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J. Biol. Chem., Vol. 281, Issue 40, 30104-30111, October 6, 2006

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Identification of -Aminobutyric Acid Receptor-interacting Factor 1 (TRAK2) as a Trafficking Factor for the K⁺ Channel Kir2.1^*

From the Department of Pharmacology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

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 -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 ⁸⁶Rb⁺ 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.

Received for publication, March 15, 2006 , and in revised form, July 28, 2006.

^* This work was supported by a beginning grant-in-aid award from the American Heart Association (to E. Z.-M.) and National Institutes of Health Grants HL80632 and HL55312 (to E. S. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Division of Pediatric Surgery, Children's Hospital of Los Angeles, Los Angeles, CA 90027.

² To whom correspondence should be addressed: Dept. of Pharmacology, University of Pittsburgh School of Medicine, 220 Lothrop St., Pittsburgh, PA15261. Tel.: 412-648-9486; Fax: 412-648-1945; E-mail: levitan{at}server.pharm.pitt.edu.

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