Flash Decaging of Tyrosine Neurotechnique Sidechains in an Ion Channel

a natural sidechain that is derivatized (" caged ") by a photosensitive group. The photochemical strategy employs the well-characterized ortho-nitrobenzyl group Oocytes synthesize and incorporate into their membranes a channel in which such a caged side-Pasadena, California 91125 chain occupies a selected position. Later, while the cell is under voltage-clamp study, a light flash photolyzes (" decages ") the unnatural sidechain to produce the wild-Summary type residue, apparently within 20 ␮s of photon absorption (Tatsu et al., 1996). We monitor the increased num-A nonsense codon suppression technique was em-ber of open channels as the receptor proteins relax from ployed to incorporate ortho-nitrobenzyl tyrosine, " caged the less responsive mutant conformation(s) to the more tyrosine, " in place of tyrosine at any of three positions responsive wild-type conformation(s). (93, 127, or 198) in the ␣ subunit of the muscle nicotinic ACh receptor (nAChR) expressed in Xenopus oocytes. The ortho-nitrobenzyl group was then removed by 1 Results ms flashes at 300–350 nm to yield tyrosine itself while macroscopic currents were recorded during steady Analysis of Relaxation Amplitudes at nAChR ␣93 ACh exposure. Responses to multiple flashes showed and ␣198 (1) that each flash decages up to 17% of the tyrosines For the initial experiments, we investigated the nicotinic and (2) that two tyrosines must be decaged per re-acetylcholine receptor (nAChR) of mouse muscle. We ceptor for a response. The conductance relaxations concentrated on two highly conserved and extensively showed multiple kinetic components; rate constants studied tyrosine residues at positions 93 and 198 in the (Ͻ0.1 s Ϫ1 to 10 3 s Ϫ1) depended on pH and the site of N-terminal extracellular region of the ␣ subunit that are incorporation, and relative amplitudes depended on thought to participate in agonist binding (summarized the number of prior flashes. This method, which is potentially quite general, (1) provides a time-resolved Nowak et al., 1995). For comparison, we also examined assay for the behavior of a protein when a mutant a less crucial tyrosine residue at position ␣127. sidechain is abruptly changed to the wild-type residue Figure 2 presents a typical experiment with an oocyte and (2) will also allow for selective decaging of side-expressing Tyr(ONB) at position ␣93; similar results were chains that are candidates for covalent modification obtained at ␣198. Figure 2A shows the raw traces from (such as phosphorylation) in specific proteins in intact cells. a chart recorder. At the start of the …