Activation mechanism of recombinant NMDA receptor and AMPA receptor
The gating reaction mechanism of fully-liganded NR1/NR2A recombinant NMDARs (expressed in Xenopus oocytes) was investigated by fitting all possible 3-closed, 2-open state non-cyclic kinetic schemes to currents elicited by saturating concentrations of glutamate plus glycine. The adequacy of each scheme was assessed by the maximum likelihood values and auto-correlation functions of single-channel currents, as well as the predicted time courses of transient, macroscopic currents. Two schemes provided the best description for NMDAR gating at both the single channel and macroscopic levels. These two schemes had coupled open states, only one gateway between the closed and open aggregates, and at least two pre-opening closed states. These two models could be condensed into a cyclic reaction mechanism. The overall 'gating' rates (from the initial, stable closed state to the final, stable open state) are 177 s -1 and 4.4 s -1 . Attempts were also made to model of AMPAR at the single channel level. We expressed a nondesensitizing mutant GluR-flip L497Y in Xenopus oocytes and recorded currents from out-side-out patches containing only one active channel. Single channel currents evoked by various concentrations of glutamate revealed several interesting properties of AMPAR at the single molecule level. First, AMPAR exhibits at least three conductance levels, the relative occupancies of which are dependent on the concentration of glutamate. Secondly, AMPAR shows multiple activation patterns with distinct mean open times. The duration distributions of closed and open events in these modes can each be well fitted by three exponential components. Autocorrelation analysis indicates that there is only one pathway between these closed and open states. Third, the single channel conductance is not stable and decreases in the presence of glutamate. In the last part of this dissertation, I describe a closed-form solution of a rate-equilibrium free energy relationship (REFER, LFER, Bronsted plot) for a linear bounded, chain of reactions of arbitrary length. The result provides information about the shape of the transition state from simple measures of forward and backward rate constants of an apparently two-state process.
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