Influence of the TARP γ8-Selective Negative Allosteric Modulator JNJ-55511118 on AMPA Receptor Gating and Channel Conductance

Abstract

AMPA-type gultamate receptors (AMPARs) mediate excitatory signaling in the brain and are therapeutic targets for the treatment of diverse neurological disorders. The receptors interact with a variety of auxiliary subunits, including the transmembrane AMPAR regulatory proteins (TARPs). The TARPs influence AMPAR biosynthesis and trafficking and enhance receptor responses by slowing desensitization and deactivation and increasing single-channel conductance. TARP γ8 has an expression pattern that is distinct from that of other TARPs, being enriched in the hippocampus. Recently, several compounds have been identified that selectivity inhibit γ8-containing AMPARs. One such inhibitor, JNJ-55511118, has shown considerable promise for the treatment of epilepsy. However, key details of its mechanism of action are still lacking. Here, using patch-clamp electrophysiological recording from heterologously expressed AMPARs, we show that JNJ-55511118 inhibits peak currents of γ8-containing AMPARs by decreasing their single-channel conductance. The drug also modifies hallmark features of AMPAR pharmacology, including the TARP-dependent actions of intracellular polyamines and the partial agonist kainate. Moreover, we find that JNJ-55511118 reduces the influence of γ8 on all biophysical measures, aside from its effect on the recovery from desensitization. The drug is also effective when applied intracellularly, suggesting it may access its binding site from within the membrane. Additionally, we find that AMPARs incorporating TARP γ2 mutated to contain the JNJ-55511118 binding site, exhibit greater block than seen with AMPARs containing γ8, potentially reflecting differences in TARP stoichiometry. Taken together, our data provide new insight into the mechanism by which γ8-selective drugs inhibit AMPARs. SIGNIFICANCE STATEMENT: Although modulation of AMPA-type glutamate receptors shows promise for the treatment various neurological conditions, the absence of subtype-selective drugs has hindered adoption of this therapeutic strategy. We made patch-clamp recordings to characterize the actions of the γ8-selective AMPAR inhibitor JNJ-55511118 on GluA2(Q) receptors expressed in HEK cells. We report that JNJ-55511118 inhibits AMPAR-mediated currents by reducing single-channel conductance, providing clear insight into the mechanism of action of this important class of AMPAR modulators.

Fig. 1. JNJ-118 decreases peak amplitude of currents from GluA2(Q)/γ8.

(A) Representative outside-out patch responses (10 mM glutamate, 200 milliseconds, –60 mV) from two HEK293 cells transfected with GluA2/γ8 (left) or GluA2/γ8.DM (right) in control conditions (black) and in the presence of 1 μM JNJ-118 in both control and glutamate solutions (gray). Only the initial part of each response is shown, with the percent peak current remaining in JNJ-118 indicated. (B) Pooled peak inhibition data (I_JNJ-118/I_Control) showing the effect of 1 μM JNJ-118 on GluA2 alone, GluA2/γ8, and GluA2/γ8.DM. Box-and-whisker plots indicate the median (black line), the 25th–75th percentiles (box), and the 10th–90th percentiles (whiskers); filled circles are data from individual patches, and open circles indicate means. Indicated P values (adjusted for multiple comparisons as described in Table 1) are from two-sided Wilcoxon rank-sum tests following a nonparametric omnibus test (Supplemental Table 1).

Fig. 2. JNJ-118 decreases the weighted mean channel conductance of GluA2(Q)/γ8.

(A) Representative outside-out patch responses (10 mM glutamate, 200 milliseconds) (black bars) recorded at –60 mV from HEK293 cells transfected with GluA2/γ8 (left) or GluA2/γ8.DM (right) in control conditions (black) or in the presence of 1 μM JNJ-118 (gray). Insets show corresponding current-variance relationships and estimated channel conductance (γ) and peak open probability (P_{o, peak}). (B) Scatter and paired plots showing the effects of 1 μM JNJ-118 on weighted mean channel conductance (γ) and P_{o, peak} values for GluA2, GluA2/γ8, and GluA2/γ8.DM. Open circles show individual values, and filled circles denote the means, with error bars indicating S.E.M. In scatter plots, dashed lines denote equality, with points below the lines indicating inhibitory effects of JNJ-118. Indicated P values (adjusted for multiple comparisons as described in Table 1) are from two-sided Wilcoxon signed-rank exact tests following a nonparametric omnibus test (Supplemental Table 1).

