Incorporation of caged cysteine and caged tyrosine into a transmembrane segment of the nicotinic ACh receptor

The nonsense codon suppression technique was used to incorporate o-nitroben zyl cysteine or o-nitrobenzyl tyrosine (caged Cys or Tyr) into the 9' posit ion of the M2 transmembrane segment of the gamma -subunit of the muscle nic otinic ACh receptor expressed in Xenopus oocytes. The caged amino acids rep laced an endogenous Leu residue that has been implicated in channel gating. ACh-induced current increased substantially after ultraviolet (UV) irradia tion to remove the caging group. This represents the first successful incor poration of caged Cys into a protein in vivo and the first incorporation of caged amino acids within a transmembrane segment of a membrane protein. Th e bulky nitrobenzyl group does not prevent the synthesis, assembly, or traf ficking of the ACh receptor. When side chains were decaged using 1-ms UV li ght flashes, the channels with caged Cys or caged Tyr responded with striki ngly different kinetics. The increase in current upon photolysis of caged C ys was too rapid for resolution by the voltage-clamp circuit [time constant (tau) <10 ms], whereas the increase in current upon photolysis of caged Ty r was dominated by a phase with <tau> similar to 500 ms. Apparently, the pr esence of a bulky o-nitrobenzyl Tyr residue distorts the receptor into an a bnormal conformation. Upon release of the caging group, the receptor relaxe s, with tau similar to 500 ms, into a conformation that allows the channel to open. Tyr at the 9' position of the gamma -subunit greatly increases the ability of ACh to block the channel by binding within the channel pore. Th is is manifested in two ways. 1) A "rebound," or increase in current, occur s upon removal of ACh from the bathing medium; and 2) at ACh concentrations >400 muM, inward currents are decreased through the mutated channel. The a bility to incorporate caged amino acids into proteins should have widesprea d utility.