Tyrosine decaging leads to substantial membrane trafficking during modulation of an inward rectifier potassium channel

Tyrosine side chains participate in several distinct signaling pathways, in cluding phosphorylation and membrane trafficking. A nonsense suppression pr ocedure was used to incorporate a caged tyrosine residue in Place of the na tural tyrosine at position 242 of the inward rectifier channel Kir2.1 er;pr essed in Xenopus oocytes. When tyrosine kinases were active, flash decaging led both to decreased K+ currents and also to substantial (15-26%) decreas es in capacitance, implying net membrane endocytosis. A dominant negative d ynamin mutant completely blocked the decaging-induced endocytosis and parti ally 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leads to massive clathrin-mediated endocytosis; in fact, membrane area equivalent to many clathrin-coated vesicles is withdrawn from the oocyte surface for eac h Kir2.1 channel inhibited. Oocyte membrane proteins were also labeled with the thiol-reactive fluorophore tetramethylrhodamine-5-maleimide, and manip ulations that decreased capacitance also decreased surface membrane fluores cence, 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 ag reement with the hypothesis that tyrosine phosphorylation results in endocy tosis of Kir2.1 channels. Despite the Kir2.1 inhibition and endocytosis sti mulated by tyrosine kinase activation, neither Western blotting nor P-32 la beling produced evidence for direct tyrosine phosphorylation of Kir2.1. The refore, it is likely that tyrosine phosphorylation affects Kir2.1 function indirectly, via interactions between clathrin adaptor proteins and a tyrosi ne-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, p ossibly via direct occlusion of the conduction pathway, and also lead to en docytosis, which further decreases Kir2.1 currents. These data establish th at side chain decaging can provide valuable time-resolved data about intrac ellular signaling systems.