Identification of a Domain which Affects Kinetics and Antagonistic Potency of Clozapine at 5-HT3 Receptors

The widely used atypical antipsychotic clozapine is a potent competitive antagonist at 5-HT3 receptors which may contribute to its unique psychopharmacological profile. Clozapine binds to 5-HT3 receptors of various species. However, the structural requirements of the respective binding site for clozapine remain to be determined. Differences in the primary sequences within the 5-HT3A receptor gene in schizophrenic patients may result in an alteration of the antipsychotic potency and/or the side effect profile of clozapine. To determine these structural requirements we constructed chimeras with different 5-HT3A receptor sequences of murine and human origin and expressed these mutants in human embryonic kidney (HEK) 293 cells. Clozapine antagonises recombinant mouse 5-HT3A receptors with higher potency compared to recombinant human 5-HT3A receptors. 5-HT activation curves and clozapine inhibition curves yielded the parameters EC50 and IC50 for all receptors tested in the range of 0.6–2.7 µM and 1.5–83.3 nM, respectively. The use of the Cheng-Prusoff equation to calculate the dissociation constant Kb values for clozapine revealed that an extracellular sequence (length 86 aa) close to the transmembrane domain M1 strongly determines the binding affinity of clozapine. Kb values of clozapine were significantly lower (0.3–1.1 nM) for receptors containing the murine sequence and higher when compared with receptors containing the respective human sequence (5.8–13.4 nM). Thus, individual differences in the primary sequence of 5-HT3 receptors may be crucial for the antipsychotic potency and/or the side effect profile of clozapine

Desipramine targets astrocytes to attenuate synaptic plasticity via modulation of the ephrinA3/EphA4 signalling

Long-term potentiation (LTP), a major cellular correlate of memory storage, depends on activation of the ERK/MAPK signalling pathway, but the cell type-specific localization of activated MAPKs remains unknown. We found that in the CM field of the hippocampus, shortly after LTP induction, an increase in the number of MAPK-positive cells occurred specifically among astrocytes of the stratum radiatum, suggesting a putative role of astrocytes for LTP. Desipramine (DMI) is an antidepressant which is used to treat major depressive disorder, but also other pathologies such as neuropathic pain or attention-deficit/hyperactivity disorder. Tricyclic antidepressants such as DMI may cause memory impairment as a side effect. However, biological underpinnings of this effect still remain unclear. Here, we show that DMI inhibited the astrocytic MAPK activation and thereby hindered synaptic potentiation. These effects correlated with a reduced neuronal activation in the stratum pyramidale, thereby prompting us to analyse a regulator of LTP located at the astrocyte-neuron interface in the stratum radiatum, namely the ephrinA3/EphA4 signalling pathway. DMI enhanced EphA4 clustering, which favoured an increased ephrinA3-mediated EphA4 phosphorylation and elevated EphA4 forward signalling. The co-administration of DMI with the Src inhibitor SU6656, which blocks EphA4 forward signalling, could partially reverse the LTP attenuation, further supporting the targeting of the ephrinA3/EphA4 pathway by DMI. Thus, our findings suggest a putative novel mechanism for DMI to modulate LTP through the regulation of the ephrinA3/EphA4 signalling pathway. A further exploration of the molecular and behavioral consequences of targeting ephrinA3/EphA4 might help to improve the clinical use of DMI. (C) 2016 Elsevier Ltd. All rights reserved

Differences in rundown with regard to charge after multiple activation for human, murine and chimeric receptors.

<p>Significant differences (p<0.05, ANOVA) are indicated by black dots.</p

Potency of clozapine for human and murine 5-HT_3A receptors and different 5-HT_3A receptor chimeras against charge.

<p>Significant differences (p<0.05, ANOVA) are indicated by black dots. Comparisons of IC₅₀ for peak charge.</p

Potency of serotonin for human, mouse and chimeric 5-HT_3A receptors against charge.

<p>Significant differences (p<0.05, ANOVA) are indicated by black dots. Comparisons of EC₅₀ for charge (B).</p

Antagonistic properties of clozapine against chimeric and mutant receptors.

<p>All 5-HT-induced currents through chimeric receptors and the P391R mutant were dose-dependently reduced by clozapine in a competitive manner, however, with different potencies. Representative traces for the control and the effects of different concentrations of clozapine on chimeric 5-HT₃ receptor-mediated currents. The application duration of 5-HT was 2 s.</p

Kinetics of human, murine and chimeric 5-HT_3A receptors after first application of 5-HT for 2 s.

<p>Kinetics of human, murine and chimeric 5-HT_3A receptors after first application of 5-HT for 2 s.</p

Potency of clozapine for human and murine 5-HT_3A receptors and different 5-HT_3A receptor chimeras against peak amplitude.

<p>Significant differences (p<0.05, ANOVA) are indicated by black dots. Comparisons of IC₅₀ for peak amplitude.</p

Figure 4

<p>(A) Rundown kinetics for chimeras. Representative current traces for human, murine receptors and chimeras showing currents after the first and 26^th application of 5-HT. Records were obtained from the same cell. All chimeras tested produced functional currents upon 5-HT activation. However, the currents through the different receptor chimeras showed a strong variation in kinetics and rundown. (B) Repeated 5-HT-applications (26 applications within 40 min) reduced peak currents (left) and charge (right). Differences were only marginally for peak currents, whereas analysis of charge showed more pronounced variation between all receptor types. (C) Charge variations between the different receptor types displayed a good correlation to kinetic parameters such as desensitization, plateau current and deactivation.</p