Furthermore low affinity sites that share no structural homo

Furthermore, low-affinity sites that share no structural homology with the aforementioned sites have been described in the transmembrane domain. It has been discovered that, in α1βγ2 receptors, potentiation of GABA-activated currents by high concentrations of diazepam is biphasic, with a high- and a low-affinity component [36]. Combined AZ 628 of the homologous residues α1S269, β2N265, and γ2S280 in the second transmembrane domains (M2) each to isoleucine abolished this micromolar component of potentiation while the high-affinity component remained unaffected [36]. These residues are part of cavities homologous to the cavity occupied by ivermectin in the crystallized C. elegans GluCl receptors [30]. The homo-pentameric GluCl receptor harbors five identical ivermectin sites, one at each subunit interface. In α1βγ2 GABAA receptors there are four different interfaces, each harboring the corresponding cavity (Figure 1B). At least three different, if not all, interfaces of α1βγ2 receptors may bind diazepam [37]. Additional ligands of this site have been identified 38, 39. It is important to note that the presence of the non-canonical sites is not limited to αβγ receptors, and δ subunit-containing receptors are also modulated via these sites 39, 40.
How Do Benzodiazepines Act at the Molecular Level? Ligands of the high-affinity site include positive allosteric modulators, negative allosteric modulators, and antagonists (Box 3 for nomenclature). The presence of positive allosteric modulators induces a shift in the GABA concentration–response curve to lower concentrations. Conversely, negative allosteric modulators shift it to higher GABA concentrations. Positive allosteric modulators shift the equilibrium between the ligand-bound resting and pre-activated states before channel opening [41], without affecting the maximal current amplitude elicited by GABA. As detected in agonist ligand-binding studies, this is paralleled by increased agonist affinity. Benzodiazepines affect channel opening of GABAA receptors induced by either agonist binding site [42]. For ligands acting at non-canonical sites a detailed analysis is lacking. At the macroscopic level, such ligands also act as positive allosteric modulators, negative allosteric modulators, or antagonists [37]. Benzodiazepine site ‘antagonists’ are being used as benzodiazepine antidotes, and ideally are null modulators – in other words they bind silently without any enhancement or reduction of GABA-elicited charge transfer. However, a perfect null modulator has not yet been described. Many compounds that act as benzodiazepine antagonists in vivo have been shown to modulate at least some receptor isoforms under such experimental conditions that enable the investigation of defined receptors. For example, the clinically used benzodiazepine antidote flumazenil is a (weak) partial negative allosteric modulator. Its action is concentration-dependent. At low concentrations it acts as a weak negative allosteric modulator, and at 1μM flumazenil is an antagonist for receptors expressed in Xenopus oocytes. At higher concentrations flumazenil acts as a weak positive allosteric modulator [43]. Occupancy of allosteric sites can promote the open, desensitized, or closed states of the receptors. Consequently ligands act as positive or negative allosteric modulators. GABAA receptors exist in at least one closed, one ligand-bound pre-activated, one open, and one desensitized conformation [44]. Each of these assumes additional different conformations in the presence of positive and negative allosteric modulators or antagonists of the benzodiazepine binding site. Thus, insight into the complexity of allosteric modulation cannot be gained from crystallographic studies alone because crystallographic structures represent static conformations that might not correspond to any of the physiological conformations.