Zinc inhibits miniature GABAergic currents by allosteric modulation of GABAA receptor gating

Abstract

Zinc is abundantly present in the CNS, and after nerve stimulation is thought to be released in sufficient quantity to modulate the synaptic transmission. Although it is known that this divalent cation inhibits the GABAergic synaptic currents, the underlying mechanisms were not fully elucidated. Here we report that zinc reduced the amplitude, slowed the rise time, and accelerated the decay of mIPSCs in cultured hippocampal neurons. The analysis of current responses to rapid GABA applications and model simulations indicated that these effects on mIPSCs are caused by zinc modulation of GABA(A) receptor gating. In particular, zinc slowed the onset of GABA-evoked currents by decreasing both the binding (k(on)) and the transition rate from closed to open state (beta(2)). Moreover, slower onset and recovery from desensitization as well as an increased unbinding rate (k(off)) were shown to underlie the accelerated deactivation kinetics in the presence of zinc. The nonequilibrium conditions of GABA(A) receptor activation were found to strongly affect zinc modulation of this receptor. In particular, an extremely fast clearance of synaptic GABA is implicated to be responsible for a stronger zinc effect on mIPSCs than on current responses to exogenous GABA. Finally, the analysis of currents evoked by GABA coapplied with zinc indicated that the interaction between zinc and GABA(A) receptors was too slow to explain zinc effects in terms of competitive antagonism. In conclusion, our results provide evidence that inhibition of mIPSCs by zinc is attributable to the allosteric modulation of GABA(A) receptor gating.

Fig. 1.

Concentration-dependent effect of Zn²⁺ on amplitude of mIPSCs and kinetics.A, Average of 64 mIPSCs in control conditions and in the presence of Zn²⁺ (100 μm).B, The two traces shown in A are normalized and superimposed (thick line, control;thin line, Zn²⁺). C,Normalized mIPSC onset in control conditions and in the presence of Zn²⁺ (100 μm). Each trace is the average of 64 mIPSCs. D, Cumulative amplitude histograms of mIPSCs in control conditions (thick line) and in the presence of Zn²⁺ (100 μm, thin line). E, Cumulative rise time histograms of mIPSCs in control conditions and in the presence of Zn²⁺ (100 and 300 μm, respectively).F, Dose-dependent effect of Zn²⁺ on mIPSC amplitude (gray columns) and on total charge transfer (white columns). Amplitudes and total charge transfers were normalized to control values. In this and the following figures, error bars indicate SEM. G,Each column represents the mean (n = 6–10) 10–90% mIPSC rise time value obtained in control (black) and in the presence of different Zn²⁺ concentrations (white).

Fig. 2.

Concentration-dependent effect of Zn²⁺ on current responses elicited by ultrafast brief GABA applications. A, Currents evoked by brief (2 msec) GABA pulses (10 mm, upward deflection in thetop trace), in control conditions, and in the presence of Zn²⁺ (100 μm). B,The two traces in A are normalized and superimposed.C, Normalized onset of current responses elicited by GABA (3 mm) in control conditions (thick) and in the presence of Zn²⁺ (100 μm,thin). D, Concentration-dependent effect of Zn²⁺ on the amplitude (gray columns) and on total charge transfer (white columns) of GABA-evoked currents. Amplitudes and total charge transfers were normalized to control values. E, Each column represents the mean (n = 13–26) 10–90% rise time value of currents elicited by GABA (10 mm) in control (black) and in the presence of different Zn²⁺ concentrations (white).

Fig. 3.

Zn²⁺ affects the rate of current onset by decreasing rate constants of binding and conformational changes. A, Normalized onset responses to saturating concentration of GABA (10 and 30 mm) in the absence (thick lines) and in the presence of Zn²⁺ (100 μm, thin lines). A clear slower rate of current onset was seen in the presence of Zn²⁺. Note that this effect was not reversed when GABA concentration was raised from 10 to 30 mm. B, Each column represents the mean (n = 14–22) 10–90% rise time of currents evoked by GABA (10–30 mm) in control conditions and in the presence of Zn²⁺ (100 μm).C, Normalized onset responses to nonsaturating concentration of GABA (300 and 500 μm) in the absence (thick lines) and in the presence of Zn²⁺ (100 μm, thin lines). D, Normalized current onset to a saturating concentration of GABA (3 mm, thick line). Zn²⁺ (300 μm) slowed the current onset that was partially restored by increasing GABA concentration to 30 mm. E, Each column represents the mean 10–90% rise time of currents evoked either by GABA (3 mm) in control (n = 6; black) or by GABA 3 and 30 mm in the presence of Zn²⁺ (300 μm, n = 19; white). F, Currents evoked by GABA pulses (3 mm) of different time duration (1, 2, 5, and 10 msec) in the presence of Zn²⁺ (300 μm). The current amplitude and rise time increased with time of GABA application.

