Ubiquitylation of the plasma membrane KCa3.1 is not required for channel endocytosis but for its lysosomal targeting

We recently demonstrated that plasma membrane KCa3.1 is ubiquitylated subsequent to endocytosis and targeted for lysosomal degradation. To further elucidate the role of ubiquitylation in KCa3.1 endocytosis and trafficking, we constructed a mutant KCa3.1 in which all 18 predicted cytoplasmic lysine residues were replaced with arginines (K‐less). This K‐less KCa3.1 trafficked normally to the plasma membrane and was functional. The K‐less KCa3.1 internalized normally, indicating that channel ubiquitylation is not required for endocytosis. However, the treatment with the ubiquitin‐activating enzyme (E1) inhibitor UBEI‐41 resulted in a reduced mutant endocytosis, suggesting that ubiquitylation of other adaptor proteins is required for KCa3.1 internalization. Next, we showed that the mutant degradation was dramatically reduced. To determine which lysine(s) are being ubiquitylated and target KCa3.1 for lysosomal degradation, we generated mutants where individual lysines were introduced back into the K‐less KCa3.1. Using tandem ubiquitin‐binding entities to isolate ubiquitylated proteins we could demonstrate that lysines in the N‐terminus and intracellular loops of KCa3.1 are not important in this process. The role of the other lysines remains to be determined. These results provide novel insights into the molecular mechanisms of endocytosis and downstream trafficking of KCa3.1 channels.