Figure 4.

Characteristics of open-channel block of NMDARs by amitriptyline (ATL). (a) An overlay of normalized currents activated by 100 μM NMDA + 30 μM Gly recorded at −70 mV in the presence of 0.25 mM Ca²⁺. 10 μM or 100 μM ATL was applied at steady state. Applications of agonist and drug are shown above the traces as bars. The onset and offset of ATL block of currents were well fitted by a single exponential function (smooth lines through corresponding currents), yielding time constants for block (τ_on) and unblock (τ_off). (b) Histogram representing a comparison of rates of block onset (1/τ_on) and offset (1/τ_off) obtained for 10 μM and 100 μM ATL at −70 mV. Values (circles) from each experiment, illustrated in panel a, as well as mean values ± S.E.M. are compared. ^***Data are significantly different (p = 0.0003, Student’s two-tailed t-tests). The K_d value for ATL, calculated using Eqs. 3, 4 and 5, is 127.5 μM. (c) Currents activated by 100 μM NMDA + 30 μM Gly recorded in 0.25 mM Ca²⁺ at −100 mV, −70 mV and −30 mV in the absence of ATL (0) and in the presence of rising ATL concentrations ([ATL]) indicated by numbers at corresponding level of current (in μM). (d) Concentration-inhibition curves for ATL for currents activated by 100 μM NMDA + 30 μM Gly recorded at −100 mV, −70 mV and −30 mV in the presence of 0.25 mM Ca²⁺. Symbols depict mean values ± S.E.M of relative amplitudes of currents (I_b/I_c) measured in the presence (I_b) and absence (I_c) of different ATL concentrations from 5–10 experiments. Solid lines are fits to the data with the Hill equation (Eq. 1). (e) Voltage dependence of the IC₅₀ value for ATL inhibition of NMDA-activated currents. Values (circles) from each experiment and mean values ± S.E.M. are shown. Dotted line depicts fit to the data with Eq. 6, which yielded IC₅₀ (0) = 220 μM and a e-fold change of the IC₅₀ of 50 mV.