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J. Biol. Chem., Vol. 283, Issue 10, 5973-5984, March 7, 2008

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AP-2-dependent Internalization of Potassium Channel Kir2.3 Is Driven by a Novel Di-hydrophobic Signal^*

From the Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201

The localization and density of Kir2.3 channels are influenced by the balance between PDZ protein interaction at the cell surface and routing into the endocytic pathway. Here, we explore mechanisms by which the Kir2.3 channel is directed into the endocytic pathway. We found that Kir2.3 channels are constitutively internalized from the cell surface in a dynamin-dependent manner, indicative of vesicle-mediated endocytosis. The rate of Kir2.3 endocytosis was dramatically attenuated following RNA interference-mediated knockdown of either adaptin (AP-2 clathrin adaptor) or clathrin heavy chain, revealing that Kir2.3 is internalized by an AP-2 clathrin-dependent mechanism. Structure-rationalized mutagenesis studies of a number of different potential AP-2 interaction motifs indicate that internalization of Kir2.3 is largely dependent on a non-canonical di-isoleucine motif (II413) embedded within the C terminus. Internalization assays using CD4-Kir2.3 chimeras demonstrate that the di-isoleucine signal acts in an autonomous and transplantable manner. Kir2.3 co-immunoprecipitates with adaptin, and disruption of the di-isoleucine motif decreased interaction of the channel with AP-2. Replacement of the di-isoleucine motif with a canonical di-leucine internalization signal actually blocked Kir2.3 endocytosis. Moreover, in yeast three-hybrid studies, the Kir2.3 di-isoleucine motif does not bind the AP-2 C-2 hemicomplex in the way that has been recently observed for canonical di-leucine signals. Altogether, the results indicate that Kir2.3 channels are marked for clathrin-dependent internalization from the plasma membrane by a novel AP-2-dependent signal.

Received for publication, November 29, 2007 , and in revised form, January 3, 2008.

^* This work was supported in part by National Institutes of Health (NIH) Grant DK063049 (to P. A. W.) and by American Heart Association Grant 0615469U (to A. K. M.). 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 Supported by NIH Training Grant T32 GM08181.

² To whom correspondence should be addressed: 655 W. Baltimore St., BRB 5-029, Baltimore, MD 21201. Tel.: 410-706-3851; Fax: 410-706-8341; E-mail: pwelling{at}umaryland.edu.

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