Synthesis and photochemistry of a photolabile precursor of N-methyl-D- aspartate (NMDA) that is photolyzed in the microsecond time region and is suitable for chemical kinetic investigations of the NMDA receptor

The amino acid L-glutamate is a major neurotransmitter at excitatory synapses within the central nervous system. Neuronal responses to glutamate are mediated by at least three receptor types, one of which is the NMDA subtype, named for its specific ligand N-methyl-D-aspartic acid. Neurotransmitter receptors are transmembrane proteins that can form ion channels upon binding a specific ligand and are involved in many physiological activities of the brain and in some neurological disorders. Elucidating the mechanisms of the formation of transmembrane receptor- channels and of receptor regulation and inhibition is necessary for understanding nervous system function and for designing potential therapeutic agents. This has been hampered by the lack of rapid reaction techniques suitable for investigating protein-mediated reactions on cell surfaces. Recently a laser-pulse photolysis technique was developed to study the chemical reactions of channel-forming receptor proteins in the microsecond- to-millisecond time region. To apply the technique to NMDA receptors a photolabile NMDA precursor (β-DNB NMDA) was synthesized. In this precursor the side chain carboxylate was protected as a photosensitive 2,2'- dinitrobenzhydryl ester. Photolysis with 308 nm laser light generated free NMDA with a time constant of 4.2 ± 0.1 μs at pH 7 and a photolysis quantum yield of 0.18 ± 0.05. In rat hippocampal neurons the β-DNB NMDA (250 μM) neither activated endogenously expressed receptors nor potentiated or inhibited the NMDA response. Equilibration of hippocampal neurons in the whole-cell current recording mode with 250 μM caged precursor followed by a pulse of 333 nm laser light resulted in a rapid current rise with a rate constant of 100 s^-1 due to opening of NMDA-activated receptor-channels. The caged NMDA precursor described here now makes it possible to investigate the mechanism of NMDA receptors in the micro- to millisecond time region.