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

Tong, Yanhe and Brandt, Gabriel S. and Li, Ming and Shapovalov, George and Slimko, Eric and Karschin, Andreas and Dougherty, Dennis A. and Lester, Henry A. (2001) Tyrosine decaging leads to substantial membrane trafficking during modulation of an inward rectifier potassium channel. Journal of General Physiology, 117 (2). pp. 103-118. ISSN 0022-1295. PMCID PMC2217249. doi:10.1085/jgp.117.2.103.

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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leads to massive clathrin-mediated endocytosis; in fact, membrane area equivalent to many clathrin-coated vesicles is withdrawn from the oocyte surface for each Kir2.1 channel inhibited.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.

Item Type:Article
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URLURL TypeDescription CentralArticle
Dougherty, Dennis A.0000-0003-1464-2461
Lester, Henry A.0000-0002-5470-5255
Additional Information:© 2001 The Rockefeller University Press. Submitted: 11 August 2000; revised: 6 December 2000; accepted: 7 December 2000. Published 16 January 2001. We thank Hai-Rong Li for help with the oocytes, Mike Walsh for assistance with instrumentation, Tatiana Ivanina and Abraham Kovoor for advice on phosphorylation assays, and members of our laboratory for much discussion. This research was supported by grants from the National Institutes of Health (GM29836 and NS34407), by National Research Service Awards to Y. Tong and M. Li, by a fellowship from the Burroughs-Wellcome Foundation Computational Molecular Biology Program to G. Shapovalov, by a Predoctoral Training Award from NIH to E. Slimko, by a Boehringer Fellowship to G.S. Brandt, and by the Alexander von Humboldt Foundation.
Funding AgencyGrant Number
NIH Predoctoral FellowshipUNSPECIFIED
Burroughs Wellcome FundUNSPECIFIED
Boehringer Ingelheim FondsUNSPECIFIED
Alexander von Humboldt FoundationUNSPECIFIED
Subject Keywords:unnatural amino acids, ion channel, Kir2.1, clathrin, endocytosis
Issue or Number:2
PubMed Central ID:PMC2217249
Record Number:CaltechAUTHORS:TONjgp01
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:5168
Deposited By: Lindsay Cleary
Deposited On:04 Oct 2006
Last Modified:08 Nov 2021 20:23

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