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, 25 (9), 2726-40

Desensitization and Binding Properties Determine Distinct alpha1beta2gamma2 and alpha3beta2gamma2 GABA(A) Receptor-Channel Kinetic Behavior

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Desensitization and Binding Properties Determine Distinct alpha1beta2gamma2 and alpha3beta2gamma2 GABA(A) Receptor-Channel Kinetic Behavior

Andrea Barberis et al. Eur J Neurosci.

Abstract

GABA(A) receptor subtypes comprising the alpha1 and alpha3 subunits change with development and have a specific anatomical localization in the adult brain. These receptor subtypes have been previously demonstrated to greatly differ in deactivation kinetics but the underlying gating mechanisms have not been fully elucidated. Therefore, we expressed rat alpha1beta2gamma2 and alpha3beta2gamma2 receptors in human embryonic kidney 293 cells and recorded current responses to ultrafast GABA applications at macroscopic and single-channel levels. We found that the slow deactivation of alpha3beta2gamma2-mediated currents is associated with a relatively small rate and extent of apparent desensitization. In contrast, responses mediated by alpha1beta2gamma2 receptors had faster deactivation and stronger desensitization. Alpha3beta2gamma2 receptors had faster recovery in the paired-pulse agonist applications than alpha1beta2gamma2 channels. The onset of currents mediated by alpha3beta2gamma2 receptors was slower than that of alpha1beta2gamma2 for a wide range of GABA concentrations. Single-channel analysis did not reveal differences in the opening/closing kinetics of alpha1beta2gamma2 and alpha3beta2gamma2 channels but burst durations were longer in alpha3beta2gamma2 receptors. Simulation with a previously reported kinetic model was used to explore the differences in respective rate constants. Reproduction of major kinetic differences required a smaller desensitization rate as well as smaller binding and unbinding rates in alpha3beta2gamma2 compared with alpha1beta2gamma2 receptors. Our work describes the mechanisms underlying the kinetic differences between two major GABA(A) receptor subtypes and provides a framework to interpret data from native GABA receptors.

