Mechanism of inhibition of the GluA1 AMPA receptor channel opening by the 2,3-benzodiazepine compound GYKI 52466 and a N-methyl-carbamoyl derivative

Abstract

2,3-Benzodiazepine derivatives, also known as GYKI compounds, represent a group of the most promising synthetic inhibitors of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Here we investigate the mechanism of inhibition of the GluA1 channel opening and the site of inhibition by GYKI 52466 and its N-3 methyl-carbamoyl derivative, which we term as BDZ-f. GluA1 is a key AMPA receptor subunit involved in the brain function. Excessive activity and elevated expression of GluA1, however, has been implicated in a number of neurological disorders. Using a laser-pulse photolysis technique, which provides ∼60 μs resolution, we measured the effect of these inhibitors on the rate of GluA1 channel opening and the amplitude of the glutamate-induced whole-cell current. We found that both compounds inhibit GluA1 channel noncompetitively. Addition of an N-3 methyl-carbamoyl group to the diazepine ring with the azomethine feature (i.e., GYKI 52466) improves the potency of the resulting compound or BDZ-f without changing the site of binding. This site, which we previously termed as the "M" site on the GluA2 AMPA receptor subunit, therefore favorably accommodates an N-3 acylating group. On the basis of the magnitude of the inhibition constants for the same inhibitors but different receptors, the "M" sites on GluA1 and GuA2 are different. Overall, the "M" site or the binding environment on GluA2 accommodates the same compounds better, or the same inhibitors show stronger potency on GluA2, as we have reported previously [ Wang et al. Biochemistry ( 2011 ) 50 , 7284 - 7293 ]. However, acylating the N-3 position to occupy the N-3 side pocket of the "M" site can significantly narrow the difference and improve the potency of a resulting compound on GluA1.

Figure 1

Chemical structures of the 2,3-benzodiazepine derivatives GYKI 52466 (1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine) and BDZ-f (GYKI 53784, LY 303070, (R)-5-(4-aminophenyl)-8-methyl-7-(N-methyl-carbamoyl)-8,9-dihydro-7H-1,3-dioxolo[4,5-h][2,3]benzodiazepine).

Figure 2

(A) Representative whole-cell current traces from the laser-pulse photolysis experiment with BDZ-f as an example. As shown, BDZ-f inhibited both the rate and amplitude of the opening of the GluA1_flip channels (lower trace with 10 μM BDZ-f; k_obs = 2,299 s^–1; A = 0.24 nA) as compared to the control (upper trace; k_obs = 3376 s^–1; A = 0.56 nA). The solid line superimposed in each trace was a single exponential fit using eq 1 (Supporting Information). For data plotting, we used every fourth point (or the point at every 100 μs); for plotting the desensitization phase, we used the data points at every 500 μs. (B) Effect of BDZ-f on k_cl was obtained at 40 μM of photolytically released glutamate and as a function of BDZ-f concentration. From this plot, a K̅_I^* of 30 ± 3 μM was determined. (C) Effect of BDZ-f on k_op obtained at 300 μM of photolytically released glutamate and as a function of BDZ-f concentration. From this plot, a K_I^* of 15 ± 1 μM was determined. Each data point shown in this plot was an average of at least three measurements collected from at least three cells.

Figure 3

(A) Effect of BDZ-f on the amplitude of the whole-cell current obtained from laser-pulse photolysis measurements. A K_I of 6 ± 1 μM was obtained from the A/A_I value as a function of BDZ-f concentration (●) for the closed-channel state at 40 μM of photolytically released glutamate. At 300 μM photolytically released glutamate concentration (◊), the K_I was determined to be 5 ± 1 μM. (B) Representative whole-cell current traces of GluA1_flip channels in the absence (left) and presence (right) of BDZ-f obtained by the flow measurement. The concentrations of glutamate and the inhibitor were 2 mM and 6 μM, respectively. (C) Effect of BDZ-f on the whole-cell current amplitude of GluA1_flip receptors obtained from the flow measurement. The inhibition constant of K_I of 5 ± 1 μM was determined for the closed-channel state (40 μM glutamate, ●), whereas a K̅_I of 9 ± 1 μM was obtained for the open-channel state (2 mM glutamate, ○).

Figure 4

A minimal mechanism of the inhibition of GluA1 by 2,3-benzodiazepine compounds. L represents ligand (glutamate) and the number of ligands that bind to and open the channel is assumed to be two. R represents the active, unliganded form of the receptor; I represents an inhibitor. For simplicity and without contrary evidence, it is assumed that glutamate binds with equal affinity or K₁, the intrinsic equilibrium dissociation constant, at all binding steps. An asterisk indicates those species in the intermediate state, i.e., loose receptor:inhibitor complexes, whereas those species bound with inhibitor but without asterisk represent those in the final state of the receptor complexes through a rapid isomerization reaction. All species related to R, RL, and RL₂, including those bound with inhibitors, are in the closed-channel state, whereas those related to ₂ refer to the open-channel state.

Figure 5

The double-inhibitor experiment for GYKI 52466 and BDZ-f on GluA1_flip is shown using the ratio of current amplitude (upper solid line), as compared with the ratio of the amplitude in the presence of a single inhibitor (lower solid line). The concentration of GYKI 52466 was fixed at 50 μM, while the concentration of BDZ-f varied from 2 to 10 μM. The upper solid line represents the best fit to a one-site, double-inhibitor model, as compared with the lower solid line (i.e., the best fit to the data with BDZ-f alone). The dashed line is the simulated data for a two-site, double-inhibitor model.

Figure 6

A comparison of the inhibition constant (K_I, in μM) between GluA1_flip and GluA2Q_flip for BDZ-f (upper panel) and GYKI 52466 (lower panel). The data for GluA2Q_flip with the same compounds, i.e., GYKI 52466 and BDZ-f, were published earlier. A dashed column represents the closed-channel state, whereas a hollow column represents the open-channel state.