Endozytose des Kaliumkanals Kir2.1
We have studied some of the mechanisms responsible for controlling the density of Kir2.1 channels at the cell surface. In particular, we have explored the mechanisms underlying the of Kir2.1 channel into the endocytic pathway. We found that 1. The antibody uptake assay showed that about 20% of the...
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|Summary:||We have studied some of the mechanisms responsible for controlling the density of Kir2.1 channels at the cell surface. In particular, we have explored the mechanisms underlying the of Kir2.1 channel into the endocytic pathway. We found that
1. The antibody uptake assay showed that about 20% of the antibodies labeled surface channels were internalized within 5 min of temperature shift; an apparent maximum was reached after 15 min. Kir2.1 undergoes constitutive internalization.
2. Co-expression of a dominant-negative mutant of dynamin K44A or AP180C enhanced the surface expression of Kir2.1 by 40% and 49% respectively. AP180C inhibited clathrin mediated internalization of Kir2.1 by 80% compared with COS-7 cells transfected with Kir2.1 and a control vector. Kir2.1 is internalized by clathrin-mediated endocytosis.
3. In both Xenopus oocytes and mammalian cells system, disruption of Y242 and Y341 motif significantly increased the amount of Kir2.1 at cell surface respectively. The mutants Y242A and Y341A also showed dramatic decrease in the rate of endocytosis as compared to wild-type Kir2.1 channels respectively. Two tyrosine based endocytic motifs 242YIPL245 and 341YSRF344 in the C-terminal domain of Kir2.1 are both involved in the endocytosis of Kir2.1.
4. Live cell imaging clearly showed that Kir2.1 channels strongly colocalized with early endosomal marker, Rab5 or late endosomal marker, Rab7 or recycling endosome marker, Rab11 or Lyso-Tracker Red. Kir2.1 is targeted to the endosome-lysosomal and endosomal- recycling pathway.
5. The two endocytosis signals studied here are in close vicinity as indicated by the tertiary structure of the cytosolic pore of Kir2.1 channels. Since mutation of both motifs had the similar effect as mutation of only one of these motifs. We hypothesize that both signals must be present for efficient constitutive endocyosis to occur.|