Agonist Trapping by GABAA Receptor Channels

Abstract

GABAergic IPSCs have a relatively slow decay (deactivation) that appears to result from GABA(A) receptor channel openings that occur well beyond the predicted duration of free GABA at central synapses. Open and desensitized states have been suggested to prevent dissociation of agonist from the receptor, thus prolonging deactivation. However, simultaneous assessment of GABA binding and channel gating has not been possible. We developed a functional assay for occupancy of the GABA binding site or sites to test the GABA "trapping" hypothesis. Deactivation currents were compared in the absence and presence of bicuculline, a competitive antagonist that also allosterically inhibits GABA(A) receptors. This provided a model-independent, functional test of the hypothesis that GABA is trapped on the receptor during gating: bicuculline could only inhibit the channel if it was open but unbound by GABA. Although bicuculline inhibited spontaneous and neurosteroid-activated GABA(A) receptor currents, it failed to alter the deactivation time course of GABA-activated GABA(A) receptor currents. Protection of deactivation current from bicuculline block indicated that GABA remained bound to the receptors while the channel was open, thus suggesting that all open states, as well as all closed and desensitized states from which channel opening can occur, must be GABA liganded states. Trapping may be specific to agonists, because the positive allosteric modulator diazepam unbound from GABA(A) receptors independent of GABA binding and channel activity.

Fig. 1.

GABA_A receptor channel openings outlast the duration of agonist exposure. A, Macroscopic current response of a lifted HEK293T cell expressing α1β3γ2L GABA_A receptors exposed to 1 mm GABA for 400 msec (solid bar). Despite precise control of the solution bathing the cell by the concentration jump technique, the current relaxation after agonist removal requires many hundreds of milliseconds. The cell was voltage clamped at −15 mV.B, Individual openings are observed for hundreds of milliseconds after a 5 msec (arrow) pulse of 1 mm GABA delivered to an excised patch containing a few α1β3γ2L GABA_A receptors. The patch was voltage clamped at −70 mV.

Fig. 2.

Bicuculline inhibited GABA_Areceptor currents in the absence of GABA. A, α1β3γ2L channels open spontaneously with low probability. A 100 mm concentration of bicuculline (hatched bar) rapidly and reversibly blocked the spontaneous activity in a lifted cell expressing α1β3γ2L GABA_A receptors voltage clamped at −30 mV. A transient “overshoot” current was observed after bicuculline washout (dotted line inA and B). B, α1β3γ2L GABA_A receptors containing the L245S mutation in the γ2L subunit have a higher spontaneous opening probability. A 100 μm concentration of bicuculline (hatched bar) rapidly and reversibly blocked the spontaneous activity in an excised patch containing the mutant GABA_A receptors voltage clamped at −30 mV. C, GABA_Areceptor currents activated by direct application of alphaxalone (solid bar) were blocked by 100 μmbicuculline (hatched bar). The cell was jumped first from control solution into 10 μm alphaxalone and then back to control solution (control wash), then the same cell was jumped from control into 10 μm alphaxalone and then into 100 μm bicuculline (bicuc wash). The dotted line (in C andD) shows the smaller holding current in the presence of bicuculline. D, Inhibition of the deactivation current was also observed when 1 μm alphaxalone was used, a concentration that did not produce a rebound current.

Fig. 3.

Bicuculline failed to inhibit deactivation currents after GABA application. A, Deactivation currents after a concentration jump into 1 mm GABA (solid bar) were identical whether the cell was washed into control solution (open bar) or into 100 μm bicuculline (hatched bar).B, A 100 μm concentration of bicuculline (hatched bar) produced a small degree of inhibition in the deactivation current after activation by a lower concentration of GABA (3 μm; EC₅₀ ∼5 μm). Thedotted line shows the smaller holding current during bicuculline wash. A portion of the trace (in the circle) is expanded to show the small effect of bicuculline wash on the deactivation current. C, The noncompetitive antagonist picrotoxin (100 μm; hatched bar) significantly inhibited deactivation currents after activation by 1 mm GABA. Note the smaller holding current during picrotoxin wash. D, The benzodiazepine diazepam (1 μm; shaded bar) potentiated deactivation currents after activation with 1 mm GABA. The dotted line shows the larger holding current in the presence of diazepam. E, Deactivation current pharmacology is summarized as the percentage of change in the weighted time constant of deactivation. The number of data points is indicated next to eachbar. Asterisks indicate significant differences compared with control deactivation rate for each condition.

Fig. 4.

Bicuculline inhibited GABA_A receptor currents under conditions in which unbinding and rebinding of GABA occurs. A, A 100 μm concentration of bicuculline (hatched bar) blocked the current during a long application of 3 μm GABA (solid bar) to a lifted cell expressing α1β3γ2L GABA_A receptors. The response to 3 μm GABA alone from the same cell is superimposed in gray. Note the larger current after removal of bicuculline. B, Current responses to 400 msec jumps into 1 mm GABA in the same cell before and after lifting the cell from the recording dish. Deactivation currents were inhibited during bicuculline wash (hatched bar) before the cell was lifted, and this deactivation current overlapped with that observed in the same cell after it was lifted. Currents were normalized to the amplitude at the offset of the GABA pulse. The larger vertical scale value applies to the intact cell current response.

Fig. 5.

Onset of bicuculline block is limited by GABA unbinding. A, Deactivation currents of α1β3γ2L (L245S) GABA_A receptors after a concentration jump into 1 mm GABA (solid bar) were partially blocked by bicuculline (hatched bar). Cells were voltage clamped at 0 to −5 mV. The onset of block was time-dependent (the separation of deactivation currents during control wash and bicuculline wash increased with time). B, To illustrate the time course of bicuculline inhibition, the traces were subtracted (middle) for comparison with the direct effect of bicuculline (left) in the same cell. The traces are normalized and overlaid to demonstrate the slow onset rate when bicuculline was applied during deactivation (right). C, Summary chart showing the onset rate of bicuculline inhibition for various conditions. Although the time course of block was often best fit by a two exponential function, only the faster time constant is shown, which accounted for >90% of the decay. The number of cells is shown for each condition. Note the logarithmic ordinate.

Fig. 6.

Diazepam unbinding is slower than GABA unbinding. Diazepam unbinds at least as slowly as GABA from α1β3γ2L GABA_A receptors. Deactivation currents are shown for applications of 3 μm GABA (solid bar) with control wash (open bar), 3 μm GABA with 1 μm diazepam wash (shaded bar), 3 μm GABA + 1 μm diazepam coapplication with control wash, and 3 μm GABA + 1 μm diazepam coapplication with 1 μm diazepam wash. Currents were normalized to the amplitude at the offset of the GABA pulse.

Fig. 7.

Diazepam unbinding is independent of channel gating. A1, In the absence of GABA, diazepam (1 μm) enhanced spontaneous activity in lifted cells expressing α1β3γ2L GABA_A receptors.A2, Diazepam enhanced the response to a steady-state application of 300 nm GABA (different cell than inB1). B1, Deactivation of α1β3γ2L and α1β3γ2L(L245S) GABA_A receptors after a 5 msec pulse of 1 mm GABA (arrow). Longer mean open times caused by this mutation slowed deactivation. B2, In the absence of GABA, 1 μm diazepam enhanced spontaneous activity in α1β3γ2L(L245S) GABA_Areceptors. C, Summary chart showing the rate of deactivation after a 1 sec application of 1 μm diazepam was indistinguishable under different GABA-binding and intrinsic gating conditions. The number of cells is indicated inparentheses. D, A 1 sec application of 1 μm diazepam applied in the absence of GABA enhances subsequent responses to 1 μm GABA in lifted cells expressing α1β3γ2L GABA_A receptors. Cells were washed with control solution for the interpulse interval.