The Conduct of Studies on Characterizing the Mechanism of Action of the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)

The Swanson laboratory will perform detailed and rigorous analyses of the mechanism of action of the two potentiators acting on recombinant and native AMPARs. Electrophysiological recordings will be made to compare the following parameters in the absence and presence of each potentiator: • Deactivation rate following a 1 ms application of 1 mM Glu • Desensitization rate during a 100 ms application of 1 mM Glu • Ratio of steady-state-to-peak current amplitude during Glu application • Rate of recovery from desensitization • Peak and steady-state EC50 values for Glu These experiments will first be performed at saturating potentiator concentrations (or at their solubility limits) as determined by data either existing within Pfizer or empirically derived from initial recording experiments. For those receptor combinations modulated by each potentiator, respective potentiator concentration-response curves will be generated at a fixed Glu concentration (1 mM) based on modulation of the most readily characterized functional parameter (i.e. desensitization rates, deactivation rates or steady-state-to-peak current amplitude ratios). Subsequently for each potentiator, the remaining biophysical parameters and Glu concentration-response curve(s) will be measured at its EC20 and EC50 to complement the datasets with saturating potentiator concentrations. The noted parameters will be measured for homomeric and heteromeric recombinant AMPARs, as well as AMPARs containing transmembrane AMPAR regulatory protein (TARP) and cornichon (CNIH) auxiliary subunits to map selectivity. The recombinant receptor cDNAs will be derived from rodent (mouse or rat), with comparisons made to those human receptor cDNAs that can be obtained without being cost-prohibitive. The specific AMPARs to be tested in the assays will be: • Homomeric GluA1, -A2(Q), -A3 and -A4 receptors in the flip (i) and flop (o) variants • Heteromeric GluA1i/2i(R), GluA2i(R)/3i and GluA1o/4o • GluA1i in combination with TARPs (2, 3, 4, 5, 7 and 8) or CNIHs (CNIH-2 and CNIH-3) Lastly, a biophysical analysis of the action of each potentiator on miniature and evoked AMPAR excitatory postsynaptic currents in CA1 pyramidal neurons in slice preparations from mouse