Assembly and trafficking of a multiprotein ROMK (Kir 1.1) channel complex by PDZ interactions.
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.
Yoo
Dana
D
Department of Physiology, University of Maryland School of Medicine, Baltimore 21201, USA.
Flagg
Thomas P
TP
Olsen
Olav
O
Raghuram
Viswanathan
V
Foskett
J Kevin
JK
Welling
Paul A
PA
D020816:Amino Acid Motifs; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry; D003598:Cytoskeletal Proteins / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004594:Electrophysiology; D000939:Epitopes / Q000737:chemistry; D005982:Glutathione Transferase / Q000378:metabolism; D015151:Immunoblotting; D007668:Kidney / Q000378:metabolism; D018613:Microscopy, Confocal; D009865:Oocytes / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry; D015221:Potassium Channels / Q000737:chemistry*; D024661:Potassium Channels, Inwardly Rectifying; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D018075:RNA, Complementary / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D012965:Sodium Chloride / Q000737:chemistry; D017923:Sodium-Hydrogen Exchangers; D014162:Transfection; D020798:Two-Hybrid System Techniques; D014982:Xenopus laevis; D001616:beta-Galactosidase / Q000378:metabolism
D003598:Cytoskeletal Proteins; D000939:Epitopes; C510692:Kcnj1 protein, rat; D010750:Phosphoproteins; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D018075:RNA, Complementary; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C498832:Slc9a3r2 protein, rat; D017923:Sodium-Hydrogen Exchangers; C093376:sodium-hydrogen exchanger regulatory factor; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D012965:Sodium Chloride; D004247:DNA; D005982:Glutathione Transferase; D001616:beta-Galactosidase