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 glutamate. Three main subtypes of glutamate-gated channels have been characterized by pharmacological studies. They have been named according to their preferred agonist, N-methyl-D-aspartate (NMDA), high affinity kainate and alpha-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 subunits and their spliced and edited variants (for review see Wisden and Seeburg, 1993). These subunits can potentially form different oligomeric complexes with diverging properties. A crucial question is therefore to determine the actual subunit composition of naturally occurring glutamate receptors. We have combined patch-clamp recording, reverse transcription (RT) and PCR to correlate, at the single cell level, the pattern of subunits expression with the functional properties of native glutamate receptors. We describe here results obtained on the AMPA receptors of hippocampal neurones and on the NMDA receptors of cerebellar granule cells which show that the subunit composition of these two types of receptors explains some of their functional properties. Furthermore, our data also indicate that the expression of NMDA receptor subunits during the postnatal development of cerebellar granule cells is regulated by an activity-dependent mechanism.