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- Title
Chloride Channel-3 (ClC-3) Modifies the Trafficking of Leucine-Rich Repeat-Containing 8A (LRRC8A) Anion Channels.
- Authors
Stark, Ryan J.; Nguyen, Hong N.; Bacon, Matthew K.; Rohrbough, Jeffrey C.; Choi, Hyehun; Lamb, Fred S.
- Abstract
Chloride channel-3 (ClC-3) Cl−/H+ antiporters and leucine-rich repeat-containing 8 (LRRC8) family anion channels have both been associated with volume-regulated anion currents (VRACs). VRACs are often altered in ClC-3 null cells but are absent in LRRC8A null cells. To explore the relationship between ClC-3, LRRC8A, and VRAC we localized tagged proteins in human epithelial kidney (HEK293) cells using multimodal microscopy. Expression of ClC-3-GFP induced large multivesicular bodies (MVBs) with ClC-3 in the delimiting membrane. LRRC8A-RFP localized to the plasma membrane and to small cytoplasmic vesicles. Co-expression demonstrated co-localization in small, highly mobile cytoplasmic vesicles that associated with the early endosomal marker Rab5A. However, most of the small LRRC8A-positive vesicles were constrained within large MVBs with abundant ClC-3 in the delimiting membrane. Dominant negative (S34A) Rab5A prevented ClC-3 overexpression from creating enlarged MVBs, while constitutively active (Q79L) Rab5A enhanced this phenotype. Thus, ClC-3 and LRRC8A are endocytosed together but independently sorted in Rab5A MVBs. Subsequently, LRRC8A-labeled vesicles were sorted to MVBs labeled by Rab27A and B exosomal compartment markers, but not to Rab11 recycling endosomes. VRAC currents were significantly larger in ClC-3 null HEK293 cells. This work demonstrates dependence of LRRC8A trafficking on ClC-3 which may explain the association between ClC-3 and VRACs.
- Subjects
ANIONS; LEUCINE; CHLORIDES; IMMOBILIZED proteins; CELL membranes; CHLORIDE channels; ENDOSOMES
- Publication
Journal of Membrane Biology, 2023, Vol 256, Issue 2, p125
- ISSN
0022-2631
- Publication type
Article
- DOI
10.1007/s00232-022-00271-9