A desensitization-selective potentiator of AMPA-type glutamate receptors

Abstract

1: We examined the effects of PEPA, an allosteric potentiator of AMPA receptors, on AMPA receptor kinetics. 2: PEPA did not affect the deactivation of glutamate responses but potently attenuated the extent of receptor desensitization without slowing the onset of desensitization in most of the recombinant AMPA receptors (GluR1-flip, GluR1-flop, GluR3-flip, GluR3-flip+GluR2-flip, and GluR3-flop+GluR2-flop) expressed in Xenopus oocytes. For the GluR3-flop subunit, PEPA attenuated the extent of desensitization and only weakly prolonged deactivation (1.3 fold). 3: PEPA did not significantly affect recovery from desensitization in oocytes expressing GluR3-flip, GluR1-flop, and GluR1-flop, but weakly accelerated (2.6 fold) recovery from desensitization in oocytes expressing GluR3-flop. 4: PEPA's effect on desensitization of GluR3-flop-containing receptors is unique in that onset is very slow. 5: Simulation studies using simplified kinetic models for AMPA receptors are utilized to explore the differential effects of PEPA on GluR3-flip and -flop. It is possible to simulate the action on GluR3-flip by modulating two rate constants in a 12-state kinetic model. For simulation of the action on GluR3-flop, the 12-state kinetic model is not enough, and it is necessary to invoke a 13th state, a PEPA-bound receptor to which glutamate cannot bind. 6: These results suggest that attenuation of extent of desensitization represents the principal mechanism underlying the potentiation of AMPA receptors by PEPA, and that PEPA exhibits different mechanisms with respect to GluR3-flip and GluR3-flop.

Figure 1

Effect of PEPA on desensitization and deactivation of recombinant AMPA receptors. The effects of 100 μM PEPA on the response of AMPA receptors to 10 mM glutamate pulses (1 ms for deactivation, and 50 or 100 ms for desensitization) in outside-out patches excised from oocytes expressing AMPA receptors. In each record in A and B, the peak amplitude of the response with PEPA (+PEPA) was scaled to that in the absence of PEPA (Control). Each record represents the average of 3–10 responses. In this and subsequent figures, the trace above each record represents the open tip junction currents which indicate the solution exchange in each experiment. The arrowhead in B indicates the slow onset phase of PEPA's action. The traces labelled by * (superimposed to actual responses) are single exponential fits (a trace in ‘deactivation' is for+PEPA). Inset to A: an example of the I–V relationship recorded by two-electrode voltage clamp recordings from an oocyte injected with GluR2-flip and GluR3-flip cRNAs (4 : 1 by weight).

Figure 2

Effect of PEPA on the deactivation time constant (τ_dea, A), the desensitization time constant (τ_des, B), and extent of desensitization (I_ss/I_peak, C), and fold increase in the I_ss/I_peak by PEPA (D), in recombinant AMPA receptors. The response of AMPA receptors to 10 mM glutamate pulses, 1 ms for deactivation, and 50, 100 (for GluR2-flop+GluR3-flop), or 150 (for GluR3-flop) ms for desensitization, was recorded with or without 100 μM PEPA in outside-out patches excised from oocytes expressing AMPA receptors (i: flip, o: flop). The τ_dea, τ_des, and I_ss/I_peak values were calculated from the records and expressed as mean±standard error of mean (s.e.m., n=4–7 patches). Fold increase in I_ss/I_peak=(I_ss/I_peak in the presence of PEPA)/(I_ss/I_peak of the control glutamate response). The amplitude of the control glutamate responses ranged from 14 to 200 pA. The amplitude in the presence of PEPA was 87±8 (n=4), 94±8 (n=5), 103±7 (n=5), 80±7 (n=7), 100±10 (n=5), and 86±7 (n=6)% of the control responses (I_pepa/I_cont) in GluR1-flip, GluR1-flop, GluR3-flip, GluR3-flop, GluR2-flip+GluR3-flip and GluR2-flop+GluR3-flop, respectively. Similar data in the presence of cyclothiazide (100 μM, n=4) and aniracetam (3 mM, n=7) were included for comparison. *P=0.0293 when compared with values for glutamate alone.

