Fig. 9. Fluctuations between agonist unbound states can reduce PO, PEAK.A, The kinetic scheme was adapted from Heckmann et al. (1996), Jonas et al. (1993), and Hausser and Roth (1997), with changes made to the channel closing rate α, channel opening rate β, rate constants for recovery from desensitizationkD-1 andkD-2
, and desensitization rate constant from the doubly liganded state kD+2. These changes resulted in a simulated response time course (10–90% rise time to step rise in agonist 110 μsec, tauDECAY 3.7 msec for 10 mmglutamate), mean channel open time (625 μsec), steady-state–peak current ratio (0.025), EC50 (0.4 mm), and tauRECOVERY from desensitization (180 msec) that are similar to those measured for homomeric GluR1(flip) receptors (Mosbacher et al., 1994; Partin et al., 1996; Wahl et al., 1998; this study). The simulated agonist EC50 is also similar to that determined for CA1 pyramidal cells (0.42 mm) (Jonas and Sakmann, 1992), and IC50 for predesensitization (10 μm) is similar to that determined for AMPA receptors in chick spinal neurons (10 μm) (Trussell and Fischbach, 1989) and CA1 hippocampal neurons (4–9 μm) (Colquhoun et al., 1992). Responses were simulated by solving a Q-matrix (SCALCS provided by D. Colquhoun) as well as by using Monte Carlo methods. Rate constants (in s−1) werek-1 = 1500,k+2 = 1,k-2 = 1 (Ser845-dephosphorylated receptor) or 25 (Ser845-phosphorylated receptor),kD+1 = 100, kD-1 = 7, kD+2= 4150, kD-2 = 7, α = 1600, β = 2 × 104.kREV was adjusted to 72.2 sec−1 to ensure microscopic reversibility, and the forward agonist binding constantk+1 was 3.6 × 106s−1m−1.B, Response time course is unchanged by partial occupancy of nonfunctional unbound state R2 despite large changes to PO, PEAK. The simulation was run with 96.2 or 50% of the channels starting in R1 fork-2 = 25 or 1 s−1, respectively; the remainder of the channels started in state R2. C, Fluctuations during the response interval in the occupancy of the unbound state R1 are shown (top panel) when the rate constants between the two unbound states (R1 and R2) favor occupancy of state R1 at rest (k+2 = 1,k-2 = 25 sec−1). Four responses at the times indicated by the arrows are superimposed to theright. Calibration: 2 msec, 30 pA. The variance of responses of 200 channels (12 pS conductance, −100 mV) was simulated by incorporating the fluctuations in the proportion of channels in state R1 at the start of agonist application. Fifty responses were simulated and analyzed as described in Materials and Methods for the model shown in A withk-2 = 25. D, Fluctuations during the response interval in the occupancy of the unbound state R1 are shown when the rate constants between the two unbound states (R1 and R2) are equal (k+2 =k-2 = 1 sec−1). Note the much larger fluctuations into and out of R1 for dephosphorylated receptor than for the phosphorylated receptor (C). Four responses at the times indicated by thearrows are superimposed to the right.Calibration: 2 msec, 20 pA. These traces illustrate the increased fluctuation of the peak current compared with C. The variance of responses of 200 channels (12 pS conductance, −100 mV) were simulated by incorporating the fluctuations in the proportion of channels in state R1 at the start of an agonist application as indicated in B. Fifty responses were simulated and analyzed as described in Materials and Methods for the model shown inA with k-2 = 1. This analysis confirms that fluctuations in the occupancy of R1before agonist application appear as a reduction inPO, PEAK, provided that fluctuations occur on a more rapid time scale (tauEQUILIBRIUM = 0.5 sec for k-2 = 1) than our stimulation protocol (inter-stimulus interval 2 sec) yet on a slower time scale than the duration of agonist application (0.2 sec).