Irreversible inhibition of high-affinity [3H]kainate binding by a novel photoactivatable analogue: (2'S,3'S,4'R)-2'-carboxy-4'-(2-diazo-1-oxo-3, 3,3-trifluoropropyl)-3'-pyrrolidinyl acetate.

A photolabile trifluoromethyldiazoketone derivative of kainate (KA), (2'S,3'S,4'R)-2'-carboxy-4'-(2-diazo-1-oxo-3, 3,3-trifluoropropyl)-3'-pyrrolidinyl acetate (DZKA), was synthesized and evaluated as an irreversible inhibitor of the high-affinity KA site on rat forebrain synaptic plasma membranes (SPMs). In the absence of UV irradiation, DZKA preferentially blocked [3H]KA binding with an IC50 of 0.63 microM, a concentration that produced little or no inhibition at AMPA or NMDA sites. At 100 microM, however, DZKA inhibited [3H]AMPA and L-[3H]glutamate binding by approximately 50%. When examined electrophysiologically in HEK293 cells expressing human KA (GluR6) or AMPA (GluR1) subtypes, DZKA acted preferentially at KA receptors as a weak agonist. DZKA also exhibited little or no excitotoxic activity in mixed rat cortical cultures. Irreversible inhibition was assessed by pretreating SPMs with DZKA (50 microM) in the presence of UV irradiation, removing unbound DZKA, and then assaying the reisolated SPMs for radioligand binding. This protocol produced a selective and irreversible loss of approximately 50% of the [3H]KA sites. The binding was recoverable in SPMs pretreated with DZKA or UV alone. Coincubation with L-glutamate prevented the loss in [3H]KA binding, suggesting that the inactivation occurred at or near the ligand binding site. These results are consistent with the action of DZKA as a photoaffinity ligand for the KA site and identify the analogue as a valuable probe for future investigations of receptor structure and function.

Site-specific incorporation of nonnatural residues during in vitro protein biosynthesis with semisynthetic aminoacyl-tRNAs.

A method is presented for the incorporation of nonnatural amino acids into proteins during in vitro cell-free translation. A combination of chemical synthesis and run-off transcription was employed to prepare a semisynthetic, nonhypermodified tRNA(Gly) nonsense suppressor acylated with L-3-[125I]iodotyrosine. The presence of this synthetic tRNA during in vitro translation of mRNA containing a nonsense suppression site (e.g., a UAG termination codon) results in the incorporation of the nonnatural amino acid L-3-iodotyrosine into the polypeptide exclusively at the position corresponding to that site. Incorporation of the nonnatural amino acid L-3-[125I]iodotyrosine into the model polypeptide was assessed by quantitative and unambiguous determination of suppression efficiency, read-through, and site specificity of incorporation. Minor modifications of the method employed in this initial experiment also allow the rapid analysis of unlabeled acylated tRNA analogues. Under optimum conditions, the unlabeled amino acid L-3-iodotyrosine was found to be incorporated with a suppression efficiency of 65%. Other nonnatural residues, including N-methylphenylalanine, D-phenylalanine, and phenyllactic acid, were tested in the assay under these same conditions. Suppression efficiencies for this series ranged from 0 to 72% depending on the structure of the residue incorporated. Several other aspects of this methodology, such as tRNA structure and context effects, are briefly discussed.