Figure 3

Effect of cyclothiazide and aniracetam on desensitization and deactivation of recombinant AMPA receptors. The effects of 3 mM aniracetam (A) and 100 μM cyclothiazide (B) on the response to 10 mM glutamate pulses (1 ms for deactivation, and 50 ms for desensitization) in outside-out patches excised from oocytes expressing GluR3-flip and GluR3-flop, respectively. In each record in A and B, the peak amplitude of the response with potentiators (+Cyclo or+Ani) was scaled to that in the absence of potentiators (Control). Each record represents the average of 3–10 responses.

Figure 4

Twin pulse application of 1 ms glutamate pulses. Two 1-ms glutamate pulses were applied (over varying interpulse intervals) to a membrane patch excised from an oocyte expressing GluR3-flip (A) or GluR3-flop (B), with or without PEPA (100 μM). The records were superimposed and scaled to the amplitude of the first response. The graphs below the superimposed records show the change in the amplitude of the second response relative to the first response as a function of the interpulse interval. The values are mean±s.e.m. from 4–5 experiments. The τ_rec values were calculated for GluR1-flip, GluR1-flop, GluR3-flip, and GluR3-flop with and without PEPA, and are compared in (C). *P<0.0001.

Figure 5

Simulation of PEPA's action on GluR3-flip. (A) The kinetic model used for simulation. R=sensitized receptor, Rd=desensitized receptor, G=glutamate, P=PEPA, Open=open state. The numbers 1 to 17 denote those reactions referred to in the text. Bold lines indicate reactions that were changed for the purpose of simulating the action of PEPA (denoted in the box). (B) The traces below ‘measured' are actual responses from outside-out patches excised from oocytes expressing GluR3-flip, and the traces below ‘calculated' are simulated responses obtained using the rate constants listed in Table 1. The traces labelled ‘1' are for glutamate alone (10 mM), and those labelled ‘2' are for glutamate (10 mM)+PEPA (100 μM). The open and closed bars for ‘calculated', respectively, show the periods of PEPA and glutamate application. Deactivation and desensitization were measured with 1 and 50 ms glutamate pulses, respectively. (C) The simulated responses for twin glutamate pulses (1 ms) with various interpulse intervals (5, 10, 20, 40 and 100 ms) are superimposed. 1: control, 2:+PEPA (the parameter set in Table 1 was used). (D) The values of τ_dea, I_pepa/I_cont, τ_des, I_ss/I_peak, and τ_rec calculated in actual (=measured) and simulated (=calculated) responses for both Control (from no. 1 traces) and+PEPA (from no. 2 traces).

Figure 6

Simulation of PEPA's action on GluR3-flop. (A) The kinetic model used for simulation. R=sensitized receptor, Rd=desensitized receptor, G=glutamate, P=PEPA, Open=open state, RP*=PEPA-bound receptor to which glutamate cannot bind. Numbers 1 to 18 denote those reactions referred to in the text. Bold lines indicate major reactions (>10 fold change in the parameter, denoted in the box) that were changed for the purpose of simulating the action of PEPA. (B) The traces below ‘measured' are actual responses from outside-out patches excised from oocytes expressing GluR3-flop, and the traces below ‘calculated' or ‘calculated 1' are simulated responses obtained using the rate constants listed in Table 1. ‘Calculated 2' represents the simulated responses when state RP* is removed from the parameter set in Table 1. The traces labelled as 1 are for glutamate alone (10 mM), those labelled as 2 are for glutamate (10 mM)+PEPA (100 μM), and those labelled by * are single exponential fits. The open and closed bars for ‘calculated' show the periods of PEPA and glutamate application, respectively. Deactivation and desensitization were measured with 1 and 150 ms glutamate pulses, respectively. In ‘measured' under desensitization, an arrowhead indicates ‘slow' onset phase, and an arrow indicates the point of maximal desensitization (max). (C) The simulated responses for twin glutamate pulses (1 ms) with various interpulse intervals (5, 10, 20, 40 and 100 ms) were superimposed. 1: control, 2:+PEPA (parameter set in Table 1). (D) The values of τ_dea, I_pepa/I_cont, τ_des, I_ss/I_peak, and τ_rec calculated in actual (=measured) and simulated (=calculated) responses for both Control (from no. 1 traces) and+PEPA (from no. 2 traces). For I_ss/I_peak, the values with ‘(max)' were calculated at the point of maximal desensitization (See Figure 6B-desensitization-‘measured').