Site-specific, photochemical proteolysis applied to ion channels in vivo (nicotinic acetylcholine receptory(2-nitrophenyl)glycineyK1 channelysignature disulfide loopysite-specific, nitrobenzyl-induced photochemical proteolysis)

A method for site-specific, nitrobenzyl- induced photochemical proteolysis of diverse proteins ex- pressed in living cells has been developed based on the chemistry of the unnatural amino acid (2-nitrophenyl)glycine (Npg). Using the in vivo nonsense codon suppression method for incorporating unnatural amino acids into proteins ex- pressed in Xenopus oocytes, Npg has been incorporated into two ion channels: the Drosophila Shaker B K 1 channel and the nicotinic acetylcholine receptor. Functional studies in vivo show that irradiation of proteins containing an Npg residue does lead to peptide backbone cleavage at the site of the novel residue. Using this method, evidence is obtained for an essential functional role of the ''signature'' Cys128-Cys142 disulfide loop of the nAChR a subunit.