We have isolated highly purified rat brain postsynaptic densities (PSDs), that are known to contain glutamate receptors of the AMPA and NMDA types. These PSDs were incorporated into liposomes, and grown, by a cycle of partial de- and rehydration in 5% ethylene glycol, intogiant (5-100 μm in diameter) liposomes. These giant liposomes were then made to form Gigaohm (10-20 GΩ) seals with conventional patch-clamp electrodes, which, when withdrawn, retain an excised patch in an inside-out configuration. When 5-10 μM L-glutamate (or 10 μM NMDA) plus 1 μM glycine were present inside the patch pipete, but not in the external fluid, a highly complex pattern of currents was seen in about 55% of the cases. This was characterized by very fast kinetics, conductances as high as 460 pS and multiple lower levels of 45, 80, 120, 230 and 340 pS. These currents, when evoked by NMDA plus glycine, were entirely suppressed by the NMDA antagonist 2-amino-5-phosphonovalerate, APV. However, those activated by L-glutamate plys glycine still appeared in the presence of APV in about 18% of the cases, but with lower conductance levels. Current kinetics similar to the latter ones were also induced by the AMPA receptor agonist quisqualate (10 μM) in 16% of the cases. This indicated that both NMDA and AMPA receptors were present, in a functionally well preserved state, in isolated postsynaptic densities. Indirect evidence also suggested that in our experiments, in which 212 seals were studie, only a single postsynaptic density was present in the patches in which channel activity was found. The complex, high-conductance currents found may thus represent the opening and closure of many individual glutamate receptor ion channels present in a single isolated postsynaptic density.
|Original language||American English|
|Number of pages||4|
|State||Published - 15 Sep 1993|
- Glutamate receptor
- Ion channel
- N-methyl-D-aspartic acid
- Postsynaptic density