Modeling kinetic modes of GABAA receptor gating: A single channel study
Lema, Gareth M. C.
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The type A γ-aminobutyric acid receptor (GABAR) is the principal mediator of fast inhibition in the mammalian brain. Upon activation by agonist, the GABAR 'gates', allowing chloride ions to permeate neuronal membranes and produce fast inhibition. The GABAR is ubiquitously expressed but the localization of GABAR subtypes by brain region, with development of the CNS and even along the cell membrane is tightly controlled. Disruption of GABAerigic inhibition is implicated in various neurological and psychological disorders. These properties make the GABAR an ideal drug target. Developing a model for GABAR gating will inform our understating of all of these processes. Despite several previous studies, a consensus has not yet formed about a comprehensive model for the GABAR gating mechanism. The goal of this thesis research has been to develop a comprehensive model for GABAR gating. I expressed human α 1 β 1 γ 2S GABARs in HEK 293 cells and recorded single-channel currents in the cell-attached configuration using various GABA concentrations (50-5000 μM). GABAR gating activity was heterogeneous and complex. Closed and open events occurred individually and as clusters that could be divided into at least three different modes that were distinguishable by open probability (P O ), mean open time, and mean closed time: High (P O = 0.73), Mid (P O = 0.50), and Low (P O = 0.21). We used a critical time to separate shorter bursts of gating-associated events from the clusters, which also contained short desensitized states. Bursts contained three closed (High-Low, in ms: 0.18-0.3, 0.69-2.28, and 3.44-7.60) and three open components (High-Low, in ms: 0.44-0.28, 2.97-0.87, 8.13-9.08). I employed maximum likelihood fitting, autocorrelation analysis and macroscopic current simulation to identify two optimal models for GABAR gating. The principal gating scheme of both models contained two closed states that the receptor was required to pass through to access the conducting states (C 1 [Lef-right arrow]C 2 [Lef-right arrow]O 1 ). I estimated rate constants for two identical, independent GABA binding steps by globally-fitting data across GABA concentrations ranging from 50-1000 μM. For our most highly ranked model, the binding rate constants were: k (+) = 2.97 μM -1 s -1 and k (-) = 272 s -1 (K D = 91.6).