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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
Main Authors: Philipson, Kenneth D, Gallivan, Justin P, Brandt, Gabriel S, Dougherty, Dennis A, Lester, Henry A
Format: Article
Language:English
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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 ( )
ISSN:0363-6143
1522-1563
DOI:10.1152/ajpcell.2001.281.1.C195