We previously found that the Ca-sensing receptor (CaR) interacts with and inactivates the inwardly-rectifying K channel, Kir4.2 that is expressed in the kidney cortex and that has a C-terminal PDZ domain. In order to identify potential scaffolding proteins that could organize a macromolecular signaling complex involving the CaR and Kir4.2, we used yeast two hybrid cloning with the C-terminal 125 AA of Kir4.2 as bait to screen a human kidney cDNA library. We identified two independent partial cDNAs corresponding to the C-terminal 900 AA of MUPP1, a protein containing 13 PDZ binding domains and that is expressed in the kidney in tight junctions and lateral borders of epithelial cells. When expressed in HEK-293 cells, Kir4.2 co-immunoprecipitates reciprocally with MUPP1 but not a Kir4.2 construct lacking the four C-terminal amino acids, Kir5.1, or the CaR. MUPP1 and Kir4.2 co-immunoprecipitate reciprocally from rat kidney cortex extracts. Co-expression of MUPP1 with Kir4.2 in HEK293 cells leads to reduced cell surface expression of Kir4.2 as assessed by cell surface biotinylation. Co-expression of MUPP1 and Kir4.2 in Xenopus oocytes results in reduced whole cell currents compared to Kir4.2 alone, while expression of Kir4.2PDZ results in minimal currents, and is not affected by co-expression with MUPP1. Immunofluorecence studies of oocytes demonstrate that MUPP1 reduces Kir4.2 membrane localization. These results indicate that Kir4.2 interacts selectively with MUPP1 to affect its cell surface expression. Thus, MUPP1 and Kir4.2 may participate in a protein complex in the nephron that could regulate transport of K⁺ as well as other ions.