Figures

F<sc>ig</sc>. 8
Fig. 8
Model simulations of α1β2γ2 and α3β2γ2 receptor-mediated currents. (A) Model of GABAA receptor gating (Jones et al., 1998). Thick arrows depict binding and unbinding transitions between singly-and doubly-bound desensitized states. These transitions were found to play an important role in shaping currents mediated by these receptors (see Model simulations and Discussion). Simulations were performed for the following rate constants for α1β2γ2 receptor: kon = 3.0/ms/mm, koff = 0.1/ms, d2 = 1.5/ms, r2 = 0.02/ms, β2 = 2.5/ms, α2 = 0.284/ms, q = 0.1/ms/mm, p = 0.005/ms, d1 = 0.013/ms, r1 = 0.00013/ms, β1 = 0.2/ms, α2 = 1.11/ms and for α3β2γ2 receptor: kon = 0.3/ms/mm, koff = 0.045/ms, d2 = 0.3/ms, r2 = 0.02/ms, β2 = 2.5/ms, α2 = 0.284/ms, q = 0.00087/ms/mm, p = 0.00013/ms, d1 = 0.013/ms, r1 = 0.00013/ms, β1 = 0.2/ms, α1 = 1.11/ms. In B–F, currents simulated for α1β2γ2 receptors are drawn with a thin line and those for α3β2γ2 channels with a thick line. In all graphs currents are normalized. (B) Simulations of current responses to brief (2 ms) applications of saturating [GABA]. A slower deactivation for α3β2γ2 receptors is well reproduced (compare with Fig. 3). (C) Simulated current responses to prolonged application of saturating [GABA]. A smaller rate and extent of apparent desensitization in α3β2γ2 receptor-mediated currents is well reproduced (compare with Fig. 4). (D) Simulated currents elicited using paired-pulse protocol (pair of 2 ms pulses of saturating [GABA] separated by a 75 ms gap) for α1β2γ2 (left) and α3β2γ2 (right) receptors. A larger recovery of the second pulse for α3β2γ2 receptors is properly reproduced (compare with Fig. 5). (E) Simulation of the rising phases of currents elicited by saturating [GABA]. A slower onset rate for α3β2γ2 receptors is correctly reproduced (compare with Fig. 2). (F) Simulations of current responses to a non-saturating GABA concentration (300 µm). A dramatically slower onset of currents mediated by α3β2γ2 receptors properly reproduces the experimental findings (compare with Fig. 1).
F<sc>ig</sc>. 1
Fig. 1
Comparison of rise time for currents elicited by non-saturating GABA in α1β2γ2 and α3β2γ2 channels. (A) Typical examples of normalized α1β2γ2- and α3β2γ2-mediated current responses to a non-saturating (300 µm) GABA concentration. (B) Summary of data derived for the assessment of the onset rate of current elicited at four increasing non-saturating GABA concentrations in at least five patches excised from human embryonic kidney cells expressing α1β2γ2 and α3β2γ2 receptors. *P < 0.05 indicates a significant difference. Note that the difference in the onset rate is so large between the two combinations that the vertical axis is shown in logarithmic scale.
F<sc>ig</sc>. 2
Fig. 2
Comparison of rise time currents elicited by high GABA concentration between α1β2γ2 and α3β2γ2 channels. (A) Typical examples of normalized α1β2γ2- and α3β2γ2-mediated current responses to high GABA concentrations. Arrows point to responses to a distinct concentration for distinct subunit combinations. Note that whereas for α1β2γ2 10 mm GABA saturates the onset rate, in α3β2γ2-mediated responses onset saturation is achieved at 50 mm GABA. (B) Summary of data deriving for the assessment of the 10–90% rise time of current elicited at three distinct concentrations as indicated in at least four patches excised from human embryonic kidney cells expressing α1β2γ2 and α3β2γ2 receptors. *P < 0.05 indicates a significant difference between subunits.
F<sc>ig</sc>. 3
Fig. 3
Comparison of deactivation kinetics for currents elicited by brief GABA pulses between α1β2γ2 and α3β2γ2 channels. (A) Normalized average of 10 responses induced by 2 ms applications of saturating GABA (GABA sat.) to a patch excised from human embryonic kidney cells expressing α1β2γ2 (10 mm GABA) and α3β2γ2 (50 mm GABA) receptors Average currents are shown superimposed with a thick line for α1β2γ2 receptors and a thin line for α3β2γ2 receptors. Inset: the same currents at an expanded time scale. (B–E) Summary of the parameters derived from a triple exponential fitting of decay of α1β2γ2 and α3β2γ2 receptor GABA-gated currents with (in B) the relative contribution of each component to peak amplitude (A1–A3) and (in C–E) each of the time constants. (F) Weighted averages of deactivation time constants are shown for α1β2γ2- and α3β2γ2-mediated currents. Each bar represents the mean ± SEM of six patches for α1β2γ2 receptors and nine patches for α3β2γ2 receptors studied. Traces are normalized to the same peak amplitude. *P < 0.05 indicates a significant difference between subunits.
F<sc>ig</sc>. 4
Fig. 4
