The activation mechanism of alpha1beta2gamma2S and alpha3beta3gamma2S GABAA receptors

Abstract

The alpha1beta2gamma2 and alpha3beta3gamma2 are two isoforms of gamma-aminobutyric acid type A (GABAA) receptor that are widely distributed in the brain. Both are found at synapses, for example in the thalamus, where they mediate distinctly different inhibitory postsynaptic current profiles, particularly with respect to decay time. The two isoforms were expressed in HEK293 cells, and single-channel activity was recorded from outside-out patches. The kinetic characteristics of both isoforms were investigated by analyzing single-channel currents over a wide range of GABA concentrations. Alpha1beta2gamma2 channels exhibited briefer active periods than alpha3beta3gamma2 channels over the entire range of agonist concentrations and had lower intraburst open probabilities at subsaturating concentrations. Activation mechanisms were constructed by fitting postulated schemes to data recorded at saturating and subsaturating GABA concentrations simultaneously. Reaction mechanisms were ranked according to log-likelihood values and how accurately they simulated ensemble currents. The highest ranked mechanism for both channels consisted of two sequential binding steps, followed by three conducting and three nonconducting configurations. The equilibrium dissociation constant for GABA at alpha3beta3gamma2 channels was approximately 2.6 microM compared with approximately 19 microM for alpha1beta2gamma2 channels, suggesting that GABA binds to the alpha3beta3gamma2 channels with higher affinity. A notable feature of the mechanism was that two consecutive doubly liganded shut states preceded all three open configurations. The lifetime of the third shut state was briefer for the alpha3beta3gamma2 channels. The longer active periods, higher affinity, and preference for conducting states are consistent with the slower decay of inhibitory currents at synapses that contain alpha3beta3gamma2 channels. The reaction mechanism we describe here may also be appropriate for the analysis of other types of GABAA receptors and provides a framework for rational investigation of the kinetic effects of a variety of therapeutic agents that activate or modulate GABAA receptors and hence influence synaptic and extrasynaptic inhibition in the central nervous system.

Figure 1.

Conductance levels of α₁β₂γ_2S and α₃β₃γ_2S GABA_A receptors. (A) Continuous sweeps of single-channel activity recorded from a patch expressing α₁β₂γ_2S GABA_A receptors in the presence of 5 mM GABA. Note the two conductance levels indicated by broken lines and the transition from the larger conductance to the smaller, and back again. (B) Amplitude histograms for the data shown in A. The two amplitudes are indicated on the plot. (C) Two separate segments of single-channel activity recorded from the same patch expressing α₃β₃γ_2S GABA_A receptors in the presence of 5 mM GABA. The two conductance levels can also occur as discrete long clusters of either the small conductance (top trace) or the large conductance (bottom trace). (D) Amplitude histograms for the data shown in C. The two amplitudes are indicated on the plot. Openings are downward deflections, and the holding potential was −70 mV in both cases.

Figure 2.

Current–voltage experiments for α₁β₂γ_2S and α₃β₃γ_2S GABA_A receptors. (A) Single-channel activity recorded from the same patch expressing α₁β₂γ_2S GABA_A receptors in the presence of 5 mM GABA. (B) Single-channel activity recorded from the same patch expressing α₃β₃γ_2S GABA_A receptors in the presence of 5 mM GABA. The holding potential for each segment of record is indicated on the far left. On the right of each family of traces are the corresponding group plots of single-channel current amplitude (only the larger conductance openings are potted) as a function of holding potential. The group plots represent data from seven patches for the α₁β₂γ_2S channels and five patches for α₃β₃γ_2S channels. Note the very slight downward concavity of the i-V plots indicating mild inward rectification.

Figure 3.

Selecting segments for kinetic analysis. (A) A low-time resolution recording from a patch expressing α₁β₂γ_2S GABA_A receptors in response to 5 mM GABA. Stretches of single-channel activity were selected by eye and subsequently divided into discrete clusters by applying a critical shut time (t_crit). The most common type of activity (a) was of a high conductance and included segments with an intermediate and high (purple) intra-cluster P_O. Segments were excluded if they exhibited low P_O (b), had inconsistent single-channel conductance, contained overlapping activity from multiple channels (c), occurred as isolated openings (d), or were mainly of the low single-channel conductance (e). (B) A low-time resolution recording from a patch expressing α₃β₃γ_2S GABA_A receptors in response to 5 mM GABA. Segments of activity (a) that were selected by eye include intermediate and high (purple) intra-cluster P_O. α₃β₃γ_2S GABA_A receptors generally activated for longer continuous periods (b) compared with α₁β₂γ_2S GABA_A receptors. Records were filtered to 1.5 kHz for display.

Figure 4.

Burst length and intraburst P_O. (A) Plot of the cluster (5 mM and 200 µM GABA) or burst (20 and 2 µM GABA) durations as a function of GABA concentration for α₁β₂γ_2S GABA_A receptors (filled circles) and α₃β₃γ_2S GABA_A receptors (open squares). (B) Plot of intraburst P_O as a function of GABA concentration for α₁β₂γ_2S GABA_A receptors (filled circles) and α₃β₃γ_2S GABA_A receptors (open squares). The data for both plots were fitted to a Hill-type equation.

Figure 5.

Single-channel activity across GABA concentrations for α₁β₂γ_2S GABA_A receptors. Pairs of continuous sweeps of single-channel activity recorded from patches expressing α₁β₂γ_2S GABA_A receptors in the presence of the GABA concentrations indicated. The records from A and B are from the same patch. On the right of each pair of traces are the corresponding dwell-time distributions for apparent shut and open times. The solid line fit represents a mixture of probability density functions, and the broken lines are the individual components.

Figure 6.

Single-channel activity across GABA concentrations for α₃β₃γ_2S GABA_A receptors. Pairs of continuous sweeps of single-channel activity recorded from patches expressing α₃β₃γ_2S GABA_A receptors in the presence of the GABA concentrations indicated. On the right of each pair of traces are the corresponding dwell-time distributions for apparent shut and open times. The solid line fit represents a mixture of probability density functions, and the broken lines are the individual components.

Figure 7.

Proposed mechanism for GABA_A receptor activation. Reaction schemes obtained from global fitting data across a range of GABA concentrations for α₁β₂γ_2S GABA_A receptors (A) and α₃β₃γ_2S GABA_A receptors (B). The mean forward and backward rate constants are indicated on either side of the double arrows between each of the states. Note the difference in binding rate constants (k_–1 and k₊₁) and backward rate constant (σ₋₁) from A₂R³ to A₂R^3* and the similarity in the flipping constants (φ₋₁ and φ₊₁) between the two channels.

Figure 8.

Simulated ensemble currents. Current simulations generated by Schemes 1, 2, and 4 for the α₁β₂γ_2S GABA_A receptors (black) and α₃β₃γ_2S GABA_A receptors (blue). (A–C) Simulated response to a 1-ms application of agonist. (D–F) Simulated responses to a pair of agonist applications, separated by 24 ms.