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

Abstract: 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 µM, a concentration that produced little or no inhibition at AMPA or NMDA sites. At 100 µM, however, DZKA inhibited [3H]AMPA and l‐[3H]glutamate binding by ∼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 µM) 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 ∼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.