Assembly and trafficking of a multiprotein ROMK (Kir 1.1) channel complex by PDZ interactions.

Abstract	The ROMK subtypes of inward rectifier K+ channels (Kir 1.1, KCNJ1) mediate potassium secretion and regulate NaCl reabsorption in the kidney. In the present study, the role of the PDZ binding motif in ROMK function is explored. Here we identify the Na/H exchange regulatory factors, NHERF-1 and NHERF-2, as PDZ domain interaction partners of the ROMK channel. Characterization of the basis and consequences of NHERF association with ROMK reveals a PDZ interaction-dependent trafficking process and a coupling mechanism for linking ROMK to a channel modifier protein, the cystic fibrosis transmembrane regulator (CFTR). As measured by antibody binding of external epitope-tagged forms of Kir 1.1 in intact cells, NHERF-1 or NHERF-2 coexpression increased cell surface expression of ROMK. Channel interaction with NHERF proteins and effects of NHERF on ROMK localization were dependent on the presence of the PDZ domain binding motif in ROMK. Both NHERF proteins contain two PDZ domains; recombinant protein-protein binding assays and yeast-two-hybrid studies revealed that ROMK preferentially associates with the second PDZ domain of NHERF-1 and with the first PDZ domain of NHERF-2, precisely opposite of what has been reported for CFTR. Consistent with the scaffolding capacity of the NHERF proteins, coexpression of NHERF-2 with ROMK and CFTR dramatically increases the amount of ROMK protein that coimmunopurifies and functionally interacts with CFTR. Thus NHERF facilitates assembly of a ternary complex containing ROMK and CFTR. These observations raise the possibility that PDZ-based interactions may underscore physiological regulation and membrane targeting of ROMK in the kidney.
Authors	Dana Yoo, Thomas P Flagg, Olav Olsen, Viswanathan Raghuram, J Kevin Foskett, Paul A Welling
Journal	The Journal of biological chemistry (J Biol Chem) Vol. 279 Issue 8 Pg. 6863-73 (Feb 20 2004) ISSN: 0021-9258 [Print] United States
PMID	14604981 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)
Chemical References	Cytoskeletal Proteins Epitopes Kcnj1 protein, rat Phosphoproteins Potassium Channels Potassium Channels, Inwardly Rectifying RNA, Complementary Recombinant Fusion Proteins Recombinant Proteins Slc9a3r2 protein, rat Sodium-Hydrogen Exchangers sodium-hydrogen exchanger regulatory factor Cystic Fibrosis Transmembrane Conductance Regulator Sodium Chloride DNA Glutathione Transferase beta-Galactosidase
Topics	Amino Acid Motifs Animals COS Cells Cell Membrane (metabolism) Cystic Fibrosis Transmembrane Conductance Regulator (chemistry) Cytoskeletal Proteins (chemistry) DNA (chemistry) Electrophoresis, Polyacrylamide Gel Electrophysiology Epitopes (chemistry) Glutathione Transferase (metabolism) Immunoblotting Kidney (metabolism) Microscopy, Confocal Oocytes (metabolism) Phosphoproteins (chemistry) Potassium Channels (chemistry) Potassium Channels, Inwardly Rectifying Precipitin Tests Protein Binding Protein Structure, Tertiary Protein Transport RNA, Complementary (metabolism) Rats Recombinant Fusion Proteins (metabolism) Recombinant Proteins (chemistry) Sodium Chloride (chemistry) Sodium-Hydrogen Exchangers Transfection Two-Hybrid System Techniques Xenopus laevis beta-Galactosidase (metabolism)

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