To identify proteins that regulate potassium (K+) 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 GABA Receptor Interacting Factor (GRIF-1) (Beck, M., Brickley, K., Wilkinson, H. L., Sharma, S., Smith, M., Chazot, P. L., Pollard, S., and Stephenson F. A., (2002), J. Biol. Chem., 277, 30079-30090). GRIF-1 specifically enhanced Kir2.1-dependent growth in yeast and Kir2.1-mediated 86Rb+ uptake 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 coimmunoprecipitation 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.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Annotation Extension||Reference|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Assay||Construct||Conditions||Strain Background||Reference|