Tyrosine Decaging Leads to Substantial Membrane Trafficking during Modulation of an Inward Rectifier Potassium Channel
| Autor: | Gabriel S. Brandt, Henry A. Lester, George Shapovalov, Dennis A. Dougherty, Yanhe Tong, Eric M. Slimko, Andreas Karschin, Ming Li |
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| Rok vydání: | 2001 |
| Předmět: |
Dynamins
Patch-Clamp Techniques Potassium Channels Kir2.1 Physiology Xenopus Biology Endocytosis Bulk endocytosis GTP Phosphohydrolases 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Animals Phosphorylation Potassium Channels Inwardly Rectifying Tyrosine Fluorescent Dyes 030304 developmental biology Dynamin 0303 health sciences Rhodamines Electric Conductivity Tyrosine phosphorylation Receptor-mediated endocytosis Protein-Tyrosine Kinases Clathrin Cell biology Protein Transport chemistry ion channel Oocytes Potassium cardiovascular system Original Article Ion Channel Gating Tyrosine kinase Caltech Library Services unnatural amino acids 030217 neurology & neurosurgery |
| Zdroj: | The Journal of General Physiology |
| ISSN: | 1540-7748 0022-1295 |
| Popis: | Tyrosine side chains participate in several distinct signaling pathways, including phosphorylation and membrane trafficking. A nonsense suppression procedure was used to incorporate a caged tyrosine residue in place of the natural tyrosine at position 242 of the inward rectifier channel Kir2.1 expressed in Xenopus oocytes. When tyrosine kinases were active, flash decaging led both to decreased K+ currents and also to substantial (15–26%) decreases in capacitance, implying net membrane endocytosis. A dominant negative dynamin mutant completely blocked the decaging-induced endocytosis and partially blocked the decaging-induced K+ channel inhibition. Thus, decaging of a single tyrosine residue in a single species of membrane protein leads to massive clathrin-mediated endocytosis; in fact, membrane area equivalent to many clathrin-coated vesicles is withdrawn from the oocyte surface for each Kir2.1 channel inhibited. Oocyte membrane proteins were also labeled with the thiol-reactive fluorophore tetramethylrhodamine-5-maleimide, and manipulations that decreased capacitance also decreased surface membrane fluorescence, confirming the net endocytosis. In single-channel studies, tyrosine kinase activation decreased the membrane density of active Kir2.1 channels per patch but did not change channel conductance or open probability, in agreement with the hypothesis that tyrosine phosphorylation results in endocytosis of Kir2.1 channels. Despite the Kir2.1 inhibition and endocytosis stimulated by tyrosine kinase activation, neither Western blotting nor 32P labeling produced evidence for direct tyrosine phosphorylation of Kir2.1. Therefore, it is likely that tyrosine phosphorylation affects Kir2.1 function indirectly, via interactions between clathrin adaptor proteins and a tyrosine-based sorting motif on Kir2.1 that is revealed by decaging the tyrosine side chain. These interactions inhibit a fraction of the Kir2.1 channels, possibly via direct occlusion of the conduction pathway, and also lead to endocytosis, which further decreases Kir2.1 currents. These data establish that side chain decaging can provide valuable time-resolved data about intracellular signaling systems. |
| Databáze: | OpenAIRE |
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