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Flash Decaging of Tyrosine Sidechains in an Ion Channel

Miller, Jeffrey C. and Silverman, Scott K. and England, Pamela M. and Dougherty, Dennis A. and Lester, Henry A. (1998) Flash Decaging of Tyrosine Sidechains in an Ion Channel. Neuron, 20 (4). pp. 619-624. ISSN 0896-6273. doi:10.1016/s0896-6273(00)81001-6.

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A nonsense codon suppression technique was employed to incorporate ortho-nitrobenzyl tyrosine, “caged tyrosine,” in place of tyrosine at any of three positions (93, 127, or 198) in the α subunit of the muscle nicotinic ACh receptor (nAChR) expressed in Xenopus oocytes. The ortho-nitrobenzyl group was then removed by 1 ms flashes at 300–350 nm to yield tyrosine itself while macroscopic currents were recorded during steady ACh exposure. Responses to multiple flashes showed (1) that each flash decages up to 17% of the tyrosines and (2) that two tyrosines must be decaged per receptor for a response. The conductance relaxations showed multiple kinetic components; rate constants (<0.1 s⁻¹ to 10³ s⁻¹) depended on pH and the site of incorporation, and relative amplitudes depended on the number of prior flashes. This method, which is potentially quite general, (1) provides a time-resolved assay for the behavior of a protein when a mutant sidechain is abruptly changed to the wild-type residue and (2) will also allow for selective decaging of sidechains that are candidates for covalent modification (such as phosphorylation) in specific proteins in intact cells.

Item Type:Article
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Dougherty, Dennis A.0000-0003-1464-2461
Lester, Henry A.0000-0002-5470-5255
Additional Information:© 1998 Cell Press. Published by Elsevier Inc. Received 17 December 1997, Revised 9 February 1998. This work was supported by grants from the National Institutes of Health (NS-11756, NS-34407). We thank Mark W. Nowak, Patrick C. Kearney, and Rory Sayres for help and Justin Gallivan for continuing advice. J. C. M. held Caltech undergraduate research fellowships; P. M. E. held an NRSA.
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NIH Predoctoral FellowshipUNSPECIFIED
Issue or Number:4
Record Number:CaltechAUTHORS:20200515-132054818
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Official Citation:Jeffrey C. Miller, Scott K. Silverman, Pamela M. England, Dennis A. Dougherty, Henry A. Lester, Flash Decaging of Tyrosine Sidechains in an Ion Channel, Neuron, Volume 20, Issue 4, 1998, Pages 619-624, ISSN 0896-6273, (
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:103239
Deposited By: Tony Diaz
Deposited On:15 May 2020 20:32
Last Modified:16 Nov 2021 18:19

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