Chemical structure of KB-R7943.

The voltage independence of inhibition of NMDA-induced current by 10 μM KBR. (A) Whole-cell currents induced by 30 μM NMDA + 30 μM Gly in the absence and presence of 10 μM KBR recorded at −85 and +45 mV. Application protocols are indicated above the records. (B) The I-V relations of NMDAR currents induced by 30 μM NMDA + 30 μM Gly in the absence and the presence of 10 μM KBR within the range of V_m values from −60 to +50 mV. The insert shows the applied voltage ramp protocol. (C) The fraction of NMDA-induced currents inhibited by 10 μM KBR at several V_m values (−85, −55, −25, and +45 mV). Protocol of measurements was similar to those indicated in (A). The mean values ± S.E.M. from five experiments are plotted.

The KBR inhibition of native and recombinant NMDARs. (A and B) Currents evoked by 30 μM NMDA + 30 μM Gly in the absence and the presence of 10 μM KBR or 1 μM ifenprodil (Ifen) in cortical neurons at V_m = −55 mV. Application protocols are indicated above the records. (C and D) Currents evoked by 30 μM NMDA + 30 μM Gly in the absence and presence of 10 μM KBR in HEK293T cells expressing GluN1/2A (C) and GluN1/2B (D) NMDARs at V_m = −35 mV. Application protocols are indicated above the records. (E) Quantitative comparison of 10 μM KBR inhibition of currents activated by 30 μM NMDA + 30 μM Gly mediated by native NMDARs (Ifen-sensitive and Ifen-resistant, n = 5 for each group) and recombinant NMDARs of GluN1/2A and GluN/2B subunit compositions (HEK293T cells, n = 3 for each group). Ordinate represents the relative inhibition of currents by 10 μM KBR measured as (1 − I_NMDA+KBR/I_NMDA), where I_NMDA (pA) is an amplitude of current in the absence of KBR and I_NMDA+KBR (pA) is an amplitude of current in the presence of KBR. The mean values ± S.E.M. are plotted. Data do not differ significantly (P > 0.2, ANOVA).

The KBR block of NMDARs depends on glycine concentration. (A) Currents elicited by 30 μM NMDA in the presence of 1 μM (left) and 300 μM glycine (right) recorded in the presence of the indicated KBR concentrations at V_m = −55 mV. Application protocols are indicated above the records. (B) KBR concentration-inhibition curves for currents activated by 30 μM NMDA in the presence of the indicated glycine concentrations (1, 30, and 300 μM). The mean values ± S.E.M. from four experiments for each condition are plotted. Solid lines indicate fits of the data with the Hill equation with the following parameters: for 1 μM Gly, KBR IC₅₀ = 5.3 ± 0.1 μM and Hill coefficient (h) = 2.7 ± 0.3; for 30 μM Gly, IC₅₀ = 15.0 ± 1.2 μM and h = 2.0 ± 0.1; and for 300 μM Gly, IC₅₀ = 41.2 ± 8.8 μM and h = 1.0 ± 0.1. The IC₅₀ values are significantly different for all Gly concentrations under study (P < 0.01, ANOVA, post-hoc Bonferroni test). (C) KBR concentration-inhibition curves for currents activated by 10, 30, and 100 μM NMDA in the presence of 30 μM glycine. The mean values ± S.E.M. from four experiments for each condition are plotted. Solid lines indicate fits of the data with the Hill equation. KBR IC₅₀ values were 12.9 ± 1.5 μM (h = 1.8 ± 0.2), 15.0 ± 1.2 μM (h = 1.8 ± 0.2), and 15.6 ± 1.2 μM (h = 1.9 ± 0.4) for 10, 30, and 100 μM NMDA, respectively, and did not differ significantly (P > 0.4, ANOVA).

Relief from KBR inhibition by increasing the Gly concentration. (A) Currents elicited by applications of 30 μM NMDA + 30 μM Gly and 1 μM Gly (1), 30 μM Gly (2), and 300 μM Gly (3) in the presence of 30 μM NMDA + 10 μM KBR recorded at V_m = −55 mV in a neuron. Application protocols are shown above the trace. The insert represents an overlay of Gly-induced currents replotted at a higher time resolution. (B) Currents elicited by applications of 30 μM NMDA + 30 μM Gly and 30 μM Gly (1), 100 μM Gly (2) in the presence of 30 μM NMDA + 10 μM KBR, and 100 μM Gly (3) in the presence of 30 μM NMDA + 33 μM KBR recorded at V_m = −55 mV in a neuron. Application protocols are shown above the trace by solid lines. The insert represents an overlay of Gly-induced currents replotted at a higher time resolution.