Fig. 4.

Zn²⁺ affects desensitization kinetics of GABA-evoked currents. A, Normalized currents evoked by GABA (10 mm, for 300 msec) in the absence (thick line) and in the presence of Zn²⁺ (100 μm, thin line). B, C, Mean (n = 5–11) time constant (τ) desensitization onset and relative area (A₁) of GABA responses (10 mm) evoked in the absence (black) and in the presence (white) of different Zn²⁺concentrations. D, Paired brief (2 msec) GABA pulses (10 mm) elicited at 5 msec interval in the absence (thick line) and in the presence of Zn²⁺ (100 μm, thin line). Note that Zn²⁺ strongly accelerated the recovery of currents evoked by the second pulse. E,Normalized recovery of the second peak evoked in the absence (open circles) or in the presence of Zn²⁺ (100 μm, closed circles). Each point represents the mean (n= 5–9). In the inset, a part of the graph has been enlarged to show effects of Zn²⁺ at shorter time intervals.

Fig. 5.

Zn²⁺ slows the recovery from desensitization. Current response elicited by a brief (2 msec) pulse of β-alanine (100 mm). B, Conditioning pulse of β-alanine (100 mm) followed by a test pulse with a time interval of 50 msec, in control (thick line) and in the presence of Zn²⁺ (100 μm,thin line). C, Normalized recovery from desensitization of β-alanine currents evoked in the absence (open circles) or in the presence of Zn²⁺ (100 μm, closed circles). Each point represents the mean (n= 7–56).

Fig. 6.

Onset and decay kinetics of current responses elicited by coapplication of GABA and Zn²⁺.A, Normalized current responses to application of GABA (10 mm, thick line) and coapplication of GABA and Zn²⁺ (100 μm, thin line). For clarity the two responses have been slightly separated. Note that the two responses almost overlap.B, The onsets of the responses shown in Aare illustrated at an expanded time scale. C,Conditioning pulse (40 msec) of GABA (10 mm) and Zn²⁺ (100 μm), followed at 40 msec interval by a short (5 msec) test pulse of GABA and Zn²⁺. The dotted line indicates perfusion with a Zn²⁺-free solution.D, Onset of the conditioning and test responses shown inC are superimposed and shown with an expanded time scale. E, Responses to brief (2 msec) GABA pulses (10 mm in the absence of Zn²⁺), applied either in control conditions (thick line) or after pre-equilibration in Zn²⁺-containing solution.

Fig. 7.

Model simulation of Zn²⁺effects on GABA_A receptors. For simulation of control recordings the parameters are k_on = 8 msec/mm, k_off = 0.13 msec⁻¹, β₂ = 8 msec⁻¹, α₂ = 1 msec⁻¹,d₂ = 1.5 msec⁻¹,r₂ = 0.15 msec⁻¹, and for recordings in Zn²⁺, k_on = 2 msec/mm, k_off = 0.25 msec⁻¹, β₂ = 1.5 msec⁻¹, α₂ = 1 msec⁻¹,d₂ = 0.5 msec⁻¹, andr₂ = 0.04 msec⁻¹. The values of the rate constants for singly bound receptors were adopted from Jones and Westbrook (1995) and were assumed not to be affected by zinc.A, Simulation of current responses to paired pulses of 10 mm GABA (2 msec pulse duration at 10 msec time interval) in control (thick line) and in the presence of Zn²⁺ (100 μm, thin line). B, Simulation of zinc effects on desensitization onset. Note the Zn²⁺ decreased the rate and increased the extent of desensitization. C,Normalized rise (left) and decay time (right) of GABA response (10 mm) in the presence or absence of Zn²⁺. Zn²⁺-induced reduction of the onset rate and acceleration of the decay is clearly reproduced. D,Current responses in the presence of Zn²⁺ evoked by “synaptic” GABA application [A · exp(−t/τ); A = 5 mm, τ = 0.2 msec] and to a 2 msec pulse of 5 mm GABA. Similar to the experimental results, a larger reduction was observed in the case of “synaptic” response. E, Simulation of rise time kinetics of the “synaptic” responses to GABA in the presence (thin line) and the absence of Zn (thick line). F, Kinetic model (Jones and Westbrook, 1995). G, Values of the rate constants reproducing the current responses in the control conditions and in the presence of 100 μm Zn.