Flash Decaging of Tyrosine Sidechains in an Ion Channel

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 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.