Fig. 3. JNJ-118 reduces the prevalence of higher conductance openings of GluA2(Q)/γ8 channels.

(A) Representative responses (10 mM glutamate, 200 milliseconds; black bars) recorded at –60 mV from an outside-out patch expressing GluA2/γ8 in the presence and absence of 1 μM JNJ-118. Five consecutive sweeps are shown in each condition; the initial peak is truncated, and the single-channel openings from these sweeps that were included in the analysis are highlighted. Note the prevalence of lower amplitude events in the presence of JNJ-118. (B) Pooled amplitude histograms of resolved GluA2/γ8 openings in the absence (top) and presence (bottom) of JNJ-118 (392 and 272 openings from 8 and 6 patches, respectively). Note the skew in amplitudes toward lower values in the presence of JNJ-118. Dotted black lines are individual gaussian fits with indicated means and proportions. Solid blue lines are the sums of the fitted gaussians.

Fig. 4. JNJ-118 increases spermine block of GluA2(Q)/γ8 receptors.

(A) Representative responses evoked by 10 mM glutamate (200 milliseconds; black bars) recorded at potentials between –110 mV and +80 mV from an outside-out patch in the absence (left) and presence (right) of 1 μM JNJ-118. In each case, the responses at –60 and +60 mV (from which RI was calculated) are shown in black. (B) Pooled normalized conductance-voltage relationships for GluA2/γ8 in the absence and presence of 1 μM JNJ-118. The filled symbols are the mean values from 6 cells (with error bars showing S.E.M.), and the solid lines are fits of double Boltzmann relationships (see Methods). (C) Scatter and paired plots (as in Fig. 2) showing the effects of 1 μM JNJ-118 on Rectification Index and V_b (from individual double Boltzmann fitted conductance-voltage relationships) for GluA2, GluA2/γ8, and GluA2/γ8.DM. The indicated P values (adjusted for multiple comparisons as described in Table 1) are from two-sided Wilcoxon signed-rank exact tests following a nonparametric omnibus test (Supplemental Table 1).

Fig. 5. Inhibition by JNJ-118 is unaffected by channel state, and the drug is effective when applied intracellularly.

(A) Representative concatenated responses from a GluA2/γ8 outside-out patch evoked by applications of 10 mM glutamate (200 milliseconds, 1 Hz; black bars) at –60 mV, showing inhibition produced by preapplications of 1 μM JNJ-118 (200 milliseconds; gray bars). Right-hand panel shows mean peak current data from 7 records (error bars indicate S.E.M.), normalized in each case to the mean of three applications delivered before the first preapplication of JNJ-118. (B) Representative response from a GluA2/γ8 outside-out patch produced by a 48-second application of 10 mM glutamate (black bar) in the constant presence of 50 μM cyclothizide. Filled gray area denotes the rapid application of 1 μM JNJ-118 for 10 seconds. White dotted lines are single exponential fits showing the timecourse of block and unblock. (C) Representative response, as in panel B, but recorded with an internal solution containing 10 μM JNJ-118. Note that in this case, the extracellular application of JNJ-118 produced a greatly reduced block. (D) Pooled data showing the degree of inhibition produced by 1 μM JNJ-118_ext when the internal solution contained either 0, 1, or 10 μM JNJ-118. Box-and-whisker plots as in Fig. 1. Indicated P values (adjusted for multiple comparisons using Holm’s sequential Bonferroni correction) are from two-sided Wilcoxon rank-sum tests following Kruskal-Wallis rank-sum test (Supplemental Table 1).

