Identification of gamma-aminobutyric acid receptor-interacting factor 1 (TRAK2) as a trafficking factor for the K+ channel Kir2.1.
To identify proteins that regulate potassium channel activity and expression, we performed functional screening of mammalian cDNA libraries in yeast that express the mammalian K(+) channel Kir2.1. Growth of Kir2.1-expressing yeast in media with low K(+) concentration is a function of K(+) uptake via Kir2.1 channels. Therefore, the host strain was transformed with a human cDNA library, and cDNA clones that rescued growth at low K(+) concentration were selected. One of these clones was identical to the protein of unknown function isolated previously as gamma-aminobutyric acid receptor-interacting factor 1 (GRIF-1) (Beck, M., Brickley, K., Wilkinson, H., Sharma, S., Smith, M., Chazot, P., Pollard, S., and Stephenson, F. (2002) J. Biol. Chem. 277, 30079-30090). GRIF-1 specifically enhanced Kir2.1-dependent growth in yeast and Kir2.1-mediated (86)Rb(+) efflux in HEK293 cells. Quantitative microscopy and flow cytometry analysis of immunolabeled surface Kir2.1 channel showed that GRIF-1 significantly increased the number of Kir2.1 channels in the plasma membrane of COS and HEK293 cells. Physical interaction of Kir2.1 channel and GRIF-1 was demonstrated by co-immunoprecipitation from HEK293 lysates and yeast two-hybrid assay. In vivo association of Kir2.1 and GRIF-1 was demonstrated by co-immunoprecipitation from brain lysate. Yeast two-hybrid assays showed that an N-terminal region of GRIF-1 interacts with a C-terminal region of Kir2.1. These results indicate that GRIF-1 binds to Kir2.1 and facilitates trafficking of this channel to the cell surface.
Grishin
Anatoly
A
Department of Pharmacology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
Li
Hui
H
Levitan
Edwin S
ES
Zaks-Makhina
Elena
E
D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D024661:Potassium Channels, Inwardly Rectifying / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000235:genetics; D020798:Two-Hybrid System Techniques
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; C489457:KCNJ2 protein, human; D009419:Nerve Tissue Proteins; D024661:Potassium Channels, Inwardly Rectifying; C465341:TRAK2 protein, human