DISULFIDE BONDING AND CYSTEINE ACCESSIBILITY IN THE LPHA-AMINO-3-HYDROXY-5-METHYLISOXAZOLE-4-PROPIONIC ACID RECEPTOR SUBUNIT GLURD - IMPLICATIONS FOR REDOX MODULATION OF GLUTAMATE RECEPTORS

Redox agents elicit a wide variety of effects on the ligand affinity a nd channel properties of ionotropic glutamate receptors and have been proposed as potential therapeutic agents for neuropathological process es. One such effect is the dithiothreitol (DTT)-induced increase in ag onist affinity of certain ionotropic glutamate receptors (GluRs), pres umably due to reduction of a disulfide bridge formed between cysteine residues conserved among all GluRs. Using biochemical techniques, this disulfide is shown to exist in the ligand-binding domain of the lpha- amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor sub unit GluRD, although GluRD homomeric receptors are not modulated by DT T. The disulfide is inaccessible to DTT, explaining the insensitivity of the intact receptor. Single mutants C260S and C315S show a 2-3-fold higher ligand affinity than wild-type, as observed for several intact GluRs, indicating that the affinity switch is completely contained wi thin the ligand-binding domain. Also, mutants lacking the native disul fide show non-native oligomerization and dramatically reduced specific activity. These facts suggest that the disulfide bridge is required f or the stability of the ligand-binding domain, explaining its conserva tion. A third cysteine residue in the ligand-binding domain exists as a free thiol, partially sequestered in a hydrophobic environment. Thes e results provide a framework for interpreting a variety of GluR redox modulatory phenomena.