The activation mechanism of α1β2γ2S and α3β3γ2S GABAA receptors

Abstract

The α1β2γ2 and α3β3γ2 are two isoforms of γ-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. α1β2γ2 channels exhibited briefer active periods than α3β3γ2 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 α3β3γ2 channels was ∼2.6 µM compared with ∼19 µM for α1β2γ2 channels, suggesting that GABA binds to the α3β3γ2 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 α3β3γ2 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 α3β3γ2 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.