Dependence of currents elicited by NMDA + KBR on extracellular Ca²⁺. (A) Currents recorded in a neuron at V_m = −55 mV elicited by application of KBR alone or NMDA alone, their simultaneous application (NMDA + KBR), and application of NMDA + Gly. Application protocols are shown above the trace. (B) NMDA + KBR elicited currents recorded in a neuron at V_m = −55 mV in the presence and absence of Ca²⁺ in the external bathing solution. Application protocols are shown above the trace by solid lines. (C) Currents recorded in the same neuron at V_m = −55 mV elicited by NMDA + KBR (left record) and NMDA + Gly (right record) in the Ca²⁺-free solution. KBR (10 µM) was added for a short episode in the middle of NMDA + Gly application. Application protocols are shown above the trace. (D) The NMDAR-mediated current activated by 100 μM NMDA + 10 μM KBR in a neuron at V_m = ;−55 mV in the Ca²⁺-free solutions. Kynurenate (Kyn; 50 µM) was added for a short episode in the middle of NMDA + KBR application. The application protocol is shown above the trace. (E) Currents activated by 30 μM NMDA + 10 μM KBR in a neuron at V_m = −55 mV in the presence of 1 mM Ca²⁺ in the external bathing solution (left record) and in the Ca²⁺-free solutions (right record). Ca²⁺ (1 mM) was added for a short episode in the middle of NMDA + KBR application. Application protocols are shown above the trace by solid lines. (F) Quantitative comparison of steady-state amplitudes of currents activated by 30 μM NMDA + 10 μM KBR in neurons in the absence (n = 7) and presence (n = 7) of 1 mM Ca²⁺ in the external bathing solution and when 1 mM Ca²⁺ is applied for a short period of time (n = 3). *Data differ significantly (P < 0.0001, ANOVA, post-hoc Bonferroni test). (G) NMDAR-mediated currents activated by 100 μM NMDA + 10 μM KBR and 100 μM NMDA + 10 μM Gly in a neuron at V_m = −55 mV loaded with BAPTA in the presence of 1 mM Ca²⁺ in the external bathing solution and in the Ca²⁺-free media. (H) Quantitative comparison of steady-state amplitudes of currents activated by 100 μM NMDA + 10 μM KBR and 100 μM NMDA + 10 μM Gly in BAPTA-loaded neurons in the absence (n = 6) and presence (n = 6) of 1 mM Ca²⁺ in the external bathing solution. Data do not differ significantly (P > 0.1, ANOVA). The mean amplitude for four recorded currents (G) in each experiment was taken for 100%.

Li⁺ enhances Ca²⁺ inactivation of NMDAR currents. (A) Currents activated by 30 μM NMDA + 10 μM Gly in a neuron at V_m = −55 mV in the presence and absence of 1 mM Ca²⁺ in Na⁺ and Li⁺ external bathing solutions. Application protocols are shown above the trace by solid lines and boxes. (B) Superposition of currents depicted in (A) under certain conditions (currents indicated by different colors) for better comparison of their time courses. (C) Quantitative comparison of amplitudes of currents activated by 30 μM NMDA + 10 μM Gly measured at the steady state in the absence (n = 5) and presence (n = 5) of 1 mM Ca²⁺ in Na⁺ and Li⁺ external bathing solutions. *Data differ significantly (P < 0.01, Student’s two-tailed t tests).

Measurements of EC₅₀ of KBR as a coagonist of NMDARs. (A) An overlay of currents elicited by NMDA + KBR at different concentrations (shown on the right of each trace) in a neuron at V_m = −55 mV in Ca²⁺-free solution. (B) Concentration-activation curve for KBR of currents elicited by 30 μM NMDA recorded in neurons at V_m = −55 mV in Ca²⁺-free external solution. The mean values ± S.E.M. from five experiments for each condition are plotted. Solid line indicates fit of the data with the Hill equation. Fit reveals the following parameters of KBR activation: EC₅₀ = 1.18 ± 0.16 µM and h = 1.5 ± 0.4. KBR concentrations above 10 μM were not used for the fit. (C) NMDAR-mediated currents activated by 100 μM NMDA + 10 μM KBR and 100 μM NMDA + 10 μM Gly in a neuron at V_m = −55 mV in the Ca²⁺-free media. (D) Quantitative comparison of amplitudes of currents activated by 100 µM NMDA + 10 µM KBR and by 100 µM NMDA + 10 µM Gly recorded on the same neurons (n = 7) during paired applications in the Ca²⁺-free media. The mean values ± S.E.M. are plotted. Data do not differ significantly (P > 0.7, two-tailed Student’s t test). (E) Currents recorded in a neuron at V_m = −55 mV in Ca²⁺-free external solution elicited by successive applications of 30 μM NMDA + KBR at 3, 10, 30, and 100 μM. Application protocol is shown above the trace.