Chlorpromazine inhibits miniature GABAergic currents by reducing the binding and by increasing the unbinding rate of GABAA receptors

Abstract

Recent studies have emphasized that nonequilibrium conditions of postsynaptic GABAA receptor (GABAAR) activation is a key factor in shaping the time course of IPSCs (Puia et al., 1994; Jones and Westbrook, 1995). Such nonequilibrium, resulting from extremely fast agonist time course, may affect the interaction between pharmacological agents and postsynaptic GABAARs. In the present study we found that chlorpromazine (CPZ), a widely used antipsychotic drug known to interfere with several ligand and voltage-gated channels, reduces the amplitude and accelerates the decay of miniature IPSCs (mIPSCs). A good qualitative reproduction of the effects of CPZ on mIPSCs was obtained when mIPSCs were mimicked by responses to ultrafast GABA applications to excised patches. Our experimental data and model simulations indicate that CPZ affects mIPSCs by decreasing the binding (kon) and by increasing the unbinding (koff) rates of GABAARs. Because of reduction of kon by CPZ, the binding reaction becomes rate-limiting, and agonist exposure of GABAARs during mIPSC is too short to activate the receptors to the same extent as in control conditions. The increase in unbinding rate is implicated as the mechanism underlying the acceleration of mIPSC decaying phase. The effect of CPZ on GABAAR binding rate, resulting in slower onset of GABA-evoked currents, provides a tool to estimate the speed of synaptic clearance of GABA. Moreover, the onset kinetics of recorded responses allowed the estimate the peak synaptic GABA concentration.

Fig. 1.

CPZ decreases the amplitude and accelerates the decay of mIPSCs. A, Average of 57 mIPSCs recorded from the same cell (V_h = −70 mV). The decaying phase of the trace was fitted with Equation 1 (Materials and Methods), assuming A_s = 0. The values of time constants are specified below the trace.B, Average of 76 mIPSCs recorded in the presence of 30 μm CPZ (from the same cell as in A). Traces were fitted with Equation 1. C, Superimposed, normalized current records shown in A (thick line) and B (thin line). A clear acceleration of the decaying phase in the presence of 30 μm CPZ is evident. D, The normalized traces shown in C are presented in an expanded time scale. Note that CPZ had no effect on the onset of mIPSC.E, Dose dependence of the effect of CPZ on mIPSC amplitude (open columns) and area (hatched columns). Amplitudes and areas were normalized (dotted line) to the control values. The CPZ concentrations are indicated above the bars. In this and in the following figures, bars above the columns represent SEM.

Fig. 2.

CPZ affects the whole-cell currents evoked by GABA using the multibarrel perfusion system. A,B, Example of whole-cell currents evoked by GABA (1 mm, bars) in control (A) and in the presence of 100 μmCPZ (B) at V_h = −30 mV. C, Same traces as in A (thick line) and B (thin line) are normalized and superimposed. Note the slower rate of current onset in the presence of CPZ. D, CPZ affects the amplitude of the whole-cell GABA-evoked currents to a much smaller extent than that of mIPSCs (0.73 vs 0.09 of the control values, respectively).

Fig. 3.

Currents evoked by brief GABA applications, using the fast perfusion system, mimic mIPSCs. A, Example of current evoked by brief GABA application (1 mm for 2 msec, see inset above the current trace). The decaying phase of GABA-evoked current was fitted with Equation 1 (Materials and Methods), assuming A_s = 0; the values of time constants are specified below the trace.B, Example of a mIPSC with a biexponential curve fitting (Eq. 1, A_s = 0) to the decaying phase.C, Example of the rising phases of currents evoked by different GABA concentrations. The duration of GABA application was long enough to reach the peak current value. The rising kinetics saturates at ∼3 mm GABA.

Fig. 4.

CPZ decreases the amplitude and accelerates the decay kinetics of the currents evoked by brief applications of GABA.A, Examples of currents evoked by brief GABA pulses (1 mm for 2 msec, inset above the current traces) in control conditions (0 CPZ) and in the presence of 30 and 100 μm CPZ. B, Superimposed, normalized current records shown in A (thick line, control; thin line, in the presence of 100 μm CPZ). A clear acceleration of the decaying phase in the presence of CPZ is evident. C, CPZ decreases the amplitude of currents evoked by brief GABA applications (hatched bars) in a dose-dependent manner. The comparison is made with the effect of CPZ on mIPSC amplitude (open bars). While at 30 μm, the effect of CPZ on amplitude of mIPSCs and of GABA-evoked currents is similar; at 100 μm CPZ, mIPSC amplitude is affected to a larger extent. D, Dose dependence of the effect of CPZ on area of GABA-evoked currents (hatched columns) and of mIPSCs (open columns). Both at 30 and 100 μm, CPZ affects the area of mIPSC to a larger extent because of a stronger effect of CPZ on mIPSC decay kinetics (at 30 μm, CPZ had almost no effect on decay of evoked currents). In C and D, the values are normalized to the controls (dotted lines).

