Slow phases of GABA(A) receptor desensitization: structural determinants and possible relevance for synaptic function

Abstract

GABA(A) receptor fast desensitization is thought to shape the time course of individual IPSCs. Although GABA(A) receptors also exhibit slower phases of desensitization, the possible role of slow desensitization in modifying synaptic function is poorly understood. In transiently transfected human embryonic kidney (HEK293T) cells, rat alpha1beta3delta and alpha1beta3gamma2L GABA(A) receptors showed distinct desensitization patterns during long (28 s) concentration jumps using a saturating (1 mM) GABA concentration. alpha1beta3gamma2L receptors desensitized extensively (approximately 90%), with four phases (tau(1) approximately 20 ms, tau(2) approximately 400 ms, tau(3) approximately 2 s, tau(4) approximately 10 s), while alpha1beta3delta receptors desensitized slowly and less extensively (approximately 35 %), with one or two slow phases with time constants similar to tau(3) and tau(4) of alpha1beta3gamma2L receptors. To determine the structural basis of subunit-specific desensitization, delta-gamma2L chimera subunits were expressed with alpha1 and beta3 subunits. Replacing the entire N-terminus of the gamma2L subunit with delta subunit sequence did not alter the number of phases or the extent of desensitization. Although extension of delta subunit sequence into transmembrane domain 1 (TM1) abolished the fast and intermediate components of desensitization, the two slow phases still accounted for substantial current loss (approximately 65 %). However, when delta subunit sequence was extended through TM2, the extent of desensitization was significantly decreased and indistinguishable from that of alpha1beta3delta receptors. The importance of TM2 sequence was confirmed by introducing gamma2 subunit TM2 residues into the delta subunit, which significantly increased the extent of desensitization, without introducing either the fast or intermediate desensitization phases. However, introducing delta subunit TM2 sequence into the gamma2L subunit had minimal effect on the rates or extent of desensitization. The results suggest that distinct delta subunit structures are responsible for its unique desensitization properties: lack of fast and intermediate desensitization and small contribution of the slow phases of desensitization. Finally, to investigate the possible role of slow desensitization in synaptic function, we used a pulse train protocol. We observed inhibition of peak current amplitude that depended on the frequency and duration of GABA pulses for receptors exhibiting extensive desensitization, whether fast phases were present or not. The minimally desensitizing alpha1beta3delta receptor exhibited negligible inhibition during pulse trains. Because receptors that desensitized without the fast and intermediate phases showed pulse train inhibition, we concluded that receptors can accumulate in slowly equilibrating desensitized states during repetitive receptor activation. These results may indicate a previously unrecognized role for the slow phases of desensitization for synaptic function under conditions of repeated GABA(A) receptor activation.

Figure 1. α1β3δ and α1β3γ2L desensitization differed in both rate and extent

A, current response of transiently expressed α1β3γ2L receptors to a 28 s concentration jump using 1 mm GABA (filled bar). The inset shows the first 3 s (open bar) on an expanded time scale. C, current response of α1β3δ receptors to the same protocol as in A. The parameters used to fit α1β3γ2L and α1β3δ currents are shown as scatter plots in B and D, respectively. The left ordinate indicates the time constants (τ1-τ4; note the logarithmic scale), and the right ordinate indicates the relative contribution of the corresponding time constants (a1-a4), as well as the constant term to account for incomplete desensitization. For each parameter, a horizontal line is drawn through the mean. Exponential fitting of α1β3γ2L receptor currents is shown for the entire 28 s application (E1) as well as the first ≈2 s expanded (E2). The time constants (1-4) and residual current (actual - fitted) are labelled in both panels. The asterisk in E2 indicates the slight deviation between the fitted curve and the actual current, as indicated by a non-zero residual.

Figure 2. Chloride shifts were not responsible for the fading of current during prolonged GABA application

A, currents were evoked by 10 s applications of 1 mm GABA to α1β3γ2L receptors at several command voltages (50, 30, 10, −10, −30, −50 mV, from the top trace to the bottom trace). The open symbols with arrows indicate the current measurements made at the peak (□), 5 s (▵) and 10 s (○) for the plot in B. B, current-voltage relation plots were derived from current measurements at three different time points of each GABA application from the cell shown in A. Similar plots were obtained in three other cells. C, calculated chloride reversal potentials were measured as the voltage corresponding to zero current from fitting the I-V relations with a straight line from −50 to +30 for each cell. Values were not significantly different among the three measurement time points (filled bars). E_Cl was also calculated from 3–4 GABA applications from −20 to +10 mV in cells where 85–90 % series resistance compensation was used (open bars). D, the cumulative charge transfer is shown for five randomly chosen α1β3γ2L receptor currents to demonstrate the typical relative magnitude of chloride flux occurring at various times throughout long (28 s) GABA applications. The time constants of desensitization (E1) and their relative contributions (E2) are plotted vs. conductance for each α1β3γ2L receptor current (from Fig. 1B). Linear regression lines are shown for each parameter; none of the eight regression lines had slopes that differed from zero. Note the log scale used in E2, where the time constants are shown with the fastest (1) on the bottom, followed by the intermediate and slow time constants, with the ultraslow (4) on the top.

