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  • The next level of possible correlation

    2024-05-23

    The next level of possible correlation and comparison between the α7 and 5HT3 receptors is at the subunit arrangement and protein folding. Crystal structures of the nicotinic and serotoninergic Cy5 TSA Fluorescence System Kit receptors [45], [46] reinforced the concept of similarities between these families of receptors. Namely, the perfect conservation of the β-sheet organization of the N-terminal domain with the formation of the ligand binding pocket at the interface between two adjacent subunits supports the hypothesis that these receptor families emerged from a common ancestor. The similarities between the transmembrane domains, their organization and structural elements are also indicative of the common mechanisms for receptor activation and the ion permeation. Some differences can be observed at the level of the intracellular domain with an extra α–helix (MA) in the 5HT3 receptors. However, as current data for the 5HT3 receptors are obtained with an engineered construct in which the intracellular domain is partially removed, further experiments will be necessary to refine our knowledge on the tridimensional organization of the receptor (see Fig. 2). Regarding the negative outcome of the EVP-6124 Phase III clinical trial it is important to note that this study was conducted using a much different dosing approach than other studies such as in the case of the PNU-282987 or the SSR-180711 trials. Namely, while most of the experiments conducted with α7 specific nAChR agonists used a dose (concentration) range that is able to stimulate α7 receptors, EVP-6124 was administered in the Ki range for this compound which is about two orders of magnitude lower than the in vitro EC50. To reconcile these large differences between the concentration used and the EC50, the physiological properties of the α7 nAChRs must be considered. The agonist activity of a compound is defined as a concentration able to evoke a response by the receptor, or in other words, to activate the receptors by stabilizing them in the open conformation. For the α7 nAChR it was shown that the half activation of the receptor or its EC50 was observed at about 200 μM for ACh, 10 μM for nicotine and 0.39 μM for EVP-6124 [47]. It should be noted; however, that one unique feature of nAChRs (and especially of α7 receptors) is their fast desensitization. This means that in the presence of continuous exposure to the ligand the receptors will not be continuously stabilized in the open conformation, but will rapidly enter a desensitized closed state. As a consequence, the α7 response is a fast and transient inward current lasting a few hundreds of milliseconds. The Ki of a receptor represents the binding affinity for a molecule measured at equilibrium. This means that the receptors must be exposed to the ligand for a prolonged period of time, generally around several minutes. In physiological terms for the nAChR this corresponds to a desensitized state. Interestingly, evaluation of the desensitization caused by a ligand closely matches the Ki value. This is exemplified for the EVP-6124, which displays a Ki of displacement of α-bungarotoxin at 4.33 nM and an IC50 at 3 nM. A superimposed activation and inhibition, or desensitization profile for nicotine at the human α7 is shown in Fig. 2.
    nAChR priming The cognitive improvement observed with α7 specific agonists such as PNU-282987 [48] SSR-180711 [49] or EVP-6124 [47], at concentrations that are below the activation threshold, constituted a real challenge for explaining their mode of action. For example, how could exposure to low concentrations of an α7 agonist attenuate the scopolamine induced deficit or be inhibited by competitive antagonists such as methyllycaconitine (MLA)? If the cognitive enhancement was attributable to the desensitization caused by the prolonged exposure to the agonist, which is generally in order of several minutes, co-application of MLA should have enhanced these effects. Examination of the effects of low doses of α7 agonists on the acetylcholine (ACh) evoked currents revealed a surprising effect. Namely, while application at regular interval of brief low ACh-test pulses showed a stable response in the control conditions, larger currents were observed in presence of subthreshold concentrations of agonist. While these effects where transient during regular ACh-application and progressively decreased, probably due to desensitization, a larger and stable increase in the ACh-evoked current was observed during irregular stimulations [47]. Referred to as priming, this enhancement of the ACh-evoked current by subthreshold concentrations of an agonist is thought to arise by the occupancy of at least one binding site that would facilitate receptor activation upon ACh exposure. In support of this hypothesis, it was shown that priming occurs at active brain concentrations [50]. Moreover, as increasing further the agonist concentration would cause a progressive receptor desensitization these data explain the inverted-U commonly observed in cognitive experiments with α7 agonists [51].