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Incorporation of caged cysteine and caged tyrosine into a transmembrane segment of the nicotinic ACh receptor
Divisions of 1 Biology and 2 Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125 The nonsense codon suppression technique was used to incorporate o -nitrobenzyl cysteine or o -nitrobenzyl tyrosine (caged Cys or Tyr) into the 9' position of the M2...
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Published in: | American Journal of Physiology: Cell Physiology 2001-07, Vol.281 (1), p.C195-C206 |
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Main Authors: | , , , , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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Summary: | Divisions of 1 Biology and 2 Chemistry and Chemical
Engineering, California Institute of Technology, Pasadena,
California 91125
The nonsense codon
suppression technique was used to incorporate o -nitrobenzyl
cysteine or o -nitrobenzyl tyrosine (caged Cys or Tyr) into
the 9' position of the M2 transmembrane segment of the -subunit of
the muscle nicotinic ACh receptor expressed in Xenopus
oocytes. The caged amino acids replaced an endogenous Leu residue that
has been implicated in channel gating. ACh-induced current increased
substantially after ultraviolet (UV) irradiation to remove the caging
group. This represents the first successful incorporation of caged Cys
into a protein in vivo and the first incorporation of caged amino acids
within a transmembrane segment of a membrane protein. The bulky
nitrobenzyl group does not prevent the synthesis, assembly, or
trafficking of the ACh receptor. When side chains were decaged using
1-ms UV light flashes, the channels with caged Cys or caged Tyr
responded with strikingly different kinetics. The increase in current
upon photolysis of caged Cys was too rapid for resolution by the
voltage-clamp circuit [time constant ( ) |
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ISSN: | 0363-6143 1522-1563 |
DOI: | 10.1152/ajpcell.2001.281.1.C195 |