Ifenprodil effects on GluN2B-containing glutamate receptors

Abstract

N-Methyl-d-aspartate (NMDA) receptors are glutamate- and glycine-gated channels that mediate fast excitatory transmission in the central nervous system and are critical to synaptic development, plasticity, and integration. They have a rich complement of modulatory sites, which represent important pharmacological targets. Ifenprodil is a well tolerated NMDA receptor inhibitor; it is selective for GluN2B-containing receptors and has neuroprotective effects. The mechanism by which ifenprodil inhibits NMDA receptor responses is not fully understood. The inhibition is incomplete and noncompetitive with other known NMDA receptor agonists or modulators, although reciprocal effects have been reported between ifenprodil potency and that of extracellular ligands including glutamate, glycine, zinc, protons, and polyamines. Recent structural studies revealed that ifenprodil binds to a unique site at the interface between the extracellular N termini of GluN1 and GluN2B subunits, supporting the view that interactions with other extracellular modulators are indirect. In this study, we examined how ifenprodil affects the gating reaction of NMDA receptors in conditions designed to minimize actions by contemporaneous ligands. We found that ifenprodil decreased NMDA receptor equilibrium open probability by raising an energetic barrier to activation and also by biasing the receptor toward low open probability gating modes. These results demonstrate intrinsic effects of ifenprodil on NMDA receptor stationary gating kinetics and provide means to anticipate how ifenprodil will affect receptor responses in defined physiological and pathological circumstances.

Fig. 1.

Effects of ifenprodil on single 2B receptors. Traces represent steady-state inward sodium fluxes recorded from cell-attached patches that contained in the recording pipette one active 2B channel. A, with ifenprodil (IFN, 150 nM). B, without ifenprodil (CTR). For each condition, a 50-s segment is illustrated at two time resolutions in the top and middle panels, respectively; the bottom panels expand the underlined segment and is displayed filtered, as for analyses, at 12 kHz. All traces represent inward Na⁺ currents as downward deflections from a zero-current baseline; P_o indicates the open probability calculated for the entire parent record.

Fig. 2.

Ifenprodil prolongs specific closed components. A, closed intervals observed in two records obtained from 2B receptors in the absence (CTR, 42,360 events) and presence of ifenprodil (IFN, 150 nM, 78,017 events). Probability density functions (thick lines) were calculated by fitting kinetic 5C4O state models to the displayed data; thin lines represent individual exponential components. Their time constants (τ, milliseconds) and areas (a, percentages) are given as insets. B, summary of closed time constants in the two conditions [CTR (gray), n = 31; IFN (blue), n = 6]. Average values for CTR are given in milliseconds below each component. **, p < 0.003 (Student's t test).

Fig. 3.

Ifenprodil favors low-activity mode gating of 2B receptors. A, open durations observed in two records obtained from 2B receptors in the absence (CTR, 82,224 events) (top) and presence of ifenprodil (IFN, 150 nM, 78,017 events) (bottom). Thick lines represent probability density functions calculated from fits to 5C4O state models; thin lines represent individual exponential components. Insets, time constants (τ, milliseconds) and areas (a, percentages) for the respective record. B, summary of changes for open event distributions. CTR, gray; IFN, white. *, p < 0.05 (Student's t test).

Fig. 4.

Kinetic mechanism of ifenprodil inhibition. A, reaction mechanisms for ifenprodil-free (CTR) and ifenprodil-bound 2B receptors (IFN); rate constants for the steps explicitly incorporated in the model were estimated from fits to one-channel records and are given in seconds⁻¹ as averages for the records in each data set. *, p < 0.05 (Student's t test). All states represent receptor conformations fully bound with glutamate and glycine. C, nonconductive; O, conductive. B, state-occupancy changes calculated from the reaction mechanisms in A. C, relative free-energy fluctuations during gating were calculated with the rate constants in A. Desensitized states (C₄ and C₅) are omitted for simplicity; profiles are arbitrarily aligned at C₃.

Fig. 5.

Ifenprodil binding kinetics tiered model represents the gating mechanisms of ifenprodil-free (upper arm) and ifenprodil-bound (lower arm) receptors; transitions between arms are allowed only between the C₃ states. The model was fit to data obtained in 150 nm ifenprodil (n = 3): rate constants in gray were fixed to the values obtained for CTR and IFN conditions in Fig. 4A, whereas the rates in blue were allowed to vary. Values for each transition represent the average of the rates estimated for each file and are given in seconds⁻¹. The calculated association rate constant (k_on) and the equilibrium dissociation constant (K_d) for ifenprodil are indicated.

Fig. 6.

Ifenprodil effects on 2B macroscopic responses. A, macroscopic responses to a long (5-s) pulse of 1 mM Glu (black line) were simulated with the models in Fig. 4A, to which glutamate binding steps were appended as described under Materials and Methods (left) and were recorded as whole-cell currents in the absence (black) or presence (blue) of 150 nM ambient ifenprodil (right); insets show the same traces normalized to peaks. B, macroscopic responses to a brief (10-ms) pulse of 1 mM Glu (black arrow) were simulated as above (left) and were recorded from outside-out patches in the absence (black) or presence (green) of 150 nM ambient ifenprodil (middle); recorded traces are normalized to peak and are overlaid for comparison (right).