ACTIVATION KINETICS OF AMPA RECEPTOR CHANNELS REVEAL THE NUMBER OF FUNCTIONAL AGONIST BINDING-SITES

AMPA and NMDA receptor channels are closely related molecules, yet the y respond to glutamate with distinct kinetics, attributable to differe nces in ligand binding and channel gating steps (for review,; see Edmo nds et al., 1995). We used two complementary approaches to investigate the number of functional binding sites on AMPA channels on outside-ou t patches from cultured hippocampal neurons. The activation kinetics o f agonist binding were measured during rapid steps into low concentrat ions of;selective AMPA receptor agonists and during steps from a compe titive AMPA receptor antagonist, 6-cyano-7-nitro-quinoxaline-2,3-dione , into a saturating concentration of agonist. Both approaches revealed sigmoidal kinetics, which suggests that multiple agonist binding step s or antagonist unbinding steps are needed for channel activation. A k inetic model with two independent binding sites gave a better fit to t he activation phase than models with one or three independent sites. A more refined analysis incorporating cooperative interaction between t he two binding sites significantly improved the fits to the responses. The affinity of the first binding step was two to three times higher than the second step. These results demonstrate that binding of two ag onist molecules are needed to activate AMPA receptors, but the two bin ding sites are not identical and independent. Because NMDA receptors r equire four ligand molecules for activation (two glycine and two gluta mate; Benveniste and Mayer, 1991; Clements and Westbrook, 1991), it ma y be that some binding sites on AMPA receptors are functionally silent .