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Irreversible Inhibition of High‐Affinity [ 3 H]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
Author(s) -
Willis Colin L.,
Wacker Dean A.,
Bartlett Richard D.,
Bleakman David,
Lodge David,
Chamberlin A. Richard,
Bridges Richard J.
Publication year - 1997
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1997.68041503.x
Subject(s) - kainate receptor , ampa receptor , glutamate receptor , chemistry , agonist , binding site , nmda receptor , kainic acid , ligand (biochemistry) , receptor , radioligand assay , biophysics , stereochemistry , biochemistry , biology
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 [ 3 H]KA binding with an IC 50 of 0.63 µ M , a concentration that produced little or no inhibition at AMPA or NMDA sites. At 100 µ M , however, DZKA inhibited [ 3 H]AMPA and l ‐[ 3 H]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 µ M ) 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 [ 3 H]KA sites. The binding was recoverable in SPMs pretreated with DZKA or UV alone. Coincubation with l ‐glutamate prevented the loss in [ 3 H]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.