Fig. 5.

CPZ affects the rising phase of currents evoked by brief pulses of GABA. A, Examples of normalized currents evoked by brief GABA applications (1 mm GABA for 2 msec) in control conditions (thick line) and in the presence of 100 μm CPZ (thin line). A clearly slower rate of current onset is seen in the presence of CPZ. B, Currents evoked in the presence of 100 μm CPZ by 1 mm GABA pulses of different time duration (1, 2, and 5 msec). The current amplitude and the time-to-peak increased with time of GABA application. C, Currents evoked by GABA (1 mm) were applied for the same time intervals as inB (1, 2, and 5 msec) but in the absence of CPZ. Time duration of GABA pulse within this range had no effect on the time course of the currents. D, Currents evoked by 300 μm GABA pulses of 1, 2, and 5 msec duration. At this GABA concentration, the time duration of GABA pulses had qualitatively similar effect on the rising phase as in the case of 1 mmGABA in the presence of 100 μm CPZ (B). The insets above current traces indicate the time course of GABA application.

Fig. 6.

Effect of CPZ on the rising phase of GABA-evoked currents can be reversed by saturating GABA concentrations. In contrast, the CPZ-induced acceleration of deactivation current cannot be compensated by increasing doses of GABA. A, Examples of currents evoked by 2 msec pulse of 1 mm (thin line) and 10 mm GABA (thick line) in the presence of 100 μm CPZ. B, The rising phases of currents evoked by 10 mm GABA in control conditions (thick line) and in the presence of 100 μm CPZ (thin line) have indistinguishable onset kinetics. C, The current traces shown inB are plotted in a different time scale. A clearly faster decay kinetics is seen in the presence of 100 μmCPZ. D, Currents evoked by 10 mm GABA pulses applied for 1, 2, and 5 msec in the presence of 100 μmCPZ. Within this range, time duration of GABA pulse had no effect on the current time course. In A–D, theinsets above current traces indicate the time course of GABA application. E, Absolute values of mean amplitudes of currents evoked by GABA pulses (2 msec) in conditions described above the columns. In particular, the current amplitudes evoked by 10 mm GABA in the presence and in the absence of CPZ were not significantly different (p > 0.4).

Fig. 7.

The onset of the fast desensitization component is not affected by CPZ. A, Example of a current evoked by GABA pulse (1 mm for 2 msec, see inset abovethe current trace) in which single-channel activity can be observed. During the deactivation phase (after removal of GABA), the single-channel openings are separated by silent periods indicating sojourns in a desensitized state. B, C, Examples of normalized current responses to long (200 msec) GABA applications (10 mm, solid bars) in control conditions (B) and in the presence of 100 μm CPZ (C). The decaying phases, during GABA applications, were fitted with Equation 1 (Materials and Methods). Time constants are specified below the traces, and the steady-state fractions of currents (A_s) are 0.21 and 0.17 for control and in the presence of 100 μm CPZ, respectively.

Fig. 8.

Quantitative model of GABA_A channel gating (from Jones and Westbrook, 1995). A, Scheme of transitions available for the channel. It is assumed that the receptorR has two independent binding sites for the agonistA (bound states: AR,A₂R). The channel may reach the open states (AR*,A₂R*) both from singly and doubly bound states. The model postulates also the singly and doubly bound desensitized states (AD,A₂D). B, Values of the rate constants reproducing the current responses to GABA in control conditions and in the presence of 100 μm CPZ.

Fig. 9.

