We have previously demonstrated that LRRC8A co‐immunoprecipitates with Nox1 and its p22phox subunit (Choi, FRBM , 2016). We now observe that immunoprecipitation of endogenous ClC‐3 pulls down LRRC8A. These findings demonstrate co‐localization and physical association between ClC‐3 and LRRC8A. This association, at sites where Nox1 is producing O 2 −• , may explain why both proteins play a role in Nox1‐mediated O 2 −• production and TNFα signaling. Support or Funding Information HC is supported by an SDG from the American Heart Association. FL is supported by R01 HL128386.