Modes and models of GABAA receptor gating

Upon activation by agonist, the type A gamma-aminobutyric acid receptor (GABAR) 'gates', allowing chloride ions to permeate membranes and produce fast inhibition of neurons. There is no consensus kinetic model for the GABAR gating mechanism. We expressed human alpha(1)beta(1)gamma(2S) GABARs in HEK 293 cells and recorded single channel currents in the cell-attached configuration using various GABA concentrations (50-5000 mu m). Closed and open events occurred individually and in clusters that had at least three different modes that were distinguishable by open probability (P-O): High (P-O= 0.73), Mid (P-O= 0.50), and Low (P-O= 0.21). We used a critical time to isolate shorter bursts of openings and to thus eliminate long-lived, desensitized events. Bursts from all three modes contained three closed and three open components. We employed maximum likelihood fitting, autocorrelation analysis and macroscopic current simulation to distinguish kinetic schemes. The 'core' gating scheme for most models contained two closed states that preceded an open state (C-1 -><- C-2 -><- O-1). The two best-fitting models had a third closed state connected to C-1 and a second open state (O-2) connected to C-2. The third open state, whose occupancy varied greatly between modes, could be connected either to O-2 or C-2. We estimated rate constants for two identical, independent GABA binding steps by globally fitting data across GABA concentrations ranging from 50 to 1000 mu m. For the most highly ranked model the binding rate constants were: k(+)= 3 mu m(-1) s(-1) and k(-)= 272 s(-1) (K-D= 91 mu m).