Kinetic modelling of current responses to brief pulses of GABA in control conditions and in the presence of CPZ. Decrease in the binding rate k_on and increase in the unbinding rate k_offreproduces qualitatively the effects of CPZ. A, Control responses evoked by 1 msec pulses of GABA at different concentrations. For these simulations, the following values of rate constants were assumed: k_on = 12 mm/msec,k_off = 0.25 msec⁻¹, β₁ = 0.2 msec⁻¹, α₁ = 1.1 msec⁻¹, d₁ = 0.013 msec⁻¹, r₁ = 0.00013 msec⁻¹, β₂ = 8 msec⁻¹, α₂ = 0.285 msec⁻¹, d₂ = 1.7 msec⁻¹, and r₂ = 0.04 msec⁻¹. B, Same traces shown inA in an expanded time scale. The onset of GABA responses saturates at ∼3 mm GABA. C, Modelling of the effect of CPZ on GABA-evoked currents by decreasingk_on rate constant (k_on = 1.5 msec⁻¹, other kinetic parameters are unchanged, thin line).Thick line, control (rate constants as indicated for simulations shown in A and B). The identical time course to the control (1 mm GABA for 1 msec) was obtained when applying 8 mm GABA (1 msec) assumingk_on = 1.5 msec⁻¹ because in these conditions k_on · [GABA] is equal to that in control conditions (k_on = 12 msec⁻¹, [GABA] = 1 mm).D, The same traces as in C, after normalization and in an expanded time scale. A clear decrease in the onset rate as well as a moderate acceleration of the decay is seen whenk_on = 1.5 msec⁻¹. However, an increase in GABA concentration (to 8 mm), reverses both effects. For controlP_AR*/P_open= 0.017 and in the case of k_on = 1.5 (other parameters unchanged),P_AR*/P_open= 0.107, where P_AR* = maximum open probability of singly bound open state (AR*) and P_open = total maximum open probability. E, Modelling the effect of CPZ by decreasing the binding rate (to k_on = 1.5 msec⁻¹) and by increasing the unbinding rate (tok_off = 0.5 msec⁻¹).Thick line, Control (1 mm GABA for 1 msec).Thin line, (k_on = 1.5 msec⁻¹, k_off = 0.5 msec⁻¹, other parameters unchanged, 10 mm GABA for 1 msec), in this case, the amplitude is very close to that in control, but the decay is clearly faster. Thick gray line, (k_on = 1.5 msec⁻¹, k_off = 0.5 msec⁻¹, other parameters unchanged, 1 mm GABA for 1 msec). F, The same traces as in E but normalized and in an expanded time scale. Control and response to 10 mm GABA application atk_on = 1.5 msec⁻¹ andk_off = 0.5 msec⁻¹ have very similar rise time, but the latter has faster decay. One micromolar GABA application (1 msec) atk_on = 1.5 msec⁻¹ andk_off = 0.5 msec⁻¹ has slower rise time and faster decay than control. Moreover, at (k_on = 1.5 msec⁻¹,k_off = 0.5 msec⁻¹) 1 mm GABA, response has faster decay than that to 10 mm GABA. The values ofP_AR*/P_openare: 0.017 (control), 0.014 (k_on = 1.5 msec⁻¹, k_off = 0.5 msec⁻¹, 10 mm GABA), 0.11 (k_on = 1.5 msec⁻¹,k_off = 0.5 msec⁻¹, 1 mm GABA). In B, D,F, insets above the curves indicate the time course of the agonist.

Fig. 10.

Kinetic modelling of mIPSCs in control conditions and in the presence of CPZ. The synaptic current is modelled by current response to “exponential application”: A· exp(−t/τ), A = 3 mm, τ = 0.1 msec. A, B, Comparison of mIPSC (thick line) to response to GABA pulse (thin line, 3 mm for 1 msec). The decaying (A) and rising phases (B) of the two currents are very similar. The peak open probability for mIPSC is slightly (<5%) smaller that that for the response elicited by 1 msec GABA pulse. C, Comparison of the effect of CPZ on mIPSC and on responses evoked by GABA pulse. Thick line, Control mIPSC; thick gray line, mIPSC in the presence of CPZ (k_on = 1.5 msec⁻¹,k_off = 0.5 msec⁻¹);thin line, response to GABA (1 mm for 1 msec) in the presence of CPZ. The CPZ treatment diminishes the mIPSC amplitude to a larger extent than that of current evoked by 1 msec GABA pulse. D, The same traces as in C after normalization. CPZ accelerates the decay both of mIPSC and of response to 1 msec GABA pulse. In the case of mIPSC, the decay acceleration is slightly larger than that of response to GABA pulse. The values ofP_AR*/P_openare: 0.009 (control mIPSC), 0.25 (mIPSC in the presence of CPZ:k_on = 1.5 msec⁻¹,k_off = 0.5 msec⁻¹), 0.11 (k_on = 1.5 msec⁻¹, k_off = 0.5 msec⁻¹, 1 mm GABA), where P_AR* = maximum open probability of singly bound open state (AR*) and P_open = total maximum open probability. E, The same traces as in Din an expanded time scale. The slowest rate of onset is predicted for GABA responses (1 mm for 1 msec) in the presence of CPZ. The rise time of mIPSC is only slightly affected by CPZ.B, E, Insets above the traces indicate the time course of agonist in the case of mIPSC (thick line) and GABA application (thin line).