Distinct Structural Changes in the GABAA Receptor Elicited by Pentobarbital and GABA

The barbiturate pentobarbital binds to γ-aminobutyric acid type A (GABAA) receptors, and this interaction plays an important role in the anesthetic action of this drug. Depending on its concentration, pentobarbital can potentiate (∼10–100 μM), activate (∼100–800 μM), or block (∼1–10 mM) the channel, but the mechanisms underlying these three distinct actions are poorly understood. To investigate the drug-induced structural rearrangements in the GABAA receptor, we labeled cysteine mutant receptors expressed in Xenopus oocytes with the sulfhydryl-reactive, environmentally sensitive fluorescent probe tetramethylrhodamine-6-maleimide (TMRM). We then used combined voltage clamp and fluorometry to monitor pentobarbital-induced channel activity and local protein movements simultaneously in real time. High concentrations of pentobarbital induced a decrease in TMRM fluorescence (FTMRM) of labels tethered to two residues in the extracellular domain (α1L127C and β2L125C) that have been shown previously to produce an increase in FTMRM in response to GABA. Label at β2K274C in the extracellular end of the M2 transmembrane helix reported a small but significant FTMRM increase during application of low modulating pentobarbital concentrations, and it showed a much greater FTMRM increase at higher concentrations. In contrast, GABA decreased FTMRM at this site. These results indicate that GABA and pentobarbital induce different structural rearrangements in the receptor, and thus activate the receptor by different mechanisms. Labels at α1L127C and β2K274C change their fluorescence by substantial amounts during channel blockade by pentobarbital. In contrast, picrotoxin blockade produces no change in FTMRM at these sites, and the pattern of FTMRM signals elicited by the antagonist SR95531 differs from that produced by other antagonists. Thus, with either channel block by antagonists or activation by agonists, the structural changes in the GABAA receptor protein differ during transitions that are functionally equivalent.