Agonist response kinetics of N-methyl-D-aspartate receptors in neurons cultured from rat cerebral cortex and cerebellum: evidence for receptor heterogeneity
Priestley, T.; Kemp, J.A.
Molecular Pharmacology 44(6): 1252-1257
1993
ISSN/ISBN: 0026-895X PMID: 7903418 Document Number: 408964
Accumulating evidence from both radioligand binding and molecular cloning 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 suggested to be different from that in higher brain regions. With these observations in mind, we considered the possibility that the NMDA receptors on cultured neurons from rat cortex and cerebellum may show differences in their affinity for the coagonists, glutamate and glycine, and the ion channel blocker ketamine. A kinetic approach was used to derive the 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 onset and decay of whole-cell currents in response to drug applications to voltage-damped neurons. In addition, differences in coagonist affinity were examined using conventional equilibrium concentration-response curve analysis. These experiments revealed that current relaxations after fast applications of either glutamate or glycine decayed more rapidly in cerebellar neurons, compared with cortical neurons. Thus, assuming two independent binding sites per receptor, the microscopic decay time constants (tau-off) for glutamate were 341 +- 47 (n = 12) and 934 +- 76 msec (n = 11, p lt 0.0001) for granule cells and cortical neurons, respectively. The resulting apparent microscopic dissociation constant (mK-d) for glutamate at cerebellar granule cells, calculated from the forward and reverse rate constants, was gt 2-fold lower than that for cerebral cortex receptors (496 nM, compared with 251 nM). The difference between the two cell types in the tau-off for glycine was more substantial, i.e., 558 +- 53 (n = 15) and 2214 +- 125 msec (n = 19, p lt 0.0001) for cerebellum and cortex, respectively. Corresponding apparent mK-d values for glycine differed by gt 4-fold, i.e., 189 nM and 45 nM for cerebellar granule and cortical neurons, respectively. Analysis of data obtained from equilibrium concentration-response curves also revealed differences in coagonist affinity between the two cell 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 were found between the two cell types with respect to the ion channel-blocking kinetics of the dissociative anesthetic ketamine. In conclusion, these functional data demonstrate differences in coagonist (glutamate and glycine) affinity for NMDA receptors associated with cultured cerebellar granule cells, compared with neurons cultured from the cerebral cortex. In view of the multisubunit structure of these ligand-gated ion channels, it must be considered likely that the observed differences in binding site affinity reflect differences in the subunit composition.