Kir4.1/5.1 heterotetramer participates in generating the negative cell membrane potential in distal convoluted tubule (DCT) and plays a critical role in determining the activity of Na-CI cotransporter (NCC). Kir5.1 contains a phosphothreonine motif at its COOH terminus (AA249-252). Coimmunoprecipitation showed that Nedd4-2 was associated with Kir5.1 in HEK293 cells cotransfected with Kir5.1 or Kir4.1/Kir5.1. GST pull-down further confirmed the association between Nedd4-2 and Kir5.1. Ubiquitination assay showed that Nedd4-2 increased the ubiquitination of Kir4.1/Kir5.l heterotetramer in the cells cotransfected with Kir4.1/Kir5.1, but it has no effect on Kir4.1 or Kir5.1 alone.
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