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Acta Physiologica Congress

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Acta Physiologica 2013; Volume 207, Supplement 694
92nd Annual Meeting of the German Physiological Society
3/2/2013-3/5/2013
Heidelberg, Germany


AN ENDOCYTIC MOTIF MEDIATING CONSTITUTIVE ENDOCYTOSIS OF THE POTASSIUM CHANNEL KIR2.1
Abstract number: P117

Tu 1  *W., Milani 1  W., Daut 1   J. , Renigunta 1   V.

1 Institut für Physiologie und Pathophysiologie, Zellphysiologie, Marburg, Germany

The cytoplasmic signals that regulate the traffic of Kir2.1 are poorly understood. Previous work has suggested that a tyrosine-based motif (YIPL242-245) of Kir2.1 is involved in clathrin mediated endocytosis. Here, we describe another tyrosine-based motif (YSRF341-344) of Kir2.1 that is also involved in clathrin mediated endocytosis. Point mutations of five putative endocytic motifs (LL231-232, YIPL242-245, YEPV326-329, YYKV 336-339, YSRF 341-344) were made in the C-terminal domain of Kir2.1 channel. These mutants were expressed in COS-7 cells and surface expression was quantified using a chemiluminescence assay. Only the mutants Y242A or Y341A showed a significantly greater cell surface expression than wild-type Kir2.1 channels. Co-expression of a dominant-negative mutant of dynamin (K44A), but not wild-type dynamin, strongly enhanced the surface expression of Kir2.1. Co-expression of C-terminus of the adaptor protein AP180 (AP180C), another inhibitor of clathrin-mediated endocytosis, also effectively inhibited the internalization of Kir2.1 and mutants (Y242A or Y341A). We also measured channel internalization directly using an antibody feeding assay. The mutants Y242A or Y341A showed dramatic decrease in the internalization as compared to wild-type Kir2.1 channels. Live-cell imaging experiments with HeLa cells transfected with EGFP-tagged Kir2.1 constructs revealed a more pronounced expression at the surface membrane of Kir2.1Y242A and Kir2.1Y341A constructs as compared to wild-type Kir2.1 channels. In conclusion, our results indicate that Kir2.1 channels show constitutive clathrin-mediated endocytosis and we describe a functional tyrosine-based endocytosis signal, YSRF, which is highly conserved in Kir2.1 channels of various species.

To cite this abstract, please use the following information:
Acta Physiologica 2013; Volume 207, Supplement 694 :P117

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