Fig. 6. A double point mutation in TARP γ2 introduces JNJ-118 sensitivity to GluA2(Q)/γ2.

(A) Representative outside-out patch responses (10 mM glutamate, 200 milliseconds) (black bars) recorded at –60 mV from HEK293 cells transfected with GluA2/γ2 (left) or GluA2/γ2.DM (right) in control conditions (black) or in the presence of 1 μM JNJ-118 (gray). Insets show corresponding current-variance relationships and estimated channel conductance (γ) and peak open probability (P_{o, peak}). (B) Pooled peak inhibition data (I_JNJ-118/I_Control) showing the effect of 1 μM JNJ-118 on GluA2 alone (from Fig. 1B), GluA2/γ2, and GluA2/γ2.DM. Box-and-whisker plots as in Fig. 1. Indicated P values are from two-sided Wilcoxon rank-sum tests (adjusted for multiple comparisons as described in Table 1) following a nonparametric omnibus test (Supplemental Table 1). (C) Scatter and paired plots (as in Fig. 2) showing the effects of 1 μM JNJ-118 on the weighted mean time constant of desensitization (τ_{w, des}), the fractional steady-state component (I_ss/I_peak), the weighted mean channel conductance, and P_{o, peak} values for GluA2/γ2 and GluA2/γ2.DM. Indicated P values are from two-sided Wilcoxon signed-rank exact tests (adjusted as described in Table 1) following a nonparametric omnibus test (Supplemental Table 1). (D) Representative responses (10 mM glutamate, 200 milliseconds; black bars) recorded at –60 mV from an outside-out patch expressing GluA2/γ2.DM in the presence and absence of 1 μM JNJ-118. Five consecutive sweeps are shown in each condition; the initial peak is truncated, and selected single-channel openings are highlighted. Note the prevalence of lower amplitude events in the presence of JNJ-118. e) Pooled amplitude histograms of resolved GluA2/γ2.DM openings in the absence (top) and presence (bottom) of JNJ-118 (228 and 289 openings from 2 and 3 patches, respectively). Note the skew in amplitudes toward lower values in the presence of JNJ-118. Dotted black lines are individual gaussian fits with indicated means and proportions. Solid blue lines are the sums of the fitted gaussians.

Fig. 7. JNJ-118 influences kainate relative efficacy but not recovery from desensitization.

(A) Glutamate- and kainate-evoked currents (−60 mV) recorded from the same representative patch in the presence of 50 μM cyclothiazide in the absence (left) and presence (right) of 1 μM JNJ-118. The glutamate responses are scaled to highlight the small decrease in the relative efficacy of kainate. (B) Scatter and paired plots (as in Fig. 2) showing the effects of 1 μM JNJ-118 on I_KA/I_Glu for GluA2, GluA2/γ8, and GluA2/γ2.DM. Indicated P values are from two-sided Wilcoxon signed-rank exact tests (adjusted for multiple comparisons as described in Table 1) following a nonparametric omnibus test (Supplemental Table 1). (C) Glutamate-evoked currents (−60 mV) from a representative GluA2/γ8 outside-out patch demonstrating the time course of recovery following desensitization with 10 mM glutamate (250 milliseconds; black bar) in the absence and presence of 1 μM JNJ-118. Recovery of peak currents was assessed using glutamate reapplication (10 milliseconds; short black bars) at intervals from 2–500 milliseconds, and single exponentials (dashed lines) were fitted to the peak currents. (D) Scatter and paired plots showing the effects of 1 μM JNJ-118 on τ_rec for GluA2, GluA2/γ8, GluA2/γ8. DM, GluA2/γ2 and GluA2/γ2.DM. Open circles show individual values, and filled circles denote the means, with error bars indicating S.E.M. In scatter plots, dashed lines denote equality, with points below the lines indicating inhibitory effects of JNJ-118. Indicated P values are from two-sided Wilcoxon signed-rank exact tests (adjusted as described in Table 1) following a nonparametric omnibus test (Supplemental Table 1).