The activation mechanism of α1β2γ2S and α3β3γ2S GABAA receptors

The alpha(1)beta(2)gamma(2) and alpha(3)beta(3)gamma(2) are two isoforms of gamma-aminobutyric acid type A (GABA(A)) 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 alpha(1)beta(2)gamma(2) channels exhibited briefer active periods than alpha(3)beta(3)gamma(2) 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 alpha(3)beta(3)gamma(2) channels was similar to 2.6 mu M compared with similar to 19 mu M for alpha(1)beta(2)gamma(2) channels, suggesting that GABA binds to the alpha(3)beta(3)gamma(2) 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 alpha(3)beta(3)gamma(2) 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 alpha(3)beta(3)gamma(2) channels. The reaction mechanism we describe here may also be appropriate for the analysis of other types of GABA(A) receptors and provides a framework for rational investigation of the kinetic effects of a variety of therapeutic agents that activate or modulate GABA(A) receptors and hence influence synaptic and extrasynaptic inhibition in the central nervous system.