AGONIST RESPONSE KINETICS OF N-METHYL-D-ASPARTATE RECEPTORS IN NEURONS CULTURED FROM RAT CEREBRAL-CORTEX AND CEREBELLUM - EVIDENCE FOR RECEPTOR HETEROGENEITY

Accumulating evidence from both radioligand binding and molecular clon ing experiments has led to the suggestion that there are heterogeneous populations of N-methyl-D-aspartate (NMDA) receptors. In particular, the NMDA receptor associated with cerebellar neurons has been suggeste d to be different from that in higher brain regions. With these observ ations in mind, we considered the possibility that the NMDA receptors on cultured neurons from rat cortex and cerebellum may show difference s in their affinity for the coagonists, glutamate and glycine, and the ion channel blocker ketamine. A kinetic approach was used to derive t he association and dissociation constants for each of the ligands from their respective macroscopic time constants of binding and unbinding. The constants were assessed electrophysiologically by measuring the o nset and decay of whole-cell currents in response to drug applications to voltage-clamped neurons. In addition, differences in coagonist aff inity were examined using conventional equilibrium concentration-respo nse curve analysis. These experiments revealed that current relaxation s after fast applications of either glutamate or glycine decayed more rapidly in cerebellar neurons, compared with cortical neurons. Thus, a ssuming two independent binding sites per receptor, the microscopic de cay time constants (tau(off)) for glutamate were 341 +/- 47 (n = 12) a nd 934 +/- 76 msec (n = 11, p < 0.0001) for granule cells and cortical neurons, respectively. The resulting apparent microscopic dissociatio n constant (mK(d)) for glutamate at cerebellar granule cells, calculat ed from the forward and reverse rate constants, was >2-fold lower than that for cerebral cortex receptors (496 nm, compared with 251 nM). Th e difference between the two cell types in the tau(off) for glycine wa s more substantial, i.e., 558 +/- 53 (n = 15) and 2214 +/- 125 msec (n = 19, p < 0.0001) for cerebellum and cortex, respectively. Correspond ing apparent mK(d) values for glycine differed by > 4-fold, i.e., 189 nm and 45 nm for cerebellar granule and cortical neurons, respectively . Analysis of data obtained from equilibrium concentration-response cu rves also revealed differences in coagonist affinity between the two c ell populations. The mean mK(d) values for glutamate at cerebellum and cortical neurons were 1260 nm and 630 nm, respectively, and those for glycine were 316 nm and 63 nm, respectively. No obvious differences w ere found between the two cell types with respect to the ion channel-b locking kinetics of the dissociative anesthetic ketamine. In conclusio n, these functional data demonstrate differences in coagonist (glutama te and glycine) affinity for NMDA receptors associated with cultured c erebellar granule cells, compared with neurons cultured from the cereb ral cortex. In view of the multi-subunit structure of these ligand-gat ed ion channels, it must be considered likely that the observed differ ences in binding site affinity reflect differences in the subunit comp osition.