(A) Domain structure of ITSN1s. It contains 2 EH domains, a central coiled-coil (CC) region, and 5 consecutive SH3 domains. The EH domains anchor ITSN to clathrin coat. The SH3 domains recruit dynamin, synaptojanin, and other endocytic proteins. (B) Amino acids 1–491 of long WNK1 interacted specifically with the ITSN SH3 C domain. GST fusion proteins were used to pull down myc-tagged WNK1_1–491 from transfected cell lysates. Western blot analysis was performed with anti-GST and anti-myc antibodies, respectively. Amphi, amphiphysin; Endo, endophilin. (C) Evidence for knockdown of endogenous ITSN1. Endogenous ITSN1s was detected by an anti-ITSN antibody. Western blot analysis of lysates from mock-transfected (control) cells and cells transfected with HA-tagged ITSN1s probed by anti-ITSN and anti-HA antibodies is shown on the right. (D) Knockdown of ITSN1 increased surface abundance of ROMK1. (E) Knockdown of ITSN1 increased baseline whole-cell ROMK1 current density and prevented the decrease caused by WNK1_1–491. Cells were transfected with ROMK1 plus indicated constructs. (F) Overexpression of dominant-negative ITSN1 increased baseline whole-cell ROMK1 current density and prevented the decrease caused by WNK1_1–491. *P < 0.05 versus ROMK1 alone.