AMPA-type glutamate receptors mediate most excitatory synaptic transmission in the brain. Receptor binding studies have shown that AMPA receptors exist in two distinct states with 20-fold different affinities. It is suggested that the two forms are differentially localized with synaptic receptors being of the low-affinity type and non-synaptic receptors of the high-affinity type. Thus, AMPA receptors in patches excised from the soma (and hence of non-synaptic origin) may have different kinetic properties than those in synapses. This would entail that somatic and synaptic receptors contribute differentially to neurological disorders involving excitotoxic damage to neurons. This proposition will be tested by examining if differences between synaptic and non-synaptic AMPA receptors are also detected with physiological measures and if they correspond to the high-low affinity distinction seen in binding. To facilitate this comparison, both physiological data and binding data will be collected under equivalent conditions.
Aim One will determine 'physiological KD' values for equilibrium currents in patches excised from hippocampal pyramidal cells and compare them with binding affinities obtained in the same buffer and at the same temperature. The results will then be compared with similar measures from recombinant AMPA receptors stably expressed in HEK293 cells that appear to be entirely of high affinity (Aim Two) and with data from synaptic receptors in autapses of cultured neurons, which presumably are of low affinity (Aim Three). It is further known that binding to the low-affinity receptors can be modulated about two-fold by biochemical manipulations such as treatment with concanavalin A, phospholipase A2 or neuroaminidase. The consequences of some of these treatments for AMPA receptor kinetics will be examined in Aim Four using the same approach. It was also observed that high affinity receptors, unlike their low-affinity counterpart, are highly unstable at 37 degrees C and it seems likely that this is related to the fast 'run-down' of AMPA receptor currents in patch experiments. Determining which cellular factor stabilizes the low-affinity receptors will constitute the Fifth Aim of this application.
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