Tyrosine decaging leads to substantial membrane trafficking during modulation of an inward rectifier potassium channel.
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leads to massive clathrin-mediated endocytosis; in fact, membrane area equivalent to many clathrin-coated vesicles is withdrawn from the oocyte surface for each Kir2.1 channel inhibited.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 (32)P 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.
Tong
Y
Y
Division of Biology, California Institute of Technology, Pasadena, California 91125, USA.
Brandt
G S
GS
Li
M
M
Shapovalov
G
G
Slimko
E
E
Karschin
A
A
Dougherty
D A
DA
Lester
H A
HA
D000818:Animals; D002966:Clathrin / Q000378:metabolism; D034281:Dynamins; D004553:Electric Conductivity; D004705:Endocytosis / Q000502:physiology; D005456:Fluorescent Dyes; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism; D015640:Ion Channel Gating / Q000502:physiology*; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D011188:Potassium / Q000378:metabolism; D015221:Potassium Channels / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying; D021381:Protein Transport / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D012235:Rhodamines; D014443:Tyrosine / Q000378:metabolism*; D014981:Xenopus
D002966:Clathrin; D005456:Fluorescent Dyes; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D012235:Rhodamines; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D020558:GTP Phosphohydrolases; D034281:Dynamins; D011188:Potassium