Endocytic trafficking of the Kir2.3 channel

Polarized trafficking and expression of different inwardly rectifying potassium (Kir) channels on opposite membrane domains of the cortical collecting duct principal cell insure an efficient and precisely controlled potassium secretion process, important for potassium homeostasis. We have previously shown that basolateral membrane localization and density of the Kir 2.3 channel is coordinated by direct interaction with the Lin7/CASK PDZ complex, possibly by limiting endocytic trafficking. Here we explore mechanisms of Kir 2.3 channel routing into the endocytic pathway. Lifetime of channels on the plasma membrane was monitored by several independent techniques in Xenopus oocytes under conditions that prevent delivery of channels to the cell surface. As measured by surface biotinylation and two‐microelectrode voltage clamp, co‐expression of dominant negative dynamin II (K44A) but not wild type dynamin II caused a dramatic increase in plasmalemma residence time, indicating the Kir 2.3 cell surface density is controlled by vesicular endocytosis. The cytoplasmic Kir 2.3 C‐terminus contains several canonical sites for clathrin adapter (AP2) interaction, raising the possibility that Kir2.3 channels are internalized by clathrin dependent endocytosis. To test this hypothesis, mutations in the candidate AP‐2 interaction residues were independently created in extracellular HA epitope tagged Kir 2.3 and cell surface lifetime was measured by antibody binding and analytical luminometry. Of the four candidate internalization sequences studied, loss of function mutations within the dileucine motif at position 184/185 limited internalization most dramatically. These results suggest that interaction of AP‐2 with Kir 2.3 at LL185 marks channels for rapid internalization from the plasma membrane.