Format

Send to

Choose Destination
See comment in PubMed Commons below
Neuron. 1998 Apr;20(4):619-24.

Flash decaging of tyrosine sidechains in an ion channel.

Author information

1
Division of Biology, California Institute of Technology, Pasadena 91125, USA.

Abstract

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 alpha 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 10(3) 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.

PMID:
9581754
[Indexed for MEDLINE]
Free full text
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Elsevier Science
    Loading ...
    Support Center