FUNCTIONAL AND MOLECULAR ANALYSIS OF GLUTAMATE-GATED CHANNELS BY PATCH-CLAMP AND RT-PCR AT THE SINGLE-CELL LEVEL

In the central nervous system (CNS) rapid excitatory neurotransmission is mainly mediated by ligand gated, cationic channels activated by gl utamate. Three main subtypes of glutamate-gated channels have been cha racterized by pharmacological studies. They have been named according to their preferred agonist, iv-methyl-D-aspartate (NMDA), high affinit y kainate and pha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA ). Furthermore, a large diversity within each class of glutamate-gated channels has been revealed by the molecular cloning of multiple subun its and their spliced and edited variants (for review see Wisden and S eeburg, 1993). These subunits can potentially form different oligomeri c complexes with diverging properties. A crucial question is therefore to determine the actual subunit composition of naturally occurring gl utamate receptors. We have combined patch-clamp recording, reverse tra nscription (RT) and PCR to correlate, at the single cell level, the pa ttern of subunits expression with the functional properties of native glutamate receptors. We describe here results obtained on the AMPA rec eptors of hippocampal neurones and on the NMDA receptors of cerebellar granule cells which show that the subunit composition of these two ty pes of receptors explains some of their functional properties. Further more, our data also indicate that the expression of NMDA receptor subu nits during the postnatal development of cerebellar granule cells is r egulated by an activity-dependent mechanism.