Flash decaging of tyrosine sidechains in an ion channel.
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.
Miller
J C
JC
Division of Biology, California Institute of Technology, Pasadena 91125, USA.
Silverman
S K
SK
England
P M
PM
Dougherty
D A
DA
Lester
H A
HA
D000109:Acetylcholine / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D018389:Codon, Nonsense; D004553:Electric Conductivity; D005260:Female; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008564:Membrane Potentials / Q000187:drug effects; D018482:Muscle, Skeletal / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000502:physiology; D011978:Receptors, Nicotinic / Q000096:biosynthesis / Q000737:chemistry / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D014443:Tyrosine; D014981:Xenopus
D018389:Codon, Nonsense; D046911:Macromolecular Substances; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins; D014443:Tyrosine; D000109:Acetylcholine