Comparison of desensitization of currents elicited by GABA applications between α1β2γ2 and α3β2γ2 channels. (A) Normalized average of five responses induced by 3 s applications of saturating GABA (GABA Sat.) to a patch excised from human embryonic kidney cells expressing α1β2γ2 (10 mm GABA) and α3β2γ2 (50 mm GABA) receptors. Average currents are shown superimposed with a thick line illustrating current from α1β2γ2 receptors and a thin line illustrating that from α3β2γ2 receptors. The initial phase of the GABA currents is shown at an expanded scale in an inset at the bottom right. Traces are normalized to the peak amplitude. (B) Summary of the relative contributions of each component to the peak amplitude (A1–A3 and steady-state) of triple exponential curves used to fit the decay of α1β2γ2 and α3β2γ2 receptor GABA-gated currents to a steady-state value (SS). (C) Each of the three time constants used for the fitting is reported for α1β2γ2 and α3β2γ2 receptors. Each bar represents the mean ± SEM of three patches for α1β2γ2 receptors and seven patches for α3β2γ2 receptors studied per each subunit combination. *P < 0.05 indicates a significant difference between subunits.
F<sc>ig</sc>. 5
Fig. 5
Recovery process in the paired-pulse experiments differs between α1β2γ2 and α3β2γ2 channels. (A) Examples of normalized averages of five traces evoked by two successive applications of 2 ms GABA (10 and 50 mm) pulses separated by 30 ms intervals in a patch excised from human embryonic kidney (HEK) cells expressing α1β2γ2 (left) and α3β2γ2 (right) receptors. (B) Comparison of the recovery time course of the second response from desensitization in at least 15 patches excised from HEK cells expressing α1β2γ2 (◊) and α3β2γ2 (•) receptors. The percent recovery at each designated separation of two brief GABA pulses is calculated as described in Materials and methods, and plotted against the interpulse interval. Each data point represents the mean ± SEM of eight patches studied. (C) The data in B are shown at an expanded time scale to better illustrate the components of the double exponential fitting of the recovery.
F<sc>ig</sc>. 6
Fig. 6
Comparison between α1β2γ2 and α3β2γ2 GABA-channel currents elicited by long-lasting GABA application. (A) Channel activity evoked by long-lasting applications of GABA to outside-out patches excised from human embryonic kidney (HEK) cells expressing α1β2γ2 (top, 100 µm GABA) and α3β2γ2 (bottom, 600 µm GABA) receptors (holding potential, −100 mV). Inset: a fraction of the single-channel current activity at an expanded time scale. Corresponding calibration bars are shown in the lower right corner of traces. (B) Distribution of open time (top) and burst durations (bottom) for the patches in A. Distributions are fitted with a double exponential function. (C) Summary of chord conductance and open times characterizing the main conductance state of single-channel current in at least 10 patches excised from HEK cells expressing α1β2γ2 and α3β2γ2 receptors. (D) A comparison of average burst length of α1β2γ2 and α3β2γ2 channels in these patches and the parameters (A2, τ1 and τ2) from double exponential fitting of burst duration distributions as in B. *P < 0.05 indicates a significant difference between subunits.
F<sc>ig</sc>. 7
Fig. 7
Comparison between α1β2γ2 and α3β2γ2 GABA-channel currents elicited by brief GABA applications. (A) Multiple records of 1 s illustrating channel activity evoked by 2 ms applications of GABA to outside-out patches in two patches excised from human embryonic kidney (HEK) cells expressing single α1β2γ2 (left, 10 mm GABA) and α3β2γ2 (right, 50 mm GABA) channels (holding potential, −100 mV). The mean current resulting from the average of the single-channel records is shown in the bottom trace (right and left panel). (B) Two records of 2 s illustrating channel activity evoked by 2 ms applications of GABA to outside-out patches in two patches excised from HEK cells expressing many α1β2γ2 (top, 10 mm GABA) and α3β2γ2 (bottom, 50 mm GABA) channels (holding potential, −100 mV). Individual channel activity is visible in the tail of these currents. (C) Comparison of mean burst length for α3β2γ2 and α3β2γ2 channels measured during the entire recording period (4 s). (D) Comparison of mean burst lengths in three epochs: 0–500 ms (Epoch 1), 500–1000 ms (Epoch 2) and >1000 ms (Epoch 3). Note that burst durations tend to decrease with time after applications. (E) Comparison of frequencies of late openings (in Epoch 3) for α1β2γ2 and α3β2γ2 receptors. Note that frequency of late openings is several times larger in α3β2γ2 receptors. *P < 0.05 indicates a significant difference between subunits.

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