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 synaps es within the central nervous system. Neuronal responses to glutamate are m ediated by at least three receptor types, one of which is the NMDA subtype, named for its specific ligand N-methyl-D-aspartic acid. Neurotransmitter r eceptors 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 th e formation of transmembrane receptor-channels and of receptor regulation a nd inhibition is necessary for understanding nervous system function and fo r designing potential therapeutic agents. This has been hampered by the lac k 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 prot eins in the microsecond-to-millisecond time region. To apply the technique to NMDA(1) receptors a photolabile NMDA precursor (beta-DNB NMDA was synthe sized. In this precursor the side chain carboxylate was protected as a phot osensitive 2,2'-dinitrobenzhydryl ester. Photolysis with 308 nm laser light generated free NMDA with a time constant of 4.2 +/- 0.1 mu s at pH 7 and a photolysis quantum yield of 0.18 +/- 0.05. In rat hippocampal neurons the beta-DNB NMDA (250 mu M) neither activated endogenously expressed receptors nor potentiated or inhibited the NMDA response. Equilibration of hippocamp al neurons in the whole-cell current recording mode with 250 mu M caged pre cursor followed by a pulse of 333 nm laser light resulted in a rapid curren t rise with a rate constant of 100 s(-1) due to opening of NMDA-activated r eceptor-channels. The caged NMDA precursor described here now makes it poss ible to investigate the mechanism of NR IDA receptors in the micro- to mill isecond time region.