Figure 3. Comparison of activation and fast desensitization among various perfusion techniques

Representative currents were obtained from α1β3γ2L receptors using a modified Y-tube (A), a stepper system applied to an intact cell (B) or a lifted cell (C), or an excised patch (D). Each current trace was obtained from a different cell, and normalized to peak amplitude for comparison. The scale bar in D applies to all four traces. E, current rise time, as indicated by the time elapsed between 10 and 90 % of the peak current, is shown for applications made with the stepper system using intact cells (open bars; n= 34), lifted cells (grey bars; n= 38) or excised patches (filled bars; n= 13) expressing α1β3γ2L receptors. Bar colouration applies to panels F and G as well. F, the fastest fitted time constant of desensitization. Note the logarithmic ordinate. G, the relative contribution of the fastest desensitization component is shown. H, a typical current obtained from an excised patch (grey traces) is shown with an overlaid fitted curve (dark line) generated by extrapolating the fit to the time of current onset. The fits were generated between the 100 ms time point (not shown in the figure) and the time point indicated by the arrow. For the top trace, the best fit was a single exponential function, while the other three traces were fitted best by a two exponential function.

Figure 4. Structural determinants of desensitization explored through δ-γ2L chimeras

A1-F1, the subunit construct is shown in schematic form (left) with N-terminus to the left, and transmembrane domains represented by boxes. Open portions of the schematics indicate γ2L subunit sequence, while grey portions indicate δ subunit sequence. Current responses to 28 s GABA applications (filled bar in A1) for each construct (expressed with α1 and β3 subunits) are shown (middle), with the first 3 s (see open bar under trace in A1) expanded for comparison of initial phases of desensitization (right). A2-F2, scatter plots of all measured parameters obtained from fitting the desensitization time courses are shown (see methods). The left ordinate indicates the time constant of each component (left half of each plot), and the right ordinate indicates the relative contribution of the corresponding time constants, as well as the constant term to account for incomplete desensitization (right half of each plot). Wild-type traces and plots (A and F) are from Fig. 1.

Figure 5. δ subunit sequence in TM2 is necessary but not sufficient to block desensitization

A, current response of α1β3δ(M2S) receptors to 28 s application of GABA (1 mm, filled bar) is shown. The four residues in the δ subunit that were exchanged for the corresponding residues in the γ2L subunit were: V264T, M278S, V279T, S280I (numbered according to the δ subunit mature peptide). The first 3 s (open bar) is expanded in the inset. B, scatter plot of fitted desensitization parameters is shown. C, current response of α1β3γ2L(M2S) receptors to the same protocol and the fitted desensitization parameters (D) are shown.

Figure 6. Summary of desensitization extent during 28 s applications of 1 mm GABA

Extent of desensitization was measured for each isoform as the following percentage: (peak current - current at offset of GABA)/peak current. * Significant difference compared to both α1β3δ and α1β3γ2L receptors.

Figure 7. Simulations predict a role for slow phases of desensitization during repetitive stimulation

A, the kinetic model presented by Haas & Macdonald (1999) to account for single channel gating and macroscopic currents for α1β3γ2L receptors is shown; rate constants were taken from that study. B, response of the model in A to an 800 ms pulse of GABA (1 mm; filled bar) is shown. The probability of Df (fast desensitization; continuous line), Di (intermediate desensitization; labelled dotted line) and Ds (slow desensitization; labelled dotted line) are shown above the current trace (downward dark line labelled as open). C1, the response of the model to repeated 2 ms GABA pulses (1 mm; arrows) every 100 ms is shown. The probability of each desensitized state is shown above the simulated current (continuous dark line). Occupancy of desensitized states is shown, as in B. C2, the same protocol as C1 was used, except that the entry rate constant for Df is set to zero. D1, the response of the model to a pair of 2 ms GABA applications separated by 800 ms is shown. A horizontal dotted line is shown for visual comparison of the small inhibition of amplitude for the second peak current. D2, when the first GABA application is extended to 200 ms, the test pulse (2 ms) occurring 800 ms later shows greater inhibition. The model suggested that the greater inhibition was due to an increase in the probability of the slower phases of desensitization. E, same protocol as in C1, except that both 2 and 20 ms pulse durations were shown. The longer pulse duration resulted in a slight increase in the occupancy of all three open states, and a slight decrease in the simulated current amplitude.

Figure 8. Fast desensitization is not required for inhibition during repetitive stimulation

A, current responses of α1β3γ2L receptors to a series of 25 applications of GABA (10 ms; 1 mm) is shown. The interval between the start of each pulse is shown above the traces. The progressive inhibition of peak current amplitude decreased as the interval between pulses increased from left to right for the first four traces. The right trace shows the effect of increasing the duration of the GABA pulse to 200 ms for a 2000 ms interval (compare to the fourth trace). B, pulse train protocol was applied to α1β3δ(M2S) receptors, except that GABA was applied for 20 ms. Inhibition of peak currents during the repetitive stimulation was observed for this isoform, which lacks the two fast phases of desensitization. The right trace indicates the effect of increasing the GABA application duration to 200 ms (compare to fourth trace). C, α1β3δ receptors show minimal inhibition during trains of GABA applications (left), even during 1000 ms applications separated by only 200 ms of wash (for a start-start interval of 1200 ms; middle). The current response to a continuous application of GABA (1 mm) is shown for comparison (right). The calibration applies only to the continuous current trace.