Pharmacological Characterization of the Zebrafish (Danio Rerio) Histamine H1 Receptor Reveals the Involvement of the Second Extracellular Loop in the Binding of Histamine.

The zebrafish (Danio rerio) histamine H1 receptor gene (zfH1R) was cloned in 2007 and reported to be involved in fish locomotion. Yet, no detailed characterization of its pharmacology and signaling properties have so far been reported. In this study, we pharmacologically characterized the zfH1R expressed in HEK-293T cells by means of [3H]-mepyramine binding and G protein-signaling assays. The zfH1R [dissociation constant (KD), 0.7 nM] displayed similar affinity for the antagonist [3H]-mepyramine as the human histamine H1 receptor (hH1R) (KD, 1.5 nM), whereas the affinity for histamine is 100-fold higher than for the human H1R. The zfH1R couples to Gαq/11 proteins and activates several reporter genes, i.e., NFAT, NFÏ°B, CRE, VEGF, COX-2, SRE, and AP-1, and zfH1R-mediated signaling is prevented by the Gαq/11 inhibitor YM-254890 and the antagonist mepyramine. Molecular modeling of the zfH1R and human H1R shows that the binding pockets are identical, implying that variations along the ligand binding pathway could underly the differences in histamine affinity instead. Targeting differentially charged residues in extracellular loop 2 (ECL2) using site-directed mutagenesis revealed that Arg21045x55 is most likely involved in the binding process of histamine in zfH1R. This study aids the understanding of the pharmacological differences between H1R orthologs and the role of ECL2 in histamine binding and provides fundamental information for the understanding of the histaminergic system in the zebrafish. SIGNIFICANCE STATEMENT: The use of the zebrafish as in vivo models in neuroscience is growing exponentially, which asks for detailed characterization of the aminergic neurotransmitter systems in this model. This study is the first to pharmacologically characterize the zebrafish histamine H1 receptor after expression in HEK-293T cells. The results show a high pharmacological and functional resemblance with the human ortholog but also reveal interesting structural differences and unveils an important role of the second extracellular loop in histamine binding.

Structural and Molecular Determinants for Isoform Bias at Human Histamine H3 Receptor Isoforms.

The human histamine H3 receptor (hH3R) is predominantly expressed in the CNS, where it regulates the synthesis and release of histamine and other neurotransmitters. Due to its neuromodulatory role, the hH3R has been associated with various CNS disorders, including Alzheimer's and Parkinson's disease. Markedly, the hH3R gene undergoes extensive splicing, resulting in 20 isoforms, of which 7TM isoforms exhibit variations in the intracellular loop 3 (IL3) and/or C-terminal tail. Particularly, hH3R isoforms that display variations in IL3 (e.g., hH3R-365) are shown to differentially signal via Gαi-dependent pathways upon binding of biased agonists (e.g., immepip, proxifan, imetit). Nevertheless, the mechanisms underlying biased agonism at hH3R isoforms remain unknown. Using a structure-function relationship study with a broad range of H3R agonists, we thereby explored determinants underlying isoform bias at hH3R isoforms that exhibit variations in IL3 (i.e., hH3R-445, -415, -365, and -329) in a Gαi-dependent pathway (cAMP inhibition). Hence, we systematically characterized hH3R isoforms on isoform bias by comparing various ligand properties (i.e., structural and molecular) to the degree of isoform bias. Importantly, our study provides novel insights into the structural and molecular basis of receptor isoform bias, highlighting the importance to study GPCRs with multiple isoforms to better tailor drugs.

Cell-based and in-silico studies on the high intrinsic activity of two boron-containing salbutamol derivatives at the human ß2-adrenoceptor.

Salbutamol is a well-known ß(2) adrenoceptor (ß(2)AR) partial agonist. We synthesized two boron-containing salbutamol derivatives (BCSDs) with greater potency and efficacy, compared to salbutamol, for inducing ß(2)AR-mediated smooth-muscle relaxation in guinea-pig tracheal rings. However, the mechanism involved in this pharmacological effect remains unclear. In order to gain insight, we carried out binding and functional assays for BCSDs in HEK-293T cells transfected with the human ß(2)AR (hß(2)AR). The transfected hß(2)AR showed similar affinity for BCSDs and salbutamol, but adenosine 3',5'-cyclic phosphate (cAMP) accumulation induced by both BCSDs was similar to that elicited by isoproterenol and greater than that induced by salbutamol. The boron-containing precursors (boric and phenylboronic acids, 100 µM) had no significant effect on salbutamol binding or salbutamol-induced cAMP accumulation. These experimental results are in agreement with theoretical docking simulations on lipid bilayer membrane-embedded hß(2)AR structures. These receptors showed slightly higher affinity for BCSDs than for salbutamol. An essential change between putative active and inactive conformational states depended on the interaction of the tested ligands with the fifth, sixth and seventh transmembrane domains. Overall, these data suggest that BCSDs induce and stabilize conformational states of the hß(2)AR that are highly capable of stimulating cAMP production.