Distinct conformation of the gamma-aminobutyric acid type A receptor alpha(1) subunit M3 transmembrane region in the presence of different ligands

The GABAA receptor is the main mechanism through which inhibitory neurotransmission occurs in the central nervous system. The receptor is a pentamer of five homologous subunits surrounding a pore. The GABAA receptor also has a diverse pharmacology including modulation by clinically important drugs such as benzodiazepines (BZs) and general intravenous and volatile anesthetics. Understanding the conformational changes that occur upon ligand binding could help elucidate the mechanisms of actions of these drugs at the GABAA receptor. First, only upon binding of the endogenous neurotransmitter GABA, a set of residues in the third transmembrane segment (M3) of the alpha1 subunit become accessible to water as assayed by the substituted-cysteine-accessibility-method (SCAM) with pCMBS - as the cysteine reactive modifier. Using this state dependence of modification as a marker, when SCAM is performed in the presence of a BZ, a subset of the residues accessible in the presence of GABA become accessible. These residues were not accessible when BZ binding site antagonists were used at equipotent concentrations. Sequential and co-applications of pCMBS - with GABA and/or BZ at the accessible residue F296C further suggested that this BZ-induced conformation involves modification of only one of two alpha subunits, likely the one adjacent to the gamma subunit. When SCAM is performed in the presence of the general anesthetic propofol, two different patterns of accessible M3 residues are found depending on whether propofol potentiates or activates the receptor. The differences between the two concentrations of propofol are likely due to modification during different states of the receptor. For potentiating propofol the state is likely a ligand-bound but closed state, different than the BZ-induced state. For activating propofol, the state is likely an open state, desensitized state or mix of the two that are different from the GABA-induced state(s) due to slightly different kinetics of propofol and GABA. These different ligand induced conformations may be important in the mechanism of action of these drugs: for example, the conformational change in one alpha subunit by BZ may increase the affinity of the receptor for GABA at its binding sites; for potentiating propofol, these changes may